Library molecule titration for tunable surface density in polony sequencing

Abstract

The present disclosure provides compositions, apparatus and methods for conducting separate sequencing batches on a support having nucleic acid template molecules immobilized thereon, where the separate sequencing batches can be conducted using any massively parallel sequencing technology. In some embodiments, a plurality of sub-populations of nucleic acid template molecules are immobilized to the support including at least a first and second sub-population. In some embodiments, the first sub-population of template molecules undergo first batch sequencing reactions and a region of the support is imaged to detect the first sequencing reactions, wherein the second sub-population of template molecules do not undergo sequencing reactions. In some embodiments, the second sub-population of template molecules undergo second batch sequencing and the same region of the support is imaged to detect the second sequencing reactions, wherein the first sub-population of template molecules do not undergo sequencing reactions.

Description

Attorney Docket No. ELEM-025 / 001WO 340101-2208LIBRARY MOLECULE TITRATION FOR TUNABLE SURFACE DENSITY IN POLONY SEQUENCINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and benefit of, U.S. Provisional Application No.63 / 573,300, filed on April 2, 2024, the contents of which are incorporated by reference in their entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (ELEM_025_001WO_SeqList_ST26.xml; Size: 32,233 bytes; and Date of Creation: March 27, 2025) are herein incorporated by reference in their entireties.TECHNICAL FIELD

[0003] The present disclosure provides compositions, apparatus and methods for conducting separate batches of nucleic acid sequencing on a support. In some embodiments, the separate batches of sequencing can be performed on a support comprising a plurality of nucleic acid template molecules immobilized to the support at high density.BACKGROUND

[0004] Massively parallel sequencing methods have applications in biomedical research and healthcare setting as they allow for analyzing large quantities of biological samples. However, the limit of optical resolution impedes the ability to perform highly multiplex sequencing. Current technologies are unable to deal with large numbers of molecules being analyzed as they lead to over-crowding signals and images during sequencing, and ultimately lead to increased costs and time when using these methods. Thus, there exists a need for improved methods of which can be used for performing highly multiplex sequencing.SUMMARY

[0005] The disclosure provides a method for nucleic acid sequencing comprising: (a) providing a support comprising a plurality of nucleic acid template molecules immobilized to the support, wherein the plurality of nucleic acid template molecules comprises at least a first and a second sub-population of template molecules, wherein individual template molecules in 1316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208the first sub-population of template molecules comprises a first batch sequencing primer binding site, a first batch barcode sequence and at least one first sequence-of-interest, wherein the individual template molecules in the second sub-population of template molecules comprises a second batch sequencing primer binding site, a second batch barcode sequence and at least one second sequence-of-interest, (b) sequencing the first sub-population of template molecules using a plurality of first batch sequencing primers, thereby generating a plurality of first batch sequencing read products and imaging a region of the support to detect the first batch sequencing read products; and (c) sequencing the second sub-population of template molecules using a plurality of second batch sequencing primers, thereby generating a plurality of second batch sequencing read products and imaging the same region of the support to detect the second batch sequencing read products.

[0006] In some embodiments of the method of the disclosure, the first batch sequencing primer binding site and the second batch sequencing primer binding site have different sequences. In some embodiments, the first batch barcode sequence and the second batch barcode sequence are different.

[0007] In some embodiments, sequencing the first sub-population of template molecules of step (b) comprises: Step (bl): conducting short read sequencing by performing up to 1000 sequencing cycles of the first sub-population of template molecules to generate a plurality of first batch sequencing read products that comprise up to 1000 bases in length; Step (b2): stopping and / or blocking the short read sequencing of step (bl); Step (b3): removing the plurality of first batch sequencing read products and retaining the first sub-population of template molecules; and optionally Step (b4): repeating steps (bl) - (b3) at least once.

[0008] In some embodiments, sequencing the second sub-population of template molecules of step (c) comprises: Step (cl): conducting short read sequencing by performing up to 1000 sequencing cycles of the second sub-population of template molecules to generate a plurality of second batch sequencing read products that comprise up to 1000 bases in length; Step (c2): stopping and / or blocking the short read sequencing of step (cl); Step (c3): removing the plurality of second batch sequencing read products and retaining the second sub-population of template molecules; and optionally Step (c4): repeating steps (cl) - (c3) at least once.

[0009] In some embodiments, the first sub-population of template molecules have the same first batch sequencing primer binding site, and have the same sequence of interest or different sequences of interest.2316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0010] In some embodiments, the individual template molecules of the second subpopulation of template molecules have the same second batch sequencing primer binding site, and have the same sequence of interest or different sequences of interest.

[0011] In some embodiments, the plurality of nucleic acid template molecules immobilized to the support are at a density of about 102- 1015template molecules per mm2. In some embodiments, the plurality of nucleic acid template molecules are immobilized to the support at a high density. In some embodiments, at least some individual template molecules of the first and second sub-populations of template molecules comprise nearest neighbor template molecules that touch each other and / or overlap each other when viewed from any angle of the support including above, below or side views of the support. In some embodiments, the support lacks partitions and / or barriers that separate regions of the support. In some embodiments, the plurality of template molecules are immobilized to the support at random and non-determined positions on the support. In some embodiments, the plurality of template molecules are immobilized to the support at pre-determined positions on the support (e.g., a patterned support).

[0012] In some embodiments, the plurality of nucleic acid template molecules comprises concatemer template molecules comprising at least a first and second sub-population of concatemer template molecules.

[0013] In some embodiments, individual concatemer template molecules in the first subpopulation of concatemer template molecules comprise a plurality of tandem polynucleotide units comprising a first sequence of interest, a first batch sequencing primer binding site sequence which corresponds to the first sequence of interest, and a first batch barcode sequence which corresponds to the first sequence of interest. In some embodiments, individual concatemer template molecules in the second sub-population of concatemer template molecules comprise a plurality of tandem polynucleotide units comprising a second sequence of interest, a second batch sequencing primer binding site sequence which corresponds to the second sequence of interest, and a second batch barcode sequence which corresponds to the second sequence of interest.

[0014] In some embodiments, the first batch sequencing read products comprise: the first batch barcode sequence; or the first batch barcode sequence and the first sequence of interest.

[0015] In some embodiments, the second batch sequencing read products comprise: the second batch barcode sequence; or the second batch barcode sequence and the second sequence of interest.3316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0016] The disclosure provides a method for re-seeding a support, comprising: (a) providing a support comprising a plurality of surface capture primers immobilized to the support; (b) distributing on the support a first plurality of circularized library molecules under a condition suitable for hybridizing individual circularized library molecules to individual surface capture primers, and conducting a first rolling circle amplification reaction thereby generating a first plurality of concatemer template molecules immobilized to the support; (c) sequencing at least a subset of the first plurality of concatemer template molecules, thereby generating a first plurality of sequencing read products; (d) distributing on the support a second plurality of circularized library molecules under a condition suitable for hybridizing individual circularized library molecules of the second plurality to individual surface capture primers, and conducting a second rolling circle amplification reaction thereby generating a second plurality of concatemer template molecules immobilized to the support; and (e) sequencing at least a subset of the second plurality of concatemer template molecules, thereby generating a second plurality of sequencing read products.

[0017] In some embodiments, the first plurality of circularized library molecules comprises: circularized padlock probes; linear library molecules circularized using singlestranded splint strands; linear library molecules circularized using double-stranded adaptors; or a mixture of any combination of circularized padlock probes, linear library molecules circularized using single-stranded splint strands and / or linear library molecules circularized using double-stranded adaptors.

[0018] In some embodiments, the plurality of surface capture primers are immobilized to the support at random and non-pre-determined positions. In some embodiments, the plurality of surface capture primers are immobilized to the support at pre-determined positions.

[0019] In some embodiments, individual circularized library molecules in the first plurality of circularized library molecules comprise a first seeding batch sequencing primer binding site, a first seeding batch barcode sequence, and a first sequence of interest.

[0020] In some embodiments, the first plurality of sequencing read products of step (c) comprises: a first seeding batch barcode sequence; or a first seeding batch barcode sequence and a first sequence of interest.

[0021] In some embodiments, second individual circularized library molecules in the second plurality of circularized library molecules comprise a second seeding batch sequencing primer binding site, a second seeding batch barcode sequence, and a second sequence of interest.4316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0022] In some embodiments, the second plurality of sequencing read products of step (e) comprises: a second seeding batch barcode sequence; or a second seeding batch barcode sequence and a second sequence of interest.

[0023] In some embodiments, sequencing at least the subset of the first plurality of concatemer template molecules of step (c) comprises: Step (cl): conducting short read sequencing by performing up to 1000 sequencing cycles of the first plurality of concatemer template molecules to generate a first plurality of sequencing read products that comprise up to 1000 bases in length; Step (c2): stopping and / or blocking the short read sequencing of step (cl); Step (c3): removing the first plurality of sequencing read products and retaining the first plurality of immobilized concatemer template molecules; and optionally Step (c4): repeating steps (cl) - (c3) at least once.

[0024] In some embodiments, the sequencing at least the subset of the second plurality of concatemer template molecules of step (e) comprises: Step (el): conducting short read sequencing by performing up to 1000 sequencing cycles of the second plurality of concatemer template molecules to generate a second plurality of sequencing read products that comprise up to 1000 bases in length; Step (e2): stopping and / or blocking the short read sequencing of step (el); Step (e3): removing the second plurality of sequencing read products and retaining the second plurality of immobilized concatemer template molecules; and optionally Step (e4): repeating steps (el) - (e3) at least once.

[0025] In some embodiments, the plurality of surface capture primers immobilized to the support are at a density of about 102- 1015capture primers per mm2. In some embodiments, at least some of the surface capture primers comprise nearest neighbor surface capture primers that touch each other and / or overlap each other when viewed from any angle of the support including above, below or side views of the support. In some embodiments, the support lacks partitions and / or barriers that separate regions of the support.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:5316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0027] FIG. 1 is a schematic of various exemplary configurations of multivalent molecules.Left (Class I): schematics of multivalent molecules having a “starburst” or “helter-skelter” configuration. Center (Class II): a schematic of a multivalent molecule having a dendrimer configuration. Right (Class III): a schematic of multiple multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS with biotin and dNTPs. Nucleotide units are designated ‘N’, biotin is designated ‘B’, and streptavidin is designated ‘SA’.

[0028] FIG. 2 is a schematic of an exemplary multivalent molecule comprising a generic core attached to a plurality of nucleotide-arms.

[0029] FIG. 3 is a schematic of an exemplary multivalent molecule comprising a dendrimer core attached to a plurality of nucleotide-arms.

[0030] FIG. 4 is a schematic of an exemplary multivalent molecule comprising a core attached to a plurality of nucleotide-arms, where the nucleotide-arms comprise biotin, spacer, linker and a nucleotide unit.

[0031] FIG. 5 is a schematic of an exemplary nucleotide-arm comprising a core attachment moiety, spacer, linker and nucleotide unit.

[0032] FIG. 6 shows the chemical structure of an exemplary spacer (top), and the chemical structures of various exemplary linkers, including an 11 -atom Linker, 16-atom Linker, 23-atom Linker, and an N3 Linker (bottom).

[0033] FIG. 7 shows the chemical structures of various exemplary linkers, including Linkers 1-9.

[0034] FIG. 8 shows the chemical structures of various exemplary linkers joined / attached to nucleotide units.

[0035] FIG. 9 shows the chemical structures of various exemplary linkers joined / attached to nucleotide units.

[0036] FIG. 10 shows the chemical structures of various exemplary linkers joined / attached to nucleotide units.

[0037] FIG. 11 shows the chemical structure of an exemplary biotinylated nucleotide-arm. In this example, the nucleotide unit is connected to the linker via a propargyl amine attachment at the 5 position of a pyrimidine base or the 7 position of a purine base.

[0038] FIG. 12 is a schematic of a guanine tetrad (e.g., a G-tetrad).

[0039] FIG. 13 is a schematic of an exemplary intramolecular G-quadruplex structure.

[0040] FIG. 14A is a pair of schematics, (i) and (ii), of an exemplary support having a plurality of nucleic acid capture primers arranged on the support in a non-predetermined and random manner. In (i), the capture primers can be attached to the support such that some of 6316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208the nearest neighbor capture primers touch each other and / or overlap each other when viewed from any angle of the support including above, below or side views of the support. The dotted lines that surround the four capture primers represents nearest neighbor capture primers that touch each other. In (ii), which is a schematic of the same support shown in (i), individual nucleic acid capture primers are attached to a nucleic acid template molecule having one of four different batch sequences. The different batch sequences of the template molecules are represented by horizontal stripes, vertical dashed, brick, or solid black. The template molecules can attach to the support (e.g., via attachment to the capture primers) such that some of the nearest neighbor template molecules touch each other and / or overlap each other when viewed from any angle of the support including above, below or side views of the support. The dotted lines that surround the four template molecules represent nearest neighbor template molecules that touch each other.

[0041] FIG. 14B is a pair of schematics, (iii) and (iv), of an exemplary support having a plurality of nucleic acid template molecules immobilized to the support (e.g., via attachment to the capture primers) where the template molecules are arranged on the support in a predetermined manner. The template molecule comprise one of four different batch sequences. The different batch sequences of the template molecules are represented by horizontal stripes, vertical dashed, brick, or solid black. For example, the template molecules can be immobilized to the support to form spots arranged in rows and columns (iii), or the template molecules can be immobilized to the support to form stripes (iv).

[0042] FIG. 14C is a schematic of an exemplary low binding support comprising a glass substrate and alternating layers of hydrophilic coatings which are covalently or non-covalently adhered to the glass, and which further comprises chemically reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., capture oligonucleotides). Alternatively, the support can be made of any material such as glass, plastic, or a polymer material.

[0043] FIG. 15A is a schematic showing an exemplary workflow for generating circularized padlock probes, comprising hybridizing first and second target-specific padlock probes to the first and second target molecules (respectively) to generate first (left schematic) and second (right schematic) circularized padlock probes (respectively) having a nick or gap, and closing the nick or gap to generate circularized padlock probes. The first padlock probe (left schematic) can comprise: (i) a batch barcode sequence (i.e., a batch-specific barcode sequence), which corresponds to the first target sequence (Batch BC-1); (ii) a batch-specific sequencing primer (also referred to herein as a “batch sequencing primer”) binding site 7316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208sequence which corresponds to the first target sequence (e.g., Batch Seq-1); (iii) a capture primer binding site; and (iv) a compaction oligonucleotide binding site. The second padlock probe (right schematic) can comprise: (i) a batch barcode sequence (i.e., a batch-specific barcode sequence), which corresponds to the second target sequence (Batch BC-2); (ii) a batch-specific sequencing primer (also referred to herein as a “batch sequencing primer”) binding site sequence which corresponds to the second target sequence (e.g., Batch Seq-2); (iii) a capture primer binding site; and (iv) a compaction oligonucleotide binding site.

[0044] FIG. 15B is a schematic showing an exemplary workflow in which the circularized padlock probes shown in FIG. 15A are subjected to rolling circle amplification (RCA) to generate first (left schematic) and second (right schematic) concatemer template molecules which are immobilized to a support having one type of immobilized capture primers. The RCA reaction can be conducted in-solution using soluble amplification primers or on-support using capture primers immobilized to a support. The first and second circularized padlock probes can be distributed onto the support essentially simultaneously, or distributed onto the support sequentially (e.g., re-seeding the support). The first and second concatemer template molecules can be subjected to a first sequencing workflow using first batch-specific sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of first sequencing read products (dashed arrows), where the first sequencing read products include the first batch barcode sequence (Batch BC-1). The first concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles, but the second concatemer template molecules do not undergo first batch sequencing. The first sequencing read products from the first concatemer template molecules can be up to 1000 bases in length. In addition, or alternatively, the first and second concatemer template molecules can be subjected to a second sequencing workflow using second batch-specific sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of second sequencing read products (dashed arrows), where the second sequencing read products include the second batch barcode sequence (Batch BC-2). The second concatemers can undergo reiterative sequencing comprising up to 1000 sequencing cycles, but the first concatemers do not undergo second batch sequencing. The second sequencing read products from the second concatemers can be up to 1000 bases in length.

[0045] FIG. 16 is a schematic of an exemplary workflow in which circularized padlock probes are subjected to rolling circle amplification (RCA) and batch sequencing. The RCA reaction can be conducted in-solution using soluble amplification primers or on-support using 8316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208capture primers immobilized to a support. The first circularized padlock probe (Left schematic) can comprise: (i) a batch barcode sequence (i.e., a batch-specific barcode sequence) which corresponds to the first target sequence (Batch BC-1); (ii) a batch-specific sequencing primer binding site sequence which corresponds to the first target sequence (e.g., Batch Seq-1); (iii) a first batch capture primer binding site; and (iv) a compaction oligonucleotide binding site. The second padlock probe (Right schematic) can comprise: (i) a batch barcode sequence (i.e., a batch-specific barcode sequence) which corresponds to the second target sequence (Batch BC-2); (ii) a batch-specific sequencing primer binding site sequence which corresponds to the second target sequence (e.g., Batch Seq-2); (iii) a second batch capture primer binding site; and (iv) a compaction oligonucleotide binding site. The first and second circularized padlock probes can be distributed onto a support having two types of immobilized capture primers which selectively hybridize to the first or second batch capture primer binding site sequences in the first or second circularized padlock probes. The circularized padlock probes can be subjected to rolling circle amplification (RCA) to generate first and second concatemer template molecules which are immobilized to the support. For instance, the first and second circularized padlock probes can be distributed onto the support essentially simultaneously, or distributed onto the support sequentially (e.g., re-seeding the support). The first and second concatemer template molecules can be subjected to a first sequencing workflow using first batch-specific sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of first sequencing read products (dashed arrows), where the first sequencing read products include the first batch barcode sequence (Batch BC-1). The first concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles, but the second concatemer template molecules do not undergo first batch sequencing. The first sequencing read products from the first concatemers can be up to 1000 bases in length. In addition, or alternatively, the first and second concatemer template molecules can be subjected to a second sequencing workflow using second batch-specific sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of second sequencing read products (dashed arrows), where the second sequencing read products include the second batch barcode sequence (Batch BC-2). The second concatemers can undergo reiterative sequencing comprising up to 1000 sequencing cycles, but the first concatemers do not undergo second batch sequencing. Alternatively, or in addition, the second sequencing read products from the second concatemers can be up to 1000 bases in length.9316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0046] FIG. 17 is a schematic of an exemplary workflow in which circularized padlock probes are subjected to rolling circle amplification (RCA) and batch sequencing. The RCA reaction can be conducted in-solution using soluble amplification primers or on-support using capture primers immobilized to a support. The first (left schematic) and second (right schematic) circularized padlock probes can comprise: (i) a batch barcode sequence (i.e., a batch-specific barcode sequence) which corresponds to the first target sequence (Batch BC-1); (ii) a batch-specific sequencing primer binding site sequence which corresponds to the first target sequence (e.g., Batch Seq-1); (iii) a first batch capture primer binding site; and (iv) a compaction oligonucleotide binding site. The first and second circularized padlock probes can be distributed onto a support having one type of immobilized capture primers which selectively hybridize to the first batch capture primer binding site sequences in the first and second circularized padlock probes. The circularized padlock probes can be subjected to rolling circle amplification (RCA) to generate first and second concatemer template molecules which are immobilized to the support. For instance, the first and second circularized padlock probes can be distributed onto the support essentially simultaneously, or distributed onto the support sequentially (e.g., re-seeding the support). The first and second concatemer template molecules can be subjected to a first sequencing workflow using first batch-specific sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of first sequencing read products (dashed arrows), where the first sequencing read products include the first batch barcode sequence (Batch BC-1). The first and second concatemer template molecules can undergo reiterative sequencing both comprising up to 1000 sequencing cycles. The first sequencing read products from the first concatemer template molecules can be up to 1000 bases in length. Alternatively, or in addition, the second sequencing read products from the second concatemer can be up to 1000 bases in length.

[0047] FIG. 18 is a schematic of an exemplary workflow in which circularized padlock probes are subjected to rolling circle amplification (RCA) and batch sequencing. The RCA reaction can be conducted in-solution using soluble amplification primers or on-support using capture primers immobilized to a support. The first circularized padlock probe (Left schematic) can comprise: (i) a batch barcode sequence (i.e., a batch-specific barcode sequence) which corresponds to the first target sequence (Batch BC-1); (ii) a batch-specific sequencing primer binding site sequence which corresponds to the first and second target sequence (e.g., Batch Seq-1); (iii) a first batch capture primer binding site; and (iv) a compaction oligonucleotide binding site. The second padlock probe (Right schematic ) can 10316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208comprise: (i) a batch barcode sequence (i.e., a batch-specific barcode sequence) which corresponds to the second target sequence (Batch BC-2); (ii) a batch-specific sequencing primer binding site sequence which corresponds to the first and second target sequence (e.g., Batch Seq-1); (iii) a first batch capture primer binding site; and (iv) a compaction oligonucleotide binding site. The first and second circularized padlock probes can be distributed onto a support having one type of immobilized capture primers which selectively hybridizes to the first batch capture primer binding site sequence in the first and second circularized padlock probes. The circularized padlock probes can be subjected to rolling circle amplification (RCA) to generate first and second concatemer template molecules which are immobilized to the support. For instance, the first and second circularized padlock probes can be distributed onto the support essentially simultaneously, or distributed onto the support sequentially (e.g., re-seeding the support). The first and second concatemer template molecules can be subjected to a first sequencing workflow using first batch-specific sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of first and second sequencing read products (dashed arrows). The first sequencing read products can include the first batch barcode sequence (Batch BC- 1). The first concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles. The first sequencing read products from the first concatemer template molecules can be up to 1000 bases in length. Alternatively, or in addition, the second sequencing read products can include the second batch barcode sequence (Batch BC- 2). The second concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles. The second sequencing read products from the second concatemer template molecules can be up to 1000 bases in length.

[0048] FIG. 19 is a schematic of an exemplary workflow in which circularized padlock probes are subjected to rolling circle amplification (RCA) and batch sequencing. The RCA reaction can be conducted in-solution using soluble amplification primers or on-support using capture primers immobilized to a support. The first circularized padlock probe (Left schematic) can comprise: (i) a batch barcode sequence (i.e., a batch-specific barcode sequence) which corresponds to the first and second target sequence (Batch BC-1); (ii) a batch-specific sequencing primer binding site sequence which corresponds to the first and second target sequence (e.g., Batch Seq-1); (iii) a first batch capture primer binding site; and (iv) a compaction oligonucleotide binding site. The second padlock probe (Right schematic) can comprise: (i) a batch barcode sequence (i.e., a batch-specific barcode sequence) which corresponds to the first and second target sequence (Batch BC-1); (ii) a batch-specific11316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208sequencing primer binding site sequence which corresponds to the first and second target sequence (e.g., Batch Seq-1); (iii) a first batch capture primer binding site; and (iv) a compaction oligonucleotide binding site. The first and second circularized padlock probes can be distributed onto a support having one type of immobilized capture primers which selectively hybridizes to the first batch capture primer binding site sequence in the first and second circularized padlock probes. The circularized padlock probes can be subjected to rolling circle amplification (RCA) to generate first and second concatemer template molecules which are immobilized to the support. For instance, the first and second circularized padlock probes can be distributed onto the support essentially simultaneously, or distributed onto the support sequentially (e.g., re-seeding the support). The first and second concatemer template molecules can be subjected to a first sequencing workflow using first batch-specific sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of first and second sequencing read products (dashed arrows). The first sequencing read products include the first batch barcode sequence (Batch BC-1) and at least a portion of the first target sequence. The first concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles. The first sequencing read products from the first concatemer template molecules can be up to 1000 bases in length. Alternatively, or in addition, the second sequencing read products include the second batch barcode sequence (Batch BC-2) and at least a portion of the second target sequence. The second concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles. The second sequencing read products from the second concatemer template molecules can be up to 1000 bases in length.

[0049] FIG. 20 is a schematic of an exemplary workflow in which circularized padlock probes are subjected to rolling circle amplification (RCA) and batch sequencing. The RCA reaction can be conducted in-solution using soluble amplification primers or on-support using capture primers immobilized to a support. The first circularized padlock probe (Left schematic) can comprise: (i) a first sample index which distinguish sequences of interest obtained from a first sample source (e.g., Sample index-1); (ii) a batch barcode sequence (i.e., a batch-specific barcode sequence) which corresponds to the first target sequence (Batch BC-1); (iii) a batch-specific sequencing primer binding site sequence which corresponds to the first target sequence (e.g., Batch Seq-1); (iv) a first batch capture primer binding site; and (v) a compaction oligonucleotide binding site. The second circularized padlock probe (Right schematic) can comprise: (i) a second sample index which distinguish sequences of interest obtained from a second sample source (e.g., Sample index-2); (ii) a batch barcode sequence 12316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208(i.e., a batch-specific barcode sequence) which corresponds to the first target sequence (Batch BC-1); (iii) a batch-specific sequencing primer binding site sequence which corresponds to the first target sequence (e.g., Batch Seq-1); (iv) a first batch capture primer binding site; and (v) a compaction oligonucleotide binding site. The first and second circularized padlock probes can be distributed onto a support having one type of immobilized capture primers which selectively hybridizes to the first batch capture primer binding site sequence in the first and second circularized padlock probes. The circularized padlock probes can be subjected to rolling circle amplification (RCA) to generate first and second concatemer template molecules which are immobilized to the support. For instance the first and second circularized padlock probes can be distributed onto the support essentially simultaneously, or distributed onto the support sequentially (e.g., re-seeding the support). The first and second concatemer template molecules can be subjected to a first sequencing workflow using first batch-specific sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of first and second sequencing read products (dashed arrows). The first sequencing read products can include the first batch barcode sequence (Batch BC-1) and the first sample index sequence. The first concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles. The first sequencing read products from the first concatemer can be up to 1000 bases in length. Alternatively, or in addition, the second sequencing read products can include the second batch barcode sequence (Batch BC-2) and the second sample index sequence. The second concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles. The second sequencing read products from the second concatemer template molecules can be up to 1000 bases in length.

[0050] FIG. 21 is a schematic of an exemplary workflow of a linear single stranded library molecule (100) hybridizing with a single-stranded splint molecule / strand (200) (ss-splint strand) thereby circularizing the library molecule to form a library-splint complex (300) with a nick which is enzymatically ligatable. The exemplary library molecule (100) can comprise: a surface pinning primer binding site sequence (120) (e.g., a batch-specific surface pinning primer binding site sequence); an optional left unique identification sequence (180) (e.g., UMI); a left sample index sequence (160); a forward sequencing primer binding site sequence (140) (e.g., a batch-specific forward sequencing primer binding site sequence); a sequence of interest (110); a reverse sequencing primer binding site sequence (150) (e.g., a batch-specific reverse sequencing primer binding site sequence); a right sample index sequence (170); and a surface capture primer binding site sequence (130) (e.g., a batch- 13316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208specific surface capture primer binding site sequence). The single-stranded splint strand (200) can comprise a first region (210) that hybridizes with the surface pinning primer binding site sequence (120) of the linear single-stranded library molecule (100), and a second region (220) that hybridizes with the surface capture primer binding site sequence (130) of the linear single-stranded library molecule (100).

[0051] FIG. 22 is a schematic of an exemplary workflow of a linear single stranded library molecule (100) hybridizing with a single-stranded splint molecule / strand (200) (ss-splint strand) thereby circularizing the library molecule to form a library-splint complex (300) with a nick which is enzymatically ligatable. The exemplary linear single stranded library molecule (100) can comprise: a surface pinning primer binding site sequence (120) (e.g., a batch-specific surface pinning primer binding site sequence); a forward sequencing primer binding site sequence (140) (e.g., a batch-specific forward sequencing primer binding site sequence); a batch barcode sequence (195); a left sample index sequence (160); a sequence of interest (110); a reverse sequencing primer binding site sequence (150) (e.g., a batch-specific reverse sequencing primer binding site sequence); a right sample index sequence (170); and a surface capture primer binding site sequence (130) (e.g., a batch-specific surface capture primer binding site sequence). The single-stranded splint strand (200) can comprise a first region (210) that hybridizes with the surface pinning primer binding site sequence (120) of the linear single-stranded library molecule (100), and a second region (220) that hybridizes with the surface capture primer binding site sequence (130) of the linear single-stranded library molecule (100).

[0052] FIG. 23A is a schematic of an exemplary workflow of a first linear single stranded library molecule (100-1) (linear single stranded library molecule-1) hybridizing with a single-stranded splint molecule / strand (200) (ss-splint strand) thereby circularizing the library molecule to form a first library-splint complex (300-1) with a nick which is enzymatically ligatable. The exemplary first linear single stranded library molecule (100-1) can comprise: a first surface pinning primer binding site sequence (120-1); a first batch forward sequencing primer binding site sequence (140-1); a first batch barcode sequence (195-1); a first sample index sequence (160-1); a first sequence of interest (insert 1, 110-1); and a first surface capture primer binding site sequence (130-1). The single-stranded splint strand (200) can comprise a first region (210) that hybridizes with the first surface pinning primer binding site sequence (120-1) of the linear single-stranded library molecule (100), and a second region (220) that hybridizes with the first surface capture primer binding site sequence (130-1) of the linear single-stranded library molecule (100).14316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0053] FIG. 23B is a schematic of an exemplary workflow of a second linear single stranded library molecule (100-2) (linear single stranded library molecule-2) hybridizing with a single-stranded splint molecule / strand (200) (ss-splint strand) thereby circularizing the library molecule to form a second library-splint complex (300-2) with a nick which is enzymatically ligatable. The exemplary second linear single stranded library molecule (100-2) can comprise: a first surface pinning primer binding site sequence (120-1); a second batch forward sequencing primer binding site sequence (140-2); a second batch barcode sequence (195-2); a first sample index sequence (160-1); a second sequence of interest (insert-2, 110-2); and a first surface capture primer binding site sequence (130-1). The single-stranded splint strand (200) can comprise a first region (210) that hybridizes with the first surface pinning primer binding site sequence (120-1) of the linear single-stranded library molecule (100), and a second region (220) that hybridizes with the first surface capture primer binding site sequence (130-1) of the linear single-stranded library molecule (100). The first sequence of interest in the library-splint complex shown in FIG. 23A (110-1) and the second sequence of interest in the library-splint complex shown in FIG. 23B (110-2) can have the same sequence or different sequences.

[0054] FIG. 24A is a schematic of an exemplary workflow in which the nick in the first library-splint complex (300-1) shown in FIG. 23A is ligated to generate a first covalently closed circular library molecule (400-1) which is shown in FIG. 24A. The first covalently closed circular library molecule (400-1) is subjected to rolling circle amplification (RCA) to generate a first concatemer template molecule, and the first concatemer template molecule is subjected to batch reiterative sequencing. The rolling circle amplification reaction can be conducted in-solution using soluble amplification primers or on-support using capture primers immobilized to a support. The first covalently closed circular library molecule (400-1) can comprise: a first surface pinning primer binding site sequence (120-1); a first batch forward sequencing primer binding site sequence (140-1) which corresponds with the first sequence of interest (insert -1, 110-1); a first batch barcode sequence (195-1) which corresponds with the first sequence of interest (110-1); a first sample index sequence (160-1); a first sequence of interest (110-1); and a first surface capture primer binding site sequence (130-1). A plurality of the first covalently closed circular library molecule (400-1) shown in FIG. 24A can be distributed onto a support having one type of immobilized capture primers which selectively hybridizes to the first capture primer binding site sequence (130-1) in the first covalently closed circular library molecules (400-1). The first covalently closed circular library molecules (400-1) can be subjected to rolling circle amplification (RCA) to generate a 15316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208plurality of first concatemer template molecules which are immobilized to the support. The first concatemer template molecules can be subjected to a sequencing workflow using first batch-specific sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of first sequencing read products (dashed arrows). The first sequencing read products can include the first batch barcode sequence (195-1) as shown in FIG. 24A. Alternatively, or in addition, the first sequencing read products can include the first batch barcode sequence (195-1) and the first sample index sequence (160-1) (not shown). Alternatively, or in addition, the first sequencing read products can include the first batch barcode sequence (195-1), the first sample index sequence (160-1), and at least a portion of the first sequence of interest (110-1) (not shown). The first concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles. The first sequencing read products from the first concatemer template molecules can be up to 1000 bases in length.

[0055] FIG. 24B is a schematic of an exemplary workflow in which the nick in the second library-splint complex (300-2) shown in FIG. 23B is ligated to generate a second covalently closed circular library molecule (400-2) which is shown in FIG. 24B. The second covalently closed circular library molecule (400-2) is subjected to rolling circle amplification (RCA) to generate a second concatemer template molecule, and the second concatemer template molecule is subjected to batch reiterative sequencing. The rolling circle amplification reaction can be conducted in-solution using soluble amplification primers or on-support using capture primers immobilized to a support. The second covalently closed circular library molecule (400-2) can comprise: a first surface pinning primer binding site sequence (120-1); a second batch forward sequencing primer binding site sequence (140-2) which corresponds with the second sequence of interest (insert-2, 110-2) ; a second batch barcode sequence (195-2) which corresponds with the second sequence of interest (110-2) ; a first sample index sequence (160-1); a second sequence of interest (110-2) ; and a first surface capture primer binding site sequence (130-1). A plurality of the second covalently closed circular library molecule (400-2) shown in FIG. 24B can be distributed onto a support having one type of immobilized capture primers which selectively hybridizes to the first capture primer binding site sequence (130-1) in the second covalently closed circular library molecules (400-2). A plurality of the first covalently closed circular library molecule (400-1) shown in FIG. 24 A and a plurality of the second covalently closed circular library molecule (400-2) shown in FIG. 24B can be distributed onto the same support. For instance, the first covalently closed circular library molecules (400-1) shown in FIG. 24 A and the second covalently closed 16316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208circular library molecules (400-2) shown in FIG. 24B can be distributed onto the support essentially simultaneously. Alternatively, or in addition, the first covalently closed circular library molecules (400-1) shown in FIG. 24A and the second covalently closed circular library molecules (400-2) shown in FIG. 24B can be distributed onto the support sequentially (e.g., re-seeding the support). The second covalently closed circular library molecules (400-2) can be subjected to rolling circle amplification (RCA) to generate a plurality of second concatemer template molecules which are immobilized to the support. The second concatemer template molecules can be subjected to a sequencing workflow using second batch sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of second sequencing read products (dashed arrows). In some cases, the second concatemer template molecules are not sequenced when first batch sequencing primers are used to sequence the first concatemer template molecules. Alternatively, or in addition, the first concatemer template molecules are not sequenced when second batch sequencing primers are used to sequence the second concatemer template molecules. The second sequencing read products can include the second batch barcode sequence (195-2) as shown in FIG. 24B. Alternatively, or in addition, the second sequencing read products can include the second batch barcode sequence (195-2) and the first sample index sequence (160-1) (not shown). Alternatively, or in addition, the second sequencing read products include the second batch barcode sequence (195-2), the first sample index sequence (160-1), and at least a portion of the second sequence of interest (110-2) (not shown). The second concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles. The second sequencing read products from the second concatemer can be up to 1000 bases in length.

[0056] FIG. 25A is a schematic of an exemplary workflow of a first linear single stranded library molecule (100-1) (linear single-stranded library molecule-1) hybridizing with a singlestranded splint molecule / strand (ss-splint strand) (200) thereby circularizing the library molecule to form a first library-splint complex (300-1) with a nick which is enzymatically ligatable. The exemplary first linear single stranded library molecule (100-1) can comprise: a first surface pinning primer binding site sequence (120-1); a first batch forward sequencing primer binding site sequence (140-1); a first batch barcode sequence (195-1); a first sequence of interest (insert-1, 110-1); and a first surface capture primer binding site sequence (130-1). The single-stranded splint strand (200) can comprise a first region (210) that hybridizes with the first surface pinning primer binding site sequence (120-1) of the linear single-stranded library molecule (100), and a second region (220) that hybridizes with the first surface17316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208capture primer binding site sequence (130-1) of the first linear single-stranded library molecule (100-1).

[0057] FIG. 25B is a schematic of an exemplary workflow of a second single-stranded library molecule (100-2) (linear single-stranded library molecule-2) hybridizing with a singlestranded splint molecule / strand (ss-splint strand) (200) thereby circularizing the library molecule to form a second library-splint complex (300-2) with a nick which is enzymatically ligatable. The exemplary second linear single stranded library molecule (100-2) can comprise: a first surface pinning primer binding site sequence (120-1); a second batch forward sequencing primer binding site sequence (140-2); a second batch barcode sequence (195-2); a second sequence of interest (insert-2, 110-2); and a first surface capture primer binding site sequence (130-1). The single-stranded splint strand (200) can comprise a first region (210) that hybridizes with the first surface pinning primer binding site sequence (120-1) of the linear single-stranded library molecule (100), and a second region (220) that hybridizes with the first surface capture primer binding site sequence (130-1) of the second linear single-stranded library molecule (100-2). The first sequence of interest (110-1) in the first library-splint complex shown in FIG. 25 A and the second sequence of interest (110-2) in the second library-splint complex shown in FIG. 25B can have the same sequence or different sequences.

[0058] FIG. 26A is a schematic of an exemplary workflow in which the nick in the first library-splint complex (300-1) shown in FIG. 25A is ligated to generate a first covalently closed circular library molecule (400-1) which is shown in FIG. 26A. The first covalently closed circular library molecule (400-1) is subjected to rolling circle amplification (RCA) to generate a first concatemer template molecule, and the first concatemer template molecule is subjected to batch reiterative sequencing. The RCA reaction can be conducted in-solution using soluble amplification primers or on-support using capture primers immobilized to a support. The first covalently closed circular library molecule (400-1) can comprise: a first surface pinning primer binding site sequence (120-1); a first batch forward sequencing primer binding site sequence (140-1) which corresponds with the first sequence of interest (insert-1, 110-1); a first batch barcode sequence (195-1) which corresponds with the first sequence of interest (110-1); a first sequence of interest (110-1); and a first surface capture primer binding site sequence (130-1). A plurality of the first covalently closed circular library molecule (400-1) shown in FIG. 26A can be distributed onto a support having one type of immobilized capture primers which selectively hybridizes to the first capture primer binding site sequence (130-1) in the first covalently closed circular library molecules (400-1). The first covalently 18316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208closed circular library molecules (400-1) can be subjected to rolling circle amplification (RCA) to generate a plurality of first concatemer template molecules which are immobilized to the support. The first concatemer template molecules can be subjected to a sequencing workflow using first batch-specific sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of first sequencing read products (dashed arrows). The first sequencing read products can include the first batch barcode sequence (195-1) as shown in FIG. 26A. Alternatively or in addition, the first sequencing read products can include the first batch barcode sequence (195-1) and at least a portion of the first sequence of interest (110-1) (not shown). The first concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles. The first sequencing read products from the first concatemer template molecules can be up to 1000 bases in length.

[0059] FIG. 26B is a schematic of an exemplary workflow in which the nick in the librarysplint complex (300-2) shown in FIG. 25B is ligated to generate a second covalently closed circular library molecule (400-2) which is shown in FIG. 26B. The second covalently closed circular library molecule (400-2) is subjected to rolling circle amplification (RCA) to generate a second concatemer template molecule, and the second concatemer template molecule is subjected to batch reiterative sequencing. The RCA reaction can be conducted insolution using soluble amplification primers or on-support using capture primers immobilized to a support. The second covalently closed circular library molecule (400-2) can comprise: a first surface pinning primer binding site sequence (120-1); a second batch forward sequencing primer binding site sequence (140-2) which corresponds with the second sequence of interest (insert-2, 110-2); a second batch barcode sequence (195-2) which corresponds with the second sequence of interest (110-2); a second sequence of interest (110-2); and a first surface capture primer binding site sequence (130-1). A plurality of the second covalently closed circular library molecule (400-2) shown in FIG. 26B can be distributed onto a support having one type of immobilized capture primers which selectively hybridizes to the first capture primer binding site sequence (130-1) in the second covalently closed circular library molecules (400-2). A plurality of the first covalently closed circular library molecule (400-1) shown in FIG. 26 A and a plurality of the second covalently closed circular library molecule (400-2) shown in FIG. 26B can be distributed onto the same support. For instance, the first covalently closed circular library molecules (400-1) shown in FIG. 26 A and the second covalently closed circular library molecules (400-2) shown in FIG. 26B can be distributed onto the support essentially simultaneously. Alternatively, or in addition, the first 19316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208covalently closed circular library molecules (400-1) shown in FIG. 26 A and the second covalently closed circular library molecules (400-2) shown in FIG. 26B can be distributed onto the support sequentially (e.g., re-seeding the support). The second covalently closed circular library molecules (400-2) can be subjected to rolling circle amplification (RCA) to generate a plurality of second concatemer template molecules which are immobilized to the support. The second concatemer template molecules can be subjected to a sequencing workflow using second batch sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of second sequencing read products (dashed arrows). In some cases, the second concatemer template molecules are not sequenced when first batch sequencing primers are used to sequence the first concatemer template molecules. Alternatively, or in addition, the first concatemer template molecules are not sequenced when second batch sequencing primers are used to sequence the second concatemer template molecules. The second sequencing read products can include the second batch barcode sequence (195-2) as shown in FIG. 26B. Alternatively, or in addition, the second sequencing read products can include the second batch barcode sequence (195-2) and at least a portion of the second sequence of interest (110-2) (not shown). Alternatively, or in addition, the second concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles. The second sequencing read products from the second concatemer template molecule can be up to 1000 bases in length.

[0060] FIG. 27 is a schematic of an exemplary workflow of a linear single-stranded library molecule (100) hybridizing with a double-stranded adaptor (ds-splint adaptor) (500) thereby circularizing the linear single-stranded library molecule to form a library-splint complex (800) with two nicks (solid arrowheads). The exemplary linear single stranded library molecule (100) can comprise: a pinning primer binding site sequence (120) (e.g., a batchspecific pinning primer binding site sequence); an optional left unique identification sequence (180) (e.g., UMI); a left sample index sequence (160); a forward sequencing primer binding site sequence (140) (e.g., a batch-specific forward sequencing primer binding site sequence); a sequence of interest (110); a reverse sequencing primer binding site sequence (150) (e.g., a batch-specific reverse sequencing primer binding site sequence); a right sample index sequence (170); and a surface capture primer binding site sequence (130) (e.g., a batchspecific surface capture primer binding site sequence). The double-stranded adaptor can comprise a first splint strand (600) hybridized to a second splint strand (700). In the doublestranded adaptor, the first splint strand (600) can comprise a first region (620), an internal region (610), and a second region (630), wherein the internal region of the first splint strand 20316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208(610) is hybridized to the second splint strand (700). The second splint strand (700) can comprise a first, a second, and a third subregion, and the internal region (610) of the first splint strand (600) can comprise a fourth, a fifth, and a sixth subregion. The first region (620) of the first splint strand (600) can hybridize to at least a portion of the surface pinning primer binding site sequence (120) of a linear single stranded library molecule (100), and the second region (630) of the first splint strand (600) can hybridize to at least a portion of the surface capture primer binding site sequence (130) of the same linear single stranded nucleic acid library molecule (100).

[0061] FIG. 28 is a schematic of an exemplary workflow of a linear single-stranded library molecule (100) hybridizing with a double-stranded adaptor (500) (ds-splint adaptor) thereby circularizing the library molecule to form a library-splint complex (800) with two nicks (solid arrowheads). The exemplary linear single-stranded library molecule (100) can comprise: a surface pinning primer binding site sequence (120) (e.g., a batch-specific pinning primer binding site sequence); a forward sequencing primer binding site sequence (140) (e.g., batchspecific forward sequencing primer binding site sequence); a batch-specific barcode sequence (195); a left sample index sequence (160); a sequence of interest (110); a reverse sequencing primer binding site sequence (150) (e.g., a batch-specific reverse sequencing primer binding site sequence); a right sample index sequence (170); and a surface capture primer binding site sequence (130) (e.g., a batch-specific surface capture primer binding site sequence). The double-stranded adaptor can comprise a first splint strand (600) hybridized to a second splint strand (700). In the double-stranded adaptor, the first splint strand (600) comprises a first region (620), an internal region (610), and a second region (630), wherein the internal region of the first splint strand (610) is hybridized to the second splint strand (700). The second splint strand (700) can comprise a first, a second, and a third subregion, and the internal region (610) of the first splint strand (600) can comprise a fourth, a fifth, and a sixth subregion. The first region (620) of the first splint strand (600) can hybridize to at least a portion of the surface pinning primer binding site sequence (120) of a linear single stranded nucleic acid library molecule (100), and the second region (630) of the first splint strand (600) can hybridize to at least a portion of the surface capture primer binding site sequence (130) of the same single-stranded nucleic acid library molecule (100).

[0062] FIG. 29 is a schematic of an exemplary workflow of a linear single stranded library molecule (100) hybridizing with a double-stranded adaptor (500) (ds-splint adaptor) thereby circularizing the linear single-stranded library molecule (100) to form a library-splint complex (800) with two nicks (solid arrowheads). The exemplary library molecule (100) can 21316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208comprise: a surface pinning primer binding site sequence (120) (e.g., a batch-specific pinning primer binding site sequence); a forward sequencing primer binding site sequence (140) (e.g., a batch-specific forward sequencing primer binding site sequence); a batch barcode sequence (195); a left sample index sequence (160); a sequence of interest (110); and a surface capture primer binding site sequence (130) (e.g., batch-specific surface capture primer binding site sequence). The double-stranded adaptor can comprise a first splint strand (600) hybridized to a second splint strand (700). In the double-stranded adaptor, the first splint strand (600) can comprise a first region (620), an internal region (610), and a second region (630), wherein the internal region of the first splint strand (610) is hybridized to the second splint strand (700). The second splint strand (700) can comprise a first, a second, and a third subregion, and the internal region (610) of the first splint strand (600) can comprise a fourth, a fifth, and a sixth subregion. The first region (620) of the first splint strand (600) can hybridize to at least a portion of the surface pinning primer binding site sequence (120) of a linear single-stranded library molecule (100), and the second region (630) of the first splint strand (600) can hybridize to at least a portion of the surface capture primer binding site sequence (130) of the same linear single-stranded library molecule (100).

[0063] FIG. 30A is a schematic of an exemplary workflow of a first linear single-stranded library molecule (100-1) hybridizing with a double-stranded adaptor (500) (ds-splint adaptor) thereby circularizing the first linear single-stranded library molecule to form a first librarysplint complex (800-1) with two nicks (solid arrowheads) that are enzymatically ligatable. The exemplary first linear single stranded library molecule (100-1) can comprise: a first pinning primer binding site sequence (120-1); a first batch forward sequencing primer binding site sequence (140-1); a first batch barcode sequence (195-1); a first sequence of interest (insert -1, 110-1); and a first surface capture primer binding site sequence (130-1). The double-stranded adaptor can comprise a first splint strand (600) hybridized to a second splint strand (700). In the double-stranded adaptor, the first splint strand (600) can comprise a first region (620), an internal region (610), and a second region (630), wherein the internal region of the first splint strand (610) is hybridized to the second splint strand (700). The second splint strand (700) can comprise a first, a second, and a third subregion, and the internal region (610) of the first splint strand (600) can comprise a fourth, a fifth, and a sixth subregion. The first region (620) of the first splint strand (600) can hybridize to at least a portion of the first pinning primer binding site sequence (120-1) of a linear single-stranded library molecule (100-1), and the second region (630) of the first splint strand (600) can22316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208hybridize to at least a portion of the first surface capture primer binding site sequence (130-1) of the same linear single-stranded library molecule (100-1).

[0064] FIG. 30B is a schematic of an exemplary workflow of a second linear singlestranded library molecule (100-2) hybridizing with a double-stranded adaptor (500) (ds-splint adaptor) thereby circularizing the library molecule to form a second library-splint complex (800-2) with two nicks (solid arrowheads) that are enzymatically ligatable. The exemplary second linear single-stranded library molecule (100-2) can comprise: a first pinning primer binding site sequence (120-1); a second batch forward sequencing primer binding site sequence (140-2); a second batch barcode sequence (195-2); a second sequence of interest (insert-2, 110-2); and a first surface capture primer binding site sequence (130-1). The double-stranded adaptor can comprise a first splint strand (600) hybridized to a second splint strand (700). In the double-stranded adaptor, the first splint strand (600) can comprise a first region (620), an internal region (610), and a second region (630), wherein the internal region of the first splint strand (610) is hybridized to the second splint strand (700). The second splint strand (700) can comprise a first, a second, and a third subregion, and the internal region (610) of the first splint strand (600) can comprise a fourth, a fifth, and a sixth subregion. The first region (620) of the first splint strand (600) can hybridize to at least a portion of the first surface pinning primer binding site sequence (120-1) of a single-stranded library molecule (100-2), and the second region (630) of the first splint strand (600) can hybridize to at least a portion of the first surface capture primer binding site sequence (130-1) of the same single-stranded library molecule (100-2). The first sequence of interest (110-1) in the first library-splint complex (800-1) shown in FIG. 30A and the second sequence of interest (110-2) in the second library-splint complex (800-2) shown in FIG. 30B can have the same sequence or different sequences.

[0065] FIG. 31A is a schematic of an exemplary workflow in which the two nicks in the first library-splint complex (800-1) shown in FIG. 30A are ligated to generate a first covalently closed circular library molecule (900-1) which is shown in FIG. 31 A. The first covalently closed circular library molecule (900-1) is subjected to rolling circle amplification (RCA) to generate a first concatemer template molecule, and the first concatemer template molecule is subjected to batch reiterative sequencing. The RCA reaction can be conducted insolution using soluble amplification primers or on-support using capture primers immobilized to a support. The first covalently closed circular library molecule (900-1) can comprise: a first surface pinning primer binding site sequence (120-1); a first batch forward sequencing primer binding site sequence (140-1) which corresponds with the first sequence of interest 23316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208(insert-1, 110-1); a first batch barcode sequence (195-1) which corresponds with the first sequence of interest (110-1); a first sequence of interest (110-1); and a first surface capture primer binding site sequence (130-1). The first covalently closed circular library molecule (900-1) can further comprise a second splint strand (700) from the double-stranded adaptor shown in FIG. 30 A. A plurality of the first covalently closed circular library molecule (900-1) shown in FIG. 31 A can be distributed onto a support having one type of immobilized capture primers which selectively hybridizes to the first surface capture primer binding site sequence (130-1) in the first covalently closed circular library molecules (900-1). The first covalently closed circular library molecules (900-1) can be subjected to rolling circle amplification (RCA) to generate a plurality of first concatemer template molecules which are immobilized to the support. The first concatemer template molecules can be subjected to a sequencing workflow using first batch-specific sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of first sequencing read products (dashed arrows). The first sequencing read products can include the first batch barcode sequence (195-1) as shown in FIG. 31 A. Alternatively, or in addition, the first sequencing read products can include the first batch barcode sequence (195-1) and at least a portion of the first sequence of interest (110-1) (not shown). The first concatemer template molecules can undergo reiterative sequencing comprising up to 1000 sequencing cycles. The first sequencing read products from the first concatemer template molecule can be up to 1000 bases in length.

[0066] FIG. 31B is a schematic of an exemplary workflow in which the nicks in the second library-splint complex (800-2) shown in FIG. 30B are ligated to generate a second covalently closed circular library molecule (900-2) which is shown in FIG. 3 IB. The second covalently closed circular library molecule (900-2) is subjected to rolling circle amplification (RCA) to generate a second concatemer template molecule, and the concatemer template molecule is subjected to batch reiterative sequencing. The RCA reaction can be conducted in-solution using soluble amplification primers or on-support using capture primers immobilized to a support. The second covalently closed circular library molecule (900-2) can comprise: a first surface pinning primer binding site sequence (120-1); a second batch forward sequencing primer binding site sequence (140-2) which corresponds with the second sequence of interest (110-2); a second batch barcode sequence (195-2) which corresponds with the second sequence of interest (insert-2, 110-2); a second sequence of interest (110-2); and a first surface capture primer binding site sequence (130-1). The second covalently closed circular library molecule (900-2) can further comprise a second splint strand (700) from the double- 24316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208stranded adaptor shown in FIG. 3 OB. A plurality of the second covalently closed circular library molecule (900-2) shown in FIG. 3 IB can be distributed onto a support having one type of immobilized capture primers which selectively hybridizes to the first surface capture primer binding site sequence (130-1) in the second covalently closed circular library molecules (900-2). A plurality of the first covalently closed circular library molecule (900-1) shown in FIG. 31 A and a plurality of the second covalently closed circular library molecule (900-2) shown in FIG. 3 IB are distributed onto the same support. For instance, the first covalently closed circular library molecules (900-1) shown in FIG. 31A and the second covalently closed circular library molecules (900-2) shown in FIG. 3 IB can be distributed onto the support essentially simultaneously. Alternatively, or in addition, the first covalently closed circular library molecules (900-1) shown in FIG. 31A and the second covalently closed circular library molecules (900-2) shown in FIG. 3 IB can be distributed onto the support sequentially (e.g., re-seeding the support). The second covalently closed circular library molecules (900-2) can be subjected to rolling circle amplification (RCA) to generate a plurality of second concatemer template molecules which are immobilized to the support. The second concatemer template molecules can be subjected to a sequencing workflow using second batch sequencing primers (solid arrows), sequencing polymerases, and a plurality of nucleotide reagents to generate a plurality of second sequencing read products (dashed arrows). In some cases, the second concatemer template molecules are not sequenced when first batch sequencing primers are used to sequence the first concatemer template molecules. Alternatively, or in addition, the first concatemer template molecules are not sequenced when second batch sequencing primers are used to sequence the second concatemer template molecules. The second sequencing read products can include the second batch barcode sequence (195-2) as shown in FIG. 3 IB. Alternatively, or in addition, the second sequencing read products include the second batch barcode sequence (195-2) and at least a portion of the second sequence of interest (110-2) (not shown). The second concatemer template molecules undergo reiterative sequencing comprising up to 1000 sequencing cycles. The second sequencing read products from the second concatemer template molecules can be up to 1000 bases in length.

[0067] FIG. 32 is a schematic showing an exemplary linear single-stranded library molecule (100) hybridizing with a single-stranded splint molecule / strand (200) (ss-split strand) thereby circularizing the library molecule to form a library-splint complex (300) with a nick. The linear single stranded library molecule (100) can comprise: a first left junction adaptor sequence (121); an adaptor sequence for a surface pinning primer binding site 25316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208sequence (120); a second left junction adaptor sequence (125); a left sample index sequence (160); a third left junction adaptor sequence (165); an adaptor sequence for a forward sequencing primer binding site sequence (140); a fourth left junction adaptor sequence (145); a sequence of interest (e.g., an insert (110)); a fourth right junction adaptor sequence (155); an adaptor sequence for a reverse sequencing primer binding site sequence (150); a third right junction adaptor sequence (175); a right sample index sequence (170); a second right junction adaptor sequence (135); an adaptor sequence for a surface capture primer binding site (130); and a first right junction adaptor sequence (131). The single-stranded splint strand (200) comprises a first region (210) that hybridizes with one end (e.g., left end or 5’ end) of the linear single stranded library molecule (100) including at least a portion of the adaptor sequence for a surface pinning primer binding site (120) and / or at least a portion of the first left junction adaptor sequence (121). The single-stranded splint strand (200) comprises a second region (220) that hybridizes with the other end (e.g., right end or 3’ end) of the linear single stranded library molecule (100) including at least a portion of the adaptor sequence for a surface capture primer binding site (130) and / or at least a portion of the first right junction adaptor sequence (131). For the sake of simplicity, the library-splint complex (300) does not show any of the junction adaptors. The skilled artisan will recognize that the library-splint complex (300) can include any one or any combination of two or more of the junction adaptors that are present in the linear single stranded library molecule (100).

[0068] FIG. 33 is a schematic showing an exemplary linear single-stranded library molecule (100) hybridizing with a double-stranded adaptor (500) (ds-splint adaptor) thereby circularizing the library molecule to form a library-splint complex (800) with two nicks (solid arrowheads). The linear single stranded library molecule (100) can comprise: a first left junction adaptor sequence (121); an adaptor sequence for a surface pinning primer binding site sequence (120); a second left junction adaptor sequence (125); a left sample index sequence (160); a third left junction adaptor sequence (165); an adaptor sequence for a forward sequencing primer binding site sequence (140); a fourth left junction adaptor sequence (145); a sequence of interest (e.g., an insert; (110)); a fourth right junction adaptor sequence (155); an adaptor sequence for a reverse sequencing primer binding site sequence (150); a third right junction adaptor sequence (175); a right sample index sequence (170); a second right junction adaptor sequence (135); an adaptor sequence for a surface capture primer binding site (130); and a first right junction adaptor sequence (131). The doublestranded splint adaptor (500) comprises a first splint strand (600) having a first region (620) that hybridizes with one end (e.g., left end or 5’ end) of the linear single stranded library 26316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208molecule (100) including at least a portion of the adaptor sequence for a surface pinning primer binding site sequence (120) and / or at least a portion of the first left junction adaptor sequence (121). The double-stranded splint adaptor (500) comprises a first splint strand (600) having a second region (630) that hybridizes with the other end (e.g., right end or 3’ end) of the linear single stranded library molecule (100) including at least a portion of the adaptor sequence for a surface capture primer binding site sequence (130) and / or at least a portion of the first right junction adaptor sequence (131). For the sake of simplicity, the library-splint complex (300) does not show any of the junction adaptors. The skilled artisan will recognize that the library-splint complex (300) can include any one or any combination of two or more of the junction adaptors that are present in the linear single stranded library molecule (100).

[0069] FIG. 34 shows sequencing images of polonies (e.g., DNA nanoballs) immobilized on a support at high density (top) and a table summarizing read count, Q30 scores and percent error (bottom). The support (e.g., a flow cell) was loaded with 20 picomolar (pM) of a 1 : 1 mixture of covalently closed circular library molecules generated from either singlestranded splint strands (right) or double-stranded splints (left). The loaded covalently closed circular library molecules were subjected to rolling circle amplification to generate immobilized concatemer template molecules. 31 cycles of first batch sequencing was conducted using first batch sequencing primers (e.g., TruSeq sequencing primers; SEQ ID NO: 2) that selectively hybridized to the concatemer template molecules generated from double-stranded splint adaptors (ds-Splint; left image was obtained at one of the 31 sequencing cycles). The first batch sequencing read products were removed. 31 cycles of second batch sequencing were conducted using second batch sequencing primers (e.g., ss-Splint sequencing primers, e.g. SEQ ID NO: 1) that selectively hybridized to the concatemer template molecules generated from single-stranded splint strands (ss-Splint; right image was obtained at one of the 31 sequencing cycles). Other loading concentrations were tested including 30 pM and 40 pM.

[0070] FIG. 35A is a bar graph showing the pass filter count (PF Count, in millions (M)) from an experiment conducted to determine the density of immobilized polonies using 8-plex batch sequencing primers. The data represented by the bar graphs shown in FIGs. 35 A, 36A and 37A were generated from the same experiment.

[0071] FIG. 35B is a Table listing the estimated loading concentrations (extrapolated pM) of the libraries corresponding to the number of batch sequencing primers used. The Table in FIG. 35B corresponds to the bar graph shown in FIG. 35 A.27316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0072] FIG. 36A is a bar graph showing the percent pass filter from an experiment conducted to determine the density of immobilized polonies using 8-plex batch sequencing primers.

[0073] FIG. 36B is a Table listing the estimated loading concentrations (extrapolated pM) of the libraries corresponding to the number of batch sequencing primers used. The Table in FIG. 36B corresponds to the bar graph shown in FIG. 36 A.

[0074] FIG. 37A is a bar graph showing the %Q30 from an experiment conducted to determine the density of immobilized polonies using 8-plex batch sequencing primers.

[0075] FIG. 37B is a Table listing the estimated loading concentrations (extrapolated pM) of the libraries corresponding to the number of batch sequencing primers used. The Table in FIG. 37B corresponds to the bar graph shown in FIG. 37 A.

[0076] FIG. 38 is a graph showing the nucleotide base diversity (A, T, C, or G) of a right sample index sequence (170) which includes a universal right sample index and a 3-mer random sequence (NNN). The graph shows a nucleotide diversity of the 3-mer random sequence (NNN) of approximately 30% for A and T base calls, and approximately 20% for C and Gbase calls.

[0077] FIG. 39 is a graph showing the nucleotide base diversity (A, T, C, or G) of a left sample index sequence (160) which lacks a 3-mer random sequence (NNN). The graph shows a nucleotide diversity of approximately 40% for A and T base calls, approximately 15% for C base calls, and approximately 5% for Gbase calls.DETAILED DESCRIPTIONIntroduction

[0078] For massively parallel sequencing, the limit of optical resolution impedes the ability to perform highly multiplex sequencing. Batch-specific sequencing enables sequencing a desired subset (e.g., a batch) of the template molecules immobilized to the same flow cell using selected batch-specific sequencing primers to reduce over-crowding signals and images which are generated during sequencing. The use of batch-specific sequencing primers produces optical images that are intense and resolvable. The batch-specific sequencing methods described herein have many uses. For example, the number of spots that are imaged and associated with sequencing can be counted. The counted spots can be used as a measure for target nucleic acid levels in a sample.

[0079] The present disclosure provides compositions, apparatus and methods for conducting separate sequencing batches on a support having nucleic acid template molecules 28316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208immobilized thereon, where the separate sequencing batches can be conducted using any massively parallel sequencing technology. In some embodiments, a plurality of subpopulations of nucleic acid template molecules are immobilized to the support including at least a first and second sub-population. In some embodiments, the first sub-population of template molecules undergo first sequencing reactions (e.g., first batch sequencing) and a region of the support is imaged to detect the first sequencing reactions, wherein the second sub-population of template molecules do not undergo sequencing reactions. In some embodiments, the second sub-population of template molecules undergo second sequencing reactions (e.g., second batch sequencing) and the same region of the support is imaged to detect the second sequencing reactions, wherein the first sub-population of template molecules do not undergo sequencing reactions. Thus, the first and second sub-populations of template molecules undergo batch sequencing.

[0080] The present disclosure also provides compositions, apparatus, and methods for conducting massively parallel sequencing methods using concatemerized template molecules that are generated by rolling circle amplification. The concatemer template molecules contain multiple copies of the target sequences and unique barcode sequences and sequencing primer binding sequences associated with the target sequences. Use of the concatemer template molecules increases the accuracy of the sequencing.

[0081] The methods described herein employ batch sequencing on high density immobilized template molecules which offers the advantage of maximizing space on a support (e.g., a flow cell). Furthermore, the same seeded support can be re-used by re-seeding the support with additional template molecules and conducting additional sequencing reactions on the re-seeded template molecules.

[0082] Batch sequencing can be conducted using template molecules arranged in a predetermined manner on the support (e.g., a patterned support). Alternatively, batch sequencing can be conducted using template molecules arranged in a random manner on the support which obviates the need to fabricate a support having organized and pre-determined features for attaching template molecules (e.g., fabrication via lithography is not needed).

[0083] By conducting short sequencing reads of the batch barcode regions of the template molecules, batch sequencing also significantly reduces sequencing run times, reagent use, and reagent costs.

[0084] When short sequencing reads of the batch barcode regions are conducted in a reiterative manner, it is not necessary to assemble the sequencing reads or to obtain a full length sequence of the sequence of interest, which reduces the need for long assembly 29316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208computations. Also, the redundant sequencing information obtained from the short sequencing reads obviates the need to sequence the complementary strand of the template molecules, thus obviating the need for pairwise sequencing.

[0085] Batch sequencing also offers the flexibility of re-seeding the support any time between sequencing different batches, or an ongoing sequencing batch can be interrupted to permit re-seeding then the ongoing batch sequencing can be resumed. The ability to re-seed the support any time increases throughput and efficiency.

[0086] Conducting batch sequencing with immobilized concatemer template molecule offers advantages over one-copy template molecules (e.g., one-copy template molecule generated via bridge amplification). For example concatemer template molecules carry multiple sequencing primer binding sites along the same concatemer template molecule. The multiple sequencing primer binding sites can be used to generate multiple sequencing reads for increased sequencing depth. Together, reiteratively sequencing one strand of the concatemer template molecules increases sequencing base coverage and sequencing depth compared to sequencing a one-copy template molecule.

[0087] Batch sequencing has many uses including but not limited to detecting specific nucleic acids of interest, mutant nucleic acid sequences, splice variants, and their abundance levels thereof.Definitions

[0088] The headings provided herein are not limitations of the various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole.

[0089] Unless defined otherwise, technical and scientific terms used herein have meanings that are commonly understood by those of ordinary skill in the art unless defined otherwise. Generally, terminologies pertaining to techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization described herein are those well-known and commonly used in the art.Techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the instant specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The30316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208nomenclatures utilized in connection with, and the laboratory procedures and techniques described herein are those well-known and commonly used in the art.

[0090] Unless otherwise required by context herein, singular terms shall include pluralities and plural terms shall include the singular. Singular forms “a”, “an” and “the”, and singular use of any word, include plural referents unless expressly and unequivocally limited on one referent.

[0091] It is understood the use of the alternative term (e.g., “or”) is taken to mean either one or both or any combination thereof of the alternatives.

[0092] The term “and / or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include: “A and B”; “A or B”; “A” (A alone); and “B” (B alone). In a similar manner, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: “A, B, and C”; “A, B, or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone); and “C” (C alone).

[0093] As used herein and in the appended claims, terms “comprising”, “including”, “having” and “containing”, and their grammatical variants, as used herein are intended to be non-limiting so that one item or multiple items in a list do not exclude other items that can be substituted or added to the listed items. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0094] As used herein, the terms “about” and “approximately” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (i.e., ±10%) or more depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg.Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that31316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208particular value or composition. Also, where ranges and / or subranges of values are provided, the ranges and / or subranges can include the endpoints of the ranges and / or subranges.

[0095] As used herein, “corresponding to” or “corresponds to” refers to two or more entities whose identities are sufficiently related such that the identity of one entity can be used to determine the identity, position and / or other properties of the other entity. As nonlimiting example, a barcode sequence can be said to correspond to a particular sequence of interest if the barcode sequence can be used to determine the identity of the sequence of interest.

[0096] The term “polymerase” and its variants, as used herein, comprises an enzyme comprising a domain that binds a nucleotide (or nucleoside) where the polymerase can form a complex having a template nucleic acid and a complementary nucleotide. The polymerase can have one or more activities including, but not limited to, base analog detection activities, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand displacement activity, and nucleotide binding and recognition. A polymerase can be any enzyme that can catalyze polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically but not necessarily such nucleotide polymerization can occur in a template-dependent fashion. Typically, a polymerase comprises one or more active sites at which nucleotide binding and / or catalysis of nucleotide polymerization can occur. In some embodiments, a polymerase includes other enzymatic activities, such as for example, 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, a polymerase has strand displacing activity. A polymerase can include without limitation naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof that retain the ability to catalyze nucleotide polymerization (e.g., catalytically active fragment). The polymerase includes catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes comprising a nucleotide binding domain. In some embodiments, a polymerase can be isolated from a cell, or generated using recombinant DNA technology or chemical synthesis methods. In some embodiments, a polymerase can be expressed in prokaryote, eukaryote, viral, or phage organisms. In some embodiments, a polymerase can be post-translationally modified proteins or fragments thereof. A polymerase can be derived from a prokaryote, eukaryote, virus or phage. A polymerase comprises DNA-directed DNA polymerase and RNA-directed DNA polymerase.32316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0097] As used herein, the term “strand displacing” refers to the ability of a polymerase to locally separate strands of double-stranded nucleic acids and synthesize a new strand in a template-based manner. Strand displacing polymerases displace a complementary strand from a template strand and catalyze new strand synthesis. Strand displacing polymerases include mesophilic and thermophilic polymerases. Strand displacing polymerases include wild type enzymes, and variants including exonuclease minus mutants, mutant versions, chimeric enzymes and truncated enzymes. Examples of strand displacing polymerases include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bea DNA polymerase (exo-), KI enow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g., MagniPhi® from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific®), or chimeric QualiPhi® DNA polymerase (e.g., from 4basebio®).

[0098] The terms “nucleic acid”, "polynucleotide" and "oligonucleotide" and other related terms used herein are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically-synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids (PNA) and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids comprise polymers of nucleotides, where the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids comprise naturally-occurring internucleosidic linkages, for example phosphodiester linkages. Nucleic acids can lack a phosphate group. Nucleic acids comprise non-natural internucleoside linkages, including phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, nucleic acids comprise a one type of polynucleotides or a mixture of two or more different types of polynucleotides.

[0099] The term “operably linked” and “operably joined” or related terms as used herein refers to juxtaposition of components. The juxtapositioned components can be linked together covalently. For example, two nucleic acid components can be enzymatically ligated together where the linkage that joins together the two components comprises phosphodiester linkage. A first and second nucleic acid component can be linked together, where the first nucleic acid component can confer a function on a second nucleic acid component. For example, linkage 33316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208between a primer binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion that can bind to a primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or a nucleic acid sequence of interest) can be ligated to a vector where the linkage permits expression or functioning of the transgene sequence contained in the vector. In some embodiments, a transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, enhancer, transcription and / or translation initiation sequence, transcription and / or translation termination sequence, polypeptide secretion signal sequences, and the like. In some embodiments, the host cell regulatory sequence controls expression of the level, timing and / or location of the transgene.

[0100] The terms “linked”, “joined”, “attached”, “appended” and variants thereof comprise any type of fusion, bond, adherence or association between any combination of compounds or molecules that is of sufficient stability to withstand use in the particular procedure. The procedure can include but are not limited to: nucleotide binding; nucleotide incorporation; de-blocking (e.g., removal of chain-terminating moiety); washing; removing; flowing; detecting; imaging and / or identifying. Such linkage can comprise, for example, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonds or associations involving van der Waals forces, mechanical bonding, and the like. In some embodiments, such linkage occurs intramolecularly, for example linking together the ends of a single-stranded or double-stranded linear nucleic acid molecule to form a circular molecule. In some embodiments,, such linkage can occur between a combination of different molecules, or between a molecule and a non-molecule, including but not limited to: linkage between a nucleic acid molecule and a solid surface; linkage between a protein and a detectable reporter moiety; linkage between a nucleotide and detectable reporter moiety; and the like. Some examples of linkages can be found, for example, in Hermanson, G., “Bioconjugate Techniques”, Second Edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).

[0101] The term “primer” and related terms used herein refer to an oligonucleotide that is capable of hybridizing with a DNA and / or RNA polynucleotide template to form a duplex molecule. Primers comprise natural nucleotides and / or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers may have any length, but typically range from 34316574825Attorney Docket No. ELEM-025 / 001WO 340101-22084-50 nucleotides. A typical primer comprises a 5’ end and 3’ end. The 3’ end of the primer can include a 3’ OH moiety which serves as a nucleotide polymerization initiation site in a polymerase-catalyzed primer extension reaction. Alternatively, the 3’ end of the primer can lack a 3’ OH moiety, or can include a terminal 3’ blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any one nucleotide, or more than one nucleotide, along the length of the primer can be labeled with a detectable reporter moiety. A primer can be in solution (e.g., a soluble primer) or can be immobilized to a support (e.g., a capture primer).

[0102] The term “template nucleic acid”, “template polynucleotide”, “target nucleic acid” “target polynucleotide”, “template strand,” “template molecule” and other variations refer to a nucleic acid strand that serves as the basis nucleic acid molecule for any of the methods describe herein, e.g. sequencing or amplification methods. The template nucleic acid can be single-stranded or double-stranded, or the template nucleic acid can have single-stranded or double-stranded portions. The template nucleic acid can be obtained from a naturally-occurring source, recombinant form, or chemically synthesized to include any type of nucleic acid analog. The template nucleic acid can be linear, circular, or other forms. The template nucleic acids can include an insert portion having an insert sequence. The template nucleic acids can also include at least one adaptor sequence. The insert portion can be isolated in any form, including chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned, amplified, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotides, whole genomic DNA, obtained from fresh frozen paraffin embedded tissue, needle biopsies, circulating tumor cells, cell free circulating DNA, or any type of nucleic acid library. The insert portion can be isolated from any source including from organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, viruses, cells, tissues, normal or diseased cells or tissues, body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, semen, environmental samples, culture samples, or synthesized nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. The insert portion can be isolated from any organ, including head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs. The template nucleic acid can be subjected to nucleic acid analysis, including sequencing and composition analysis. The template molecules disclosed herein can be concatemer template molecules, which comprise two or more copies of a particular sequence.35316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208For example, a concatemer template molecule can comprise two or more tandem copies of a polynucleotide unit, where each polynucleotide unit comprises a sequence of interest and at least one other sequence feature, such as any of the barcode sequences, index sequences, or sequencing, surface capture or surface pinning primer binding sequences disclosed herein.

[0103] The term “adaptor” and related terms refers to oligonucleotides that can be operably linked to a target polynucleotide, where the adaptor confers a function to the cojoined adaptor-target molecule. Adaptors comprise DNA, RNA, chimeric DNA / RNA, or analogs thereof. Adaptors can include at least one ribonucleoside residue. Adaptors can be single-stranded, double-stranded, or have single-stranded and / or double-stranded portions. Adaptors can be configured to be linear, stem-looped, hairpin, or Y-shaped forms. Adaptors can be any length, including 4-100 nucleotides or longer. Adaptors can have blunt ends, overhang ends, or a combination of both. Overhang ends include 5’ overhang and 3’ overhang ends. The 5’ end of a single-stranded adaptor, or one strand of a double-stranded adaptor, can have a 5’ phosphate group or lack a 5’ phosphate group. Adaptors can include a 5’ tail that does not hybridize to a target polynucleotide (e.g., tailed adaptor), or adaptors can be non-tailed. An adaptor can include a sequence that is complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., soluble or immobilized capture primers). Adaptors can include a random sequence or degenerate sequence. Adaptors can include at least one inosine residue. Adaptors can include at least one phosphorothioate, phosphorothiolate and / or phosphoramidate linkage. Adaptors can include a barcode sequence which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. Adaptors can include a unique identification sequence (e.g., unique molecular index, UMI; or a unique molecular tag) that can be used to uniquely identify a nucleic acid molecule to which the adaptor is appended. In some embodiments, a unique identification sequence can be used to increase error correction and accuracy, reduce the rate of false-positive variant calls and / or increase sensitivity of variant detection. Adaptors can include at least one restriction enzyme recognition sequence, including any one or any combination of two or more selected from a group consisting of type I, type II, type III, type IV, type Hs or type IIB.

[0104] In some embodiments, primer sequences, such as any of the amplification primer sequences, sequencing primer sequences, surface capture primer sequences, surface pinning primer sequences, and any of the sample barcode sequences, can be about 3-50 nucleotides in length, or about 5-40 nucleotides in length, or about 5-25 nucleotides in length.36316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0105] The term “universal sequence” and related terms refer to a sequence in a nucleic acid molecule that is common among two or more polynucleotide molecules. For example, an adaptor having a universal sequence can be operably joined to a plurality of polynucleotides so that the population of co-joined molecules carry the same universal adaptor sequence. Examples of universal adaptor sequences include an amplification primer sequence, a sequencing primer sequence or a capture primer sequence (e.g., soluble or immobilized capture primers).

[0106] When used in reference to nucleic acid molecules, the terms “hybridize” or “hybridizing” or “hybridization” or other related terms refers to hydrogen bonding between two different nucleic acids to form a duplex nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule having a duplex region. Hybridization can comprise Watson-Crick or Hoogstein binding to form a duplex double-stranded nucleic acid, or a double-stranded region within a nucleic acid molecule. The double-stranded nucleic acid, or the two different regions of a single nucleic acid, may be wholly complementary, or partially complementary. Complementary nucleic acid strands need not hybridize with each other across their entire length. The complementary base pairing can be the standard A-T or C-G base pairing, or can be other forms of base-pairing interactions. Duplex nucleic acids can include mismatched base-paired nucleotides.

[0107] When used in reference to nucleic acids, the terms “extend”, “extending”, “extension” and other variants, refers to incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation comprises polymerization of one or more nucleotides into the terminal 3’ OH end of a nucleic acid strand, resulting in extension of the nucleic acid strand. Nucleotide incorporation can be conducted with natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent fashion. Any suitable method of extending a nucleic acid molecule may be used, including primer extension catalyzed by a DNA polymerase or RNA polymerase.

[0108] The term “nucleotides” and related terms refers to a molecule comprising an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Canonical or non-canonical nucleotides are consistent with use of the term. The phosphate in some embodiments comprises a monophosphate, diphosphate, or triphosphate, or corresponding phosphate analog. The term “nucleoside” refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides can be non-labeled or labeled with a detectable reporter moiety.37316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0109] Nucleotides (and nucleosides) typically comprise a hetero cyclic base including substituted or unsubstituted nitrogen-containing parent heteroaromatic ring which are commonly found in nucleic acids, including naturally-occurring, substituted, modified, or engineered variants, or analogs of the same. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and / or Hoogstein hydrogen bonds with an appropriate complementary base. Exemplary bases include, but are not limited to, purines and pyrimidines such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N6-A2-isopentenyladenine (6iA), N6-A2-isopentenyl-2-methylthioadenine (2ms6iA), N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O6-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O4-methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines; hydroxymethylcytosines; 5-methycytosines; base (Y); as well as methylated, glycosylated, and acylated base moieties; and the like. Additional exemplary bases can be found in Fasman, 1989, in “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Boca Raton, Fla.

[0110] Nucleotides (and nucleosides) typically comprise a sugar moiety, such as carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Letters vol. 7: 3013-3016), and other sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No. 5,558,991). The sugar moiety comprises: ribosyl; 2'-deoxyribosyl; 3 '-deoxyribosyl; 2', 3 '-dideoxyribosyl; 2', 3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthioribosyl; 3 '-alkoxyribosyl; 3 '-azidoribosyl; 3 '-aminoribosyl; 3 '-fluororibosyl; 3'-mercaptoriboxyl; 3 '-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[0111] In some embodiments, nucleotides comprise a chain of one, two or three phosphorus atoms where the chain is typically attached to the 5’ carbon of the sugar moiety via an ester or phosphoramide linkage. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in 38316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208the chain include substituted side groups including O, S or BH3. In some embodiments, the chain includes phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphorodithioate, and O-methylphosphoramidite groups.

[0112] The term “reporter moiety”, “reporter moieties” or related terms refer to a compound that generates, or causes to generate, a detectable signal. A reporter moiety is sometimes called a “label”. Any suitable reporter moiety may be used, including luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent, chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten, affinity tag, atom, or an enzyme. A reporter moiety generates a detectable signal resulting from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events). A proximity event includes two reporter moieties approaching each other, or associating with each other, or binding each other. It is well known to one skilled in the art to select reporter moieties so that each absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from the other reporter moieties to permit monitoring the presence of different reporter moieties in the same reaction or in different reactions. Two or more different reporter moieties can be selected having spectrally distinct emission profiles, or having minimal overlapping spectral emission profiles. Reporter moieties can be linked (e.g., operably linked) to nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerases or reverse transcriptases), or support (e.g., surfaces).

[0113] A reporter moiety (or label) comprises a fluorescent label or a fluorophore.Exemplary fluorescent moieties which may serve as fluorescent labels or fhiorophores include, but are not limited to fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 39316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanine and derivatives such as indolium based cyanine dyes, benzo-indolium based cyanine dyes, pyridium based cyanine dyes, thiozolium based cyanine dyes, quinolinium based cyanine dyes, imidazolium based cyanine dyes, Cy 3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, Europium chelates, Terbium chelates, Alexa Fluor® dyes, DyLight® dyes, Atto™ dyes, LightCycler® Red dyes, CAL Flour dyes, JOE and derivatives thereof, Oregon Green™ dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, Malachite green, stilbene, DEG dyes, NR dyes, nearinfrared dyes and others known in the art such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. Cyanine dyes may exist in either sulfonated or non-sulfonated forms, and consist of two indolenin, benzo-indolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3, (which may comprise l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-3,3-dimethyl-l,3-dihydro-2H-indol-2-ylidenejprop- 1 -en- 1 -yl)-3 ,3 -dimethyl-3H-indolium or 1 - [6-(2, 5-dioxopyrrolidin- 1 -yloxy)-6-oxohexyl]-2-(3-{l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene}prop-l-en-l-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may comprise l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-l,3-dien- 1 -yl)-3 ,3 -dimethyl-3H-indol- 1 -ium or 1 -(6-((2, 5-dioxopyrrolidin- 1 -yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-l,3-dien-l-yl)-3,3-dimethyl-3H-indol-l-ium-5-sulfonate), and Cy7 (which may comprise l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-l,3-dihydro-2H-indol-2-ylidene)hepta-l,3,5-trien-l-yl]-3H-indolium or l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene)hepta-l,3,5-trien-l-yl]-3H-indolium-5-sulfonate), where “Cy” stands for 'cyanine', and the first digit identifies the number of carbon atoms between two indolenine groups. Cy2 which is an oxazole derivative rather than indolenin, and the benzo-derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to40316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208this rule. Additional suitable dyes are described, for example, in U.S. 2024 / 0240249A1, the contents of which are incorporated by reference in their entirety herein.

[0114] In some embodiments, the reporter moiety can be a FRET pair, such that multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET may comprise excitation exchange (Forster) transfers, or electron-exchange (Dexter) transfers.

[0115] When used in reference to nucleic acids, the terms “amplify”, “amplifying”, “amplification”, and other related terms include producing multiple copies of an original polynucleotide template molecule, where the copies comprise a sequence that is complementary to the template sequence, or the copies comprise a sequence that is the same as the template sequence. In some embodiments, the copies comprise a sequence that is substantially identical to a template sequence, or is substantially identical to a sequence that is complementary to the template sequence.

[0116] The term “support” as used herein refers to a substrate that is designed for deposition of biological molecules or biological samples for assays and / or analyses.Examples of biological molecules to be deposited onto a support include nucleic acids (e.g., DNA, RNA), polypeptides, saccharides, lipids, a single cell or multiple cells. Examples of biological samples include but are not limited to saliva, phlegm, mucus, blood, plasma, serum, urine, stool, sweat, tears and fluids from tissues or organs.

[0117] A “capture primer” or “surface capture primer” and the like refers to an oligonucleotide immobilized to a support that is complementary to a portion of, and capable of hybridizing with a given oligonucleotide, such as the library molecules and / or template molecules described herein. A “pinning primer” or “surface pinning primer” and the like refers to an oligonucleotide immobilized to a support that is complementary to a portion of, and capable of hybridizing with the concatemer template molecules described herein, thereby “pinning” down a portion of the concatemer template molecule to the support.

[0118] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porosity. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example comprising a capillary or interior surface of a capillary.

[0119] In some embodiments, the surface of the support can be substantially smooth. In some embodiments, the support can be regularly or irregularly textured, including bumps, etched, pores, three-dimensional scaffolds, or any combination thereof.41316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0120] In some embodiments, the support comprises a bead having any shape, including spherical, hemi-spherical, cylindrical, barrel-shaped, toroidal, disc-shaped, rod-like, conical, triangular, cubical, polygonal, tubular or wire-like.

[0121] The support can be fabricated from any material, including but not limited to glass, fused-silica, silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[0122] The support can have a plurality (e.g., two or more) of nucleic acid templates immobilized thereon. The plurality of immobilized nucleic acid templates have the same sequence or have different sequences. In some embodiments, individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized to a different site on the support. In some embodiments, two or more individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized to a site on the support.

[0123] The term “array” refers to a support comprising a plurality of sites located at predetermined locations on a support described herein to form an array of sites. The sites can be discrete and separated by interstitial regions. In some embodiments, the pre-determined sites on the support can be arranged in one dimension in a row or a column, or arranged in two dimensions in rows and columns. In some embodiments, the plurality of pre-determined sites is arranged on the support in an organized fashion. In some embodiments, the plurality of pre-determined sites is arranged in any organized pattern, including rectilinear, hexagonal patterns, grid patterns, patterns having reflective symmetry, patterns having rotational symmetry, or the like. The pitch between different pairs of sites can be that same or can vary. In some embodiments, the support comprises at least 102sites, at least 103sites, at least 104sites, at least 105sites, at least 106sites, at least 107sites, at least 108sites, at least 109sites, at least 1010sites, at least 1011sites, at least 1012sites, at least 1013sites, at least 1014sites, at least 1015sites, or more, where the sites are located at pre-determined locations on the support. In some embodiments, the support comprises between about 102sites and about 1015sites, between about 105sites and about 1015sites, between about 1010sites and about 1015sites, between about 103sites and about 1014sites, between about 104sites and about 1013sites, between about 105sites and about 1012sites, between about 106sites and about 1011sites, between about 107sites and about 1010sites, between about 108sites and about 1010sites, or any range therebetween located at pre-determined locations on the support. In some 42316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208embodiments, a plurality of pre-determined sites on the support (e.g., 102- 1015sites or more) are immobilized with nucleic acid templates to form a nucleic acid template array. In some embodiments, the nucleic acid templates that are immobilized at a plurality of predetermined sites by hybridization to immobilized surface capture primers, or the nucleic acid templates are covalently attached to the surface capture primer. In some embodiments, the nucleic acid templates that are immobilized at a plurality of pre-determined sites, for example immobilized at 102- 1015sites or more. In some embodiments, the immobilized nucleic acid templates are clonally-amplified to generate immobilized nucleic acid clusters at the plurality of pre-determined sites. In some embodiments, individual immobilized nucleic acid clusters comprise linear clusters, or comprise single-stranded or double-stranded concatemers.

[0124] In some embodiments, a support comprising a plurality of sites located at random locations on the support is referred to herein as a support having randomly located sites thereon. The location of the randomly located sites on the support are not pre-determined. The plurality of randomly-located sites is arranged on the support in a disordered and / or unpredictable fashion. In some embodiments, the support comprises at least 102sites, at least 103sites, at least 104sites, at least 105sites, at least 106sites, at least 107sites, at least 108sites, at least 109sites, at least IO10sites, at least 1011sites, at least 1012sites, at least 1013sites, at least 1014sites, at least 1015sites, or more, where the sites are randomly located on the support. In some embodiments, the support comprises between about 102sites and about 1015sites, between about 105sites and about 1015sites, between about IO10sites and about 1015sites, between about 103sites and about 1014sites, between about 104sites and about 1013sites, between about 105sites and about 1012sites, between about 106sites and about 1011sites, between about 107sites and about IO10sites, or between about 108sites and about IO10sites, or any range therebetween located at random locations on the support. In some embodiments, a plurality of randomly located sites on the support (e.g., 102- 1015sites or more) are immobilized with nucleic acid templates to form a support immobilized with nucleic acid templates. In some embodiments, the nucleic acid templates that are immobilized at a plurality of randomly located sites by hybridization to immobilized surface capture primers, or the nucleic acid templates are covalently attached to the surface capture primer. In some embodiments, the nucleic acid templates that are immobilized at a plurality of randomly located sites, for example immobilized at 102- 1015sites or more. In some embodiments, the template molecules are immobilized at between about 102sites and about 1015sites, between about 105sites and about 1015sites, between about IO10sites and about 1015sites, between about 103sites and about 1014sites, between about 104sites and about 1013sites, between 43316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208about 105sites and about 1012sites, between about 106sites and about 1011sites, between about 107sites and about IO10sites, or between about 108sites and about IO10sites, or any range therebetween, on the support. In some embodiments, the immobilized nucleic acid templates are clonally-amplified to generate immobilized nucleic acid clusters at the plurality of randomly located sites. In some embodiments, individual immobilized nucleic acid clusters comprise linear clusters, or comprise single-stranded or double-stranded concatemers.

[0125] In some embodiment, the plurality of immobilized surface capture primers on the support are in fluid communication with each other to permit flowing a solution of reagents (e.g., nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, and the like) onto the support so that the plurality of immobilized surface capture primers on the support can be essentially simultaneously reacted with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized surface capture primers can be used to conduct nucleic acid amplification reactions (e.g., RCA, MDA, PCR and bridge amplification) essentially simultaneously on the plurality of immobilized surface capture primers.

[0126] In some embodiment, the plurality of immobilized nucleic acid clusters on the support are in fluid communication with each other to permit flowing a solution of reagents (e.g., enzymes, nucleotides, divalent cations, and the like) onto the support so that the plurality of immobilized nucleic acid clusters on the support can be essentially simultaneously reacted with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized nucleic acid clusters can be used to conduct nucleotide binding assays and / or conduct nucleotide polymerization reactions (e.g., primer extension or sequencing) essentially simultaneously on the plurality of immobilized nucleic acid clusters, and optionally to conduct detection and imaging for massively parallel sequencing.

[0127] When used in reference to immobilized enzymes, the term “immobilized” and related terms refer to enzymes (e.g., polymerases) that are attached to a support through covalent bond or non-covalent interaction, or attached to a coating on the support, or buried within a matrix formed by a coating on the support.

[0128] When used in reference to immobilized nucleic acids, the term “immobilized” and related terms refer to nucleic acid molecules that are attached to a support through covalent bond or non-covalent interaction, or attached to a coating on the support, or buried within a matrix formed by a coating on the support, where the nucleic acid molecules include surface 44316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208capture primers, nucleic acid template molecules and extension products of capture primers. Extension products of capture primers includes nucleic acid concatemers (e.g., nucleic acid clusters).

[0129] In some embodiments, one or more nucleic acid templates are immobilized on the support, for example immobilized at the sites on the support. In some embodiments, the one or more nucleic acid templates are clonally-amplified. In some embodiments, the one or more nucleic acid templates are clonally-amplified off the support (e.g., in-solution) and then deposited onto the support and immobilized on the support. In some embodiments, the clonal amplification reaction of the one or more nucleic acid templates is conducted on the support resulting in immobilization on the support. In some embodiments, the one or more nucleic acid templates are clonally-amplified (e.g., in solution or on the support) using a nucleic acid amplification reaction, including any one or any combination of: polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, and / or single-stranded binding (SSB) protein-dependent amplification.

[0130] As used herein, the term “binding complex” refers to a complex formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or a nucleotide unit of a multivalent molecule, where the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer. In the binding complex, the free nucleotide or nucleotide unit may or may not be bound to the 3’ end of the nucleic acid primer at a position that is opposite a complementary nucleotide in the nucleic acid template molecule. A “ternary complex” is an example of a binding complex which is formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide unit of a multivalent molecule, where the free nucleotide or nucleotide unit is bound to the 3’ end of the nucleic acid primer (as part of the nucleic acid duplex) at a position that is opposite a complementary nucleotide in the nucleic acid template molecule.

[0131] The term “persistence time” and related terms refer to the length of time that a binding complex, which is formed between the target nucleic acid, a primer, a polymerase, a conjugated or unconjugated nucleotide, remains stable without any binding component dissociates from the binding complex. The persistence time is indicative of the stability of the binding complex and strength of the binding interactions. Persistence time can be measured 45316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208by observing the onset and / or duration of a binding complex, such as by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides may be present in a binding complex, thus allowing the signal from the label to be detected during the persistence time of the binding complex. One exemplary label is a fluorescent label.

[0132] The present disclosure provides various reagents, and methods that employ the reagents for conducting a trapping reaction, an imaging reaction, a nucleic acid denaturation (de-hybridization) and / or a stepping reaction. The various reagents can include at least one pH buffering agent. The full name of the pH buffering agents is listed herein.

[0133] The term “Tris” refers to a pH buffering agent Tris(hydroxymethyl)-aminomethane. The term “Tris-HCl” refers to a pH buffering agent Tris(hydroxymethyl)-aminomethane hydrochloride. The term “Tris-acetate” refers to a pH buffering agent comprising an acetate salt of Tris (hydroxymethyl)-aminomethane.

[0134] The term “Tricine” refers to a pH buffering agent N-[tris(hydroxymethyl) methyl]glycine.

[0135] The term “Bicine” refers to a pH buffering agent N,N-bis(2-hydroxyethyl)glycine.

[0136] The term “Bis-Tris propane” refers to a pH buffering agent 1,3 Bis[tris(hydroxymethyl).methylamino]propane.

[0137] The term “HEPES” refers to a pH buffering agent 4-(2-hy droxy ethyl)- 1-piperazineethanesulfonic acid.

[0138] The term “MES” refers to a pH buffering agent 2-(7V-morpholino)ethanesulfonic acid).

[0139] The term “MOPS” refers to a pH buffering agent 3-(N-morpholino)propanesulfonic acid.

[0140] The term “MOPSO” refers to a pH buffering agent 3-(N-morpholino)-2-hydroxypropanesulfonic acid.

[0141] The term “BES” refers to a pH buffering agent N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid.

[0142] The term “TES” refers to a pH buffering agent 2-[(2-Hydroxy- 1, lbis(hydroxymethyl)ethyl)amino]ethanesulfonic acid).

[0143] The term “CAPS” refers to a pH buffering agent 3 -(cyclohexylamino)- 1-propanesuhinic acid.

[0144] The term “TAPS” refers to a pH buffering agent N-[Tris(hydroxymethyl)methyl]-3 -amino propane sulfonic acid.46316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0145] The term “TAPSO” refers to a pH buffering agent N-[Tris(hydroxymethyl)methyl]-3-amino-2-hyidroxypropansulfonic acid.

[0146] The term “ACES” refers to a pH buffering agent 7V-(2-Acetamido)-2-aminoethanesulfonic acid.

[0147] The term “PIPES” refers to a pH buffering agent piperazine- l,4-bis(2-ethanesulfonic acid.

[0148] The term “ethanolamine” refers to a pH buffering agent that is also known as 2-aminoethanol.

[0149] Throughout this application various publications, patents, and / or patent applications are referenced. The disclosures of the publications, patents and / or patent applications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0150] The present disclosure provides compositions, apparatus and methods for conducting separate sequencing batches on a support having nucleic acid template molecules immobilized thereon, where the separate sequencing batches can be conducted using any massively parallel sequencing technology. In some embodiments, a plurality of subpopulations of nucleic acid template molecules are immobilized to the support including at least a first and second sub-population. In some embodiments, the first sub-population of template molecules undergo first sequencing reactions (e.g., first batch sequencing) and a region of the support is imaged to detect the first sequencing reactions, wherein the second sub-population of template molecules do not undergo sequencing reactions. In some embodiments, the second sub-population of template molecules undergo second sequencing reactions (e.g., second batch sequencing) and the same region of the support is imaged to detect the second sequencing reactions, wherein the first sub-population of template molecules do not undergo sequencing reactions. Thus, the first and second sub-populations of nucleic acid template molecules undergo batch sequencing.

[0151] In some embodiments, the plurality of sub-populations of nucleic acid template molecules are immobilized to the support at a high density. In some embodiments, at least some of the immobilized template molecules in the first and second sub-populations comprise nearest neighbor template molecules that touch each other and / or overlap each other when viewed from any angle of the support including above, below or side views of the support. For example, the plurality of sub-populations of nucleic acid template molecules are immobilized to the support at a density of about 102- 1015template molecules per mm2. In some embodiments, the template molecules are at density of between about 1010and about 47316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208IO15template molecules per mm2, between about 105and about 1015template molecules per mm2, between about 103and about 1014template molecules per mm2, between about 104and about 1013template molecules per mm2, between about 105and about 1012template molecules per mm2, between about 106and about 1011template molecules per mm2, between about 107and about IO10template molecules per mm2, or between about 108and about IO10template molecules per mm2on the support, or any range therebetween.

[0152] In some embodiments, the support comprises a plurality of template molecules immobilized at pre-determined positions on the support (e.g., a patterned support). In some embodiments, the support comprises a plurality of template molecules immobilized at random and non-pre-determined positions on the support. In some embodiments, the support comprises a mixture of at least two sub-populations of template molecules immobilized at random and non-pre-determined positions on the support.

[0153] In some embodiments, the support lacks any contours (e.g., wells, protrusions, and the like) arranged in a pre-determined pattern. In some embodiments, the support lacks contours which include features as sites for attachment of the nucleic acid template molecules. In some embodiments, the support lacks interstitial regions arranged in a predetermined pattern where the interstitial regions are sites designed to have no attached surface capture primers and / or template molecules. In some embodiments, the support lacks features that can be prepared using photo-chemical, photo-lithography, or micron-scale or nano-scale printing.

[0154] In some embodiments, individual template molecules in a given sub-population of template molecules comprise a sequence of interest, a batch barcode sequence that corresponds to the sequence of interest, and a batch sequencing primer binding site sequence that corresponds to the sequence of interest. In some embodiments, a pre-determined batch barcode sequence can be linked to a given sequence of interest, thus the pre-determined batch barcode sequence corresponds to a given sequence of interest. In some embodiments, a predetermined batch sequencing primer binding site sequence can be linked to a given sequence of interest, thus the pre-determined batch sequencing primer binding site sequence corresponds to a given sequence of interest. In some embodiments, template molecules within a given sub-population have the same or different sequences of interest. In some embodiments, template molecules within a given sub-population have the same batch barcode sequence. In some embodiments, template molecules within a given sub-population have the same sequencing primer binding site sequence. Thus, the different sub-populations of template molecules can undergo batch sequencing using a batch-specific sequencing primer.48316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0155] In some embodiments, the sequence of interest region need not undergo sequencing. Instead, the batch barcode can be sequenced by conducting a small number of sequencing cycles to reveal the batch barcode which corresponds to its sequence of interest. In some embodiments, the batch barcode and the sequence of interest can be sequenced.

[0156] In some embodiments, individual template molecules in a given sub-population of template molecules further comprise a sample index sequence that can be used to distinguish sequences of interest obtained from different sample sources in a multiplex assay. In some embodiments, template molecules within a given sub-population have the same or different sample index sequences.

[0157] In some embodiments, the sequence of interest region need not undergo sequencing. Instead, the batch barcode and the sample index can be sequenced by conducting a small number of sequencing cycles to reveal the batch barcode which corresponds to its sequence of interest and to reveal the sample index which corresponds to the sample source of the sequence of interest. In some embodiments, the template molecules lack a sample index and the batch barcode can serve as a sample index.

[0158] In some embodiments, the same portion of individual template molecules can be re-sequenced (e.g., reiterative sequencing) from the same start position to generate overlapping sequencing reads that can be aligned to a reference sequence. For example, the same portion of individual template molecules can be sequenced at least two, three, four, five, up to 50 times, up to 100 times, or more than 100 times. The start sequencing site can be any location of the template molecule and is dictated by the sequencing primers which are designed to anneal to a selected position within the template molecule. In some embodiments, the batch barcodes (or the batch barcodes and sample indexes) can be reiteratively sequenced by repeatedly conducting a short number of sequencing cycles of the batch barcode region (or the batch barcode and sample index regions) of a given template molecule. The reiterative sequencing reads increase the redundancy of sequencing information for individual bases in the template molecule. Reiteratively sequencing one strand of the template molecule can provide enough base coverage so that pairwise sequencing of the complementary strand is not necessary.

[0159] In some embodiments, after sequencing the first and / or second sub-populations of template molecules, the support can be re-seeded at least once with additional sub-population of template molecules (e.g., a third sub-population) which can undergo additional batch sequencing. In some embodiments, an ongoing batch sequencing run can be stopped prior to completion (e.g., interrupted) to permit re-seeding the support with an additional sub- 49316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208population of template molecules (e.g., the third sub-population) and then the interrupted batch sequencing can be resumed. Thus, the support can be re-seeded any time and / or before a previous sequencing batch is completed.

[0160] In some embodiments, the support comprises a plurality of template molecules immobilized at an initial low density where most of the nearest neighbor template molecules do not touch each other and / or do not overlap each other. In some embodiments, the initial low density support comprises a plurality of template molecules having interstitial space between the template molecules.

[0161] In some embodiments, the same support can undergo a first re-seeding with additional template molecules immobilized to the support so that the first re-seeded density has some nearest template molecules (e.g., 10 - 30% of the first immobilized re-seeded template molecules) that touch each other and / or overlap each other. In some embodiments, the resulting first re-seeded support comprises a plurality of template molecules having a reduced number of interstitial space (and / or having a reduced size of interstitial space) between the template molecules compared to the initial low density support.

[0162] In some embodiments, the same support can undergo a second re-seeding with additional template molecules immobilized to the support so that the second re-seeded density has an increase in nearest neighbor template molecules (e.g., 25 - 50% or more of the first re-seeded template molecules) that touch each other and / or overlap each other. In some embodiments, the resulting second re-seeded support comprises a plurality of template molecules having a further reduced number of interstitial space (and / or having a further reduced size of interstitial space) between the template molecules compared to the first reseeded density support. In some embodiments, the support can undergo multiple re-seeding workflows to generate increasing nearest neighbor template molecules that touch each other and / or overlap each other.

[0163] In some embodiments, individual template molecules comprise nucleic acid concatemer template molecules. In some embodiments, a concatemer template molecule can be generated by conducting rolling circle amplification of a circularized nucleic acid library molecule. In some embodiments, a concatemer template molecule comprises a singlestranded nucleic acid strand carrying numerous tandem copies of a polynucleotide unit, where each polynucleotide unit comprises a sequence of interest region and at least one batch sequencing primer binding site. In some embodiments, each polynucleotide unit further comprises at least one batch barcode sequence. In some embodiments, each polynucleotide unit further comprises at least one sample index sequence. Individual polynucleotide units 50316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208can bind a sequencing primer, a sequencing polymerase and a detectably-labeled nucleotide reagent (e.g., detectably labeled multivalent molecules or nucleotide analogs), to form a detectable sequencing complex. In some embodiments, individual concatemer template molecules can collapse into a compact DNA nanoball, where individual nanoballs carry numerous tandem copies of a polynucleotide unit along their lengths. During batch sequencing, individual nanoballs carry numerous detectable sequencing complexes. Thus, the compact nature of the nanoballs increases the local concentration of detectably-labeled nucleotide reagents that are used during batch sequencing which increases the signal intensity emitted from a nanoball to give a discrete detectable signal which can be imaged as a fluorescent spot. In some embodiments, a spot corresponds to a concatemer and each concatemer corresponds to a sequence of interest. Multiple spots can be detected and imaged simultaneously on a support having high density concatemer template molecules immobilized thereon.Batch Sequencing

[0164] The present disclosure provides methods for sequencing comprising step (a): providing a support comprising a plurality of nucleic acid template molecules immobilized to the support. In some embodiments, the plurality of template molecules comprises a plurality of sub-populations of template molecules including at least a first and a second subpopulation of template molecules. In some embodiments, the first sub-population of template molecules comprises a first batch sequencing primer binding site and at least one first sequence-of-interest. In some embodiments, the second sub-population of template molecules comprises a second batch sequencing primer binding site and at least one second sequence-of-interest. In some embodiments, template molecules within the first sub-population have the same first batch sequencing primer binding site. In some embodiments, template molecules within the first sub -population have the same sequence of interest or different sequences of interest. In some embodiments, the sequence of the first batch sequencing primer binding site sequence corresponds to the first sequence of interest, or the first batch sequencing primer binding site sequence corresponds to one of the first sequences of interest in the first sub-population. In some embodiments, a pre-determined first batch sequencing primer binding site sequence can be linked to a given sequence of interest in the first subpopulation (or can be linked to different sequences of interest in a first sub-population), thus the pre-determined first batch sequencing primer binding site sequence corresponds to a given sequence of interest in the first sub-population.51316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0165] In some embodiments, the sequences of interest in the first sub-population are about 50-250 bases in length, about 250-500 bases in length, about 500-800 bases in length, about 800-1200 bases in length, about 1200-2000 bases in length, or up to 2000 bases in length, or any range therebetween.

[0166] In some embodiments, template molecules within the second sub-population have the same second batch sequencing primer binding site, and have the same sequence of interest or different sequences of interest. In some embodiments, the sequence of the second batch sequencing primer binding site sequence corresponds to the second sequence of interest. In some embodiments, the sequence of the second batch sequencing primer binding site sequence corresponds to one of the second sequences of interest in the second subpopulation. In some embodiments, a pre-determined second batch sequencing primer binding site sequence can be linked to a given sequence of interest in the second sub-population (or can be linked to different sequences of interest in a second sub-population), thus the predetermined second batch sequencing primer binding site sequence corresponds to a given sequence of interest in the second sub-population.

[0167] In some embodiments, the sequences of interest in the second sub-population are about 50-250 bases in length, about 250-500 bases in length, about 500-800 bases in length, about 800-1200 bases in length, about 1200-2000 bases in length, or up to 2000 bases in length, or any range therebetween.

[0168] In some embodiments, the first and second batch sequencing primer binding sites have different sequences.

[0169] In some embodiments, the plurality of nucleic acid template molecules can be immobilized to the support at random and non-pre-determined positions on the support, or at pre-determined positions on the support (e.g., a patterned support).

[0170] In some embodiments, in the methods for sequencing of step (a), the support comprises a plurality of nucleic acid template molecules immobilized thereon at a density of about 102- 1015template molecules per mm2, e.g. between about 1010and about 1015template molecules per mm2, between about 105and about 1015template molecules per mm2, between about 103and about 1014template molecules per mm2, between about 104and about 1013template molecules per mm2, between about 105and about 1012template molecules per mm2, between about 106and about 1011template molecules per mm2, between about 107and about 1010template molecules per mm2, or between about 108and about 1010per mm2, or any range therebetween. In some embodiments, the template molecules comprise a mixture of at least two sub-populations of template molecules including at least a first and second sub- 52316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208population of template molecules. In some embodiments, the plurality of sub-populations of template molecules are immobilized to the support at a high density where at least some of the template molecules in the first and second sub-populations comprise nearest neighbor template molecules that touch each other and / or overlap each other when viewed from any angle of the support including above, below or side views of the support. In some embodiments, the support comprises up to 500 million template molecules immobilized thereon, or up to 1 billion template molecules immobilized thereon, or up to 2 billion template molecules immobilized thereon, or up to 3 billion template molecules immobilized thereon, or up to 4 billion template molecules immobilized thereon, or up to 5 billion template molecules immobilized thereon, or up to 6 billion template molecules immobilized thereon. In some embodiments, the support comprises up to 7 billion template molecules immobilized thereon, or up to 8 billion template molecules immobilized thereon, or up to 9 billion template molecules immobilized thereon, or up to 10 billion template molecules immobilized thereon, or up to 20 billion template molecules immobilized thereon. In some embodiments, the support comprises between about 500 million and about 20 billion template molecules immobilized thereon, between about 1 billion and about 10 billion template molecules immobilized thereon, between about 2 billion and about 9 billion template molecules immobilized thereon, between about 3 billion and about 8 billion template molecules immobilized thereon, between about 4 billion and about 7 billion template molecules immobilized thereon, or between about 5 billion and about 6 billion template molecules immobilized thereon, or any range therebetween.

[0171] In some embodiments, in the methods for sequencing of step (a), the support comprises features that are located in a random and non-pre-determined manner, where the features are sites for attachment of the template molecules.

[0172] In some embodiments, the support is passivated with at least one polymer layer comprising a plurality of surface capture primers covalently tethered to the at least one polymer layer.

[0173] In some embodiments, the support is passivated with multiple polymer layers. In some embodiments, at least one of the polymer layers comprises oligonucleotide primers including capture primers, pinning primers, or a mixture of capture and pinning primers. In some embodiments, the plurality of oligonucleotide primers comprise one type of capture primer (e.g., having that same batch capture primer sequence). In some embodiments, the plurality of oligonucleotide primers comprises a mixture of 2-500 different types of capture primers (e.g., having between about 2-500, between about 50-400, between about 100-300 or 53316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208between about 20-150 different batch capture primer sequences, or any range therebetween). In some embodiments, the plurality of oligonucleotide primers comprises one type of pinning primer (e.g., having the same batch pinning primer sequence). In some embodiments, the plurality of oligonucleotide primers comprise a mixture of 2-500 different types of pinning primers (e.g., having between about 2-500, between about 50-400, between about 100-300 or between about 20-150 different batch pinning primer sequences, or any range therebetween). In some embodiments, the plurality of oligonucleotide types comprises between 2 and 500, between 10 and 400, between 20 and 300, between 50 and 200, between 100 and 500, between 200 and 400, between 2 and 250, between 10 and 150, between 20 and 200, or between 20 and 100 or between 5 and 50 different capture primers and / or pinning primers, or any range therebetween.

[0174] In some embodiments, the plurality of surface capture primers comprise a plurality of sub-populations of surface capture primers including at least a first and second sub-population of surface capture primers. In some embodiments, the surface capture primers in the at least first and second sub-population have different sequences. In some embodiments, the surface capture primers in the at least first and second sub-population can hybridize to and thereby capture different circularized library molecules carrying different surface capture primer binding site sequences.

[0175] In some embodiments, the plurality of surface capture primers are randomly distributed throughout and embedded within the at least one polymer layer.

[0176] In some embodiments, the support lacks any contours (e.g., wells, protrusions, and the like) arranged in a pre-determined pattern where the contours have features that are sites for attachment of the nucleic acid template molecules. In some embodiments, the support lacks interstitial regions arranged in a pre-determined pattern where the interstitial regions are sites designed to have no attached template molecules.

[0177] In some embodiments, in the methods for sequencing of step (a), the support lacks partitions and / or barriers that would create separate regions of the support. Thus, the template molecules immobilized to the support are in fluid communication with each other in a massively parallel manner with no barriers to physically separate different batches of template molecules.

[0178] In some embodiments, the plurality of surface capture primers are located at predetermined positions on the at least one polymer layer and / or the plurality of surface capture primers are embedded within the at least one polymer layer at pre-determined locations.54316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0179] In some embodiments, the support includes contours (e.g., wells, protrusions, and the like) arranged in a pre-determined pattern where the contours have features that are sites for attachment of the nucleic acid template molecules (e.g., by localizing capture primers thereto). In some embodiments, the support includes interstitial regions arranged in a predetermined pattern where the interstitial regions are sites designed to have no attached template molecules.

[0180] In some embodiments, in the methods for sequencing of step (a), individual template molecules in the first sub-population further comprise a first batch barcode sequence which corresponds to the first sequence of interest. In some embodiments, the first batch barcode sequence corresponds to one of the first sequences of interest in the first subpopulation. In some embodiments, a pre-determined first batch barcode sequence can be linked to a given sequence of interest in the first sub-population, thus the pre-determined first batch barcode sequence corresponds to a given sequence of interest in the first subpopulation. In some embodiments, a pre-determined first batch barcode sequence can be linked to different sequences of interest in a first sub-population.

[0181] In some embodiments, individual template molecules in the second subpopulation further comprise a second batch barcode sequence which corresponds to the second sequence of interest. In some embodiments, the second batch barcode sequence corresponds to one of the second sequences of interest in the second sub-population. In some embodiments, a pre-determined second batch barcode sequence can be linked to a given sequence of interest in the second sub-population, thus the pre-determined second batch barcode sequence corresponds to a given sequence of interest in the second sub-population. In some embodiments, a pre-determined second batch barcode sequence can be linked to different sequences of interest in a second sub-population.

[0182] In some embodiments, in the methods for sequencing of step (a), individual template molecules in the first sub-population further comprise at least one sample index sequence that can be used in a multiplex assay to distinguish the first sequences of interest obtained from different sample sources. In some embodiments, individual template molecules in the second sub-population further comprises at least one sample index sequence that can be used in a multiplex assay to distinguish the second sequences of interest obtained from different sample sources.

[0183] In some embodiments, the first batch barcode sequence can include a short random sequence (e.g., NNN) that is 3-20 in length. In some embodiments, the first batch sample index sequence can include a short random sequence (e.g., NNN) that is 3-20 in 55316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208length. In some embodiments, both the first batch barcode sequence and the first batch sample index sequence both include a short random sequence (e.g., NNN) that is 3-20 in length. In some embodiments, sequencing the short random sequence can provide nucleotide diversity and color balance. In some embodiments, sequencing and imaging the short random sequence can be used for polony mapping, location, and template registration because the short random sequence provides sufficient nucleotide diversity and color balance.

[0184] In some embodiments, in the first sub-population of library molecules the short random sequence (e.g., NNN) has an overall base composition of about 25% or about 20-30% of all four nucleotide bases (e.g., A, G, C and T / U) to provide nucleotide diversity at each sequencing cycle during sequencing the short random sequence (e.g., NNN).

[0185] In some embodiments, in the first sub-population of library molecules, the proportion of adenine (A) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%. In some embodiments, in the first sub-population of library molecules, the proportion of guanine (G) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%. In some embodiments, in the first sub-population of library molecules, the proportion of cytosine (C) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%. In some embodiments, in the first sub-population of library molecules, the proportion of thymine (T) or uracil (U) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%.

[0186] In some embodiments, in the first sub-population of library molecules, the proportion of adenine (A) and thymine (T), or the proportion of adenine (A) and uracil (U), at any given position in the short random sequence is about 10-65%. In some embodiments, in the first sub-population of library molecules, the proportion of guanine (G) and cytosine (C) at any given position in the short random sequence is about 10-65%.

[0187] In some embodiments, the second batch barcode can include a short random sequence (e.g., NNN) that is 3-20 in length. In some embodiments, the second batch sample index can include a short random sequence (e.g., NNN) that is 3-20 in length. In some embodiments, both the second batch barcode sequence and the second batch sample index sequence both include a short random sequence (e.g., NNN) that is 3-20 in length. In some embodiments, sequencing the short random sequence can provide nucleotide diversity and color balance. In some embodiments, sequencing and imaging the short random sequence can be used for polony mapping, location, and template registration because the short random sequence provides sufficient nucleotide diversity and color balance.56316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0188] In some embodiments, in the second sub-population of library molecules, the short random sequence (e.g., NNN) has an overall base composition of about 25% or about 20-30% of all four nucleotide bases (e.g., A, G, C and T / U) to provide nucleotide diversity at each sequencing cycle during sequencing the short random sequence (e.g., NNN).

[0189] In some embodiments, in the second sub-population of library molecules, the proportion of adenine (A) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%. In some embodiments, in the second sub-population of library molecules, the proportion of guanine (G) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%. In some embodiments, in the second sub-population of library molecules, the proportion of cytosine (C) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%. In some embodiments, in the second sub-population of library molecules, the proportion of thymine (T) or uracil (U) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%.

[0190] In some embodiments, in the second sub-population of library molecules, the proportion of adenine (A) and thymine (T), or the proportion of adenine (A) and uracil (U), at any given position in the short random sequence is about 10-65%. In some embodiments, in the second sub-population of library molecules, the proportion of guanine (G) and cytosine (C) at any given position in the short random sequence is about 10-65%.

[0191] In some embodiments, in the methods for sequencing of step (a), the plurality of template molecules comprise concatemer template molecules. In some embodiments, the concatemer template molecules comprise at least first and second sub-populations of concatemer template molecules. In some embodiments, the concatemer template molecules can be generated by conducting rolling circle amplification (RCA) using circularized library molecules and amplification primers. In some embodiments, a concatemer template molecule comprises numerous tandem copies of a polynucleotide unit. In some embodiments, each polynucleotide unit comprises a sequence of interest and at least one sequencing primer binding site. In some embodiments, concatemer template molecules immobilized to a support can be generated using circularized library molecules and conducting rolling circle amplification. In some embodiments, the circularized library molecules can be generated using padlock probes, single-stranded splint strands, or double-stranded adaptors. In some embodiments, the circularized library molecules comprise a mixture of any combination of circularized padlock probes, linear library molecules (single-stranded linear library molecules), circularized using single-stranded splint strands, and / or linear library molecules 57316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208circularized using double-stranded adaptors. Methods for generating circularized library molecules are described herein. Methods for generating circularized library molecules are described in WO2023168444, WO2023168443, W02024011145, W02024059550, WO2025024465, the contents of each of which are incorporated by reference in their entirety herein.

[0192] In some embodiments, individual concatemer template molecules in the first subpopulation comprise a plurality of tandem polynucleotide units. In some embodiments, each polynucleotide unit comprises a first sequence of interest and a first batch sequencing primer binding site sequence which corresponds to the first sequence of interest. In some embodiments, the polynucleotide unit further comprises a first batch barcode sequence which corresponds to the first sequence of interest. In some embodiments, the polynucleotide unit further comprises at least one sample index sequence that can be used in a multiplex assay to distinguish sequences of interest obtained from different sample sources. In some embodiments, concatemer template molecules in the first sub-population have the same first batch sequencing primer binding site. In some embodiments, concatemer template molecules in the first sub-population have the same sequence of interest or different sequences of interest.

[0193] In some embodiments, individual concatemer template molecules in the second sub-population comprise a plurality of tandem polynucleotide units. In some embodiments, each polynucleotide unit comprises a second sequence of interest and a second batch sequencing primer binding site sequence which corresponds to the second sequence of interest. In some embodiments, the polynucleotide unit further comprises a second batch barcode sequence which corresponds to the second sequence of interest. In some embodiments, the polynucleotide unit further comprises at least one sample index sequence that can be used in a multiplex assay to distinguish sequences of interest obtained from different sample sources. In some embodiments, concatemer template molecules in the second sub-population have the same second batch sequencing primer binding site. In some embodiments, concatemer template molecules in the second sub-population have the same sequence of interest or different sequences of interest.

[0194] In some embodiments, in the methods for sequencing of step (a), the plurality of concatemer template molecules can be generated by conducting a rolling circle amplification reaction in the presence of a plurality of compaction oligonucleotides. Exemplary compaction oligonucleotides are described in W02024040058, the contents of which are incorporated by reference herein in their entirety. In some embodiments, in the methods for sequencing of 58316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208step (a), the plurality of concatemer template molecules can be generated by conducting a rolling circle amplification reaction in the absence of a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides can hybridize to two different locations on the same the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the template molecule to form a DNA nanoball. In some embodiments, individual concatemer template molecules collapse into a polony or nucleic acid nanoball having a compact size and shape compared to a non-collapsed concatemer template molecule.

[0195] In some embodiments, the methods for sequencing further comprise step (b): sequencing the first sub-population of template molecules using a plurality of first batch sequencing primers, thereby generating a plurality of first batch sequencing read products. In some embodiments, the sequencing of step (b) comprises imaging a region of the support to detect the sequencing reactions of the first sub-population of template molecules.

[0196] In some embodiments, the sequencing of step (b) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprise nucleotides, nucleotide analogs and / or multivalent molecules. Exemplary methods are described in WO2022266470, US20240191278A1 and WO2024159166, the contents of which are incorporated by reference in their entirety herein.

[0197] In some embodiments, the sequencing of step (b) comprises conducting a two-stage sequencing method. In some embodiments, the first stage comprises contacting the first sub-population of template molecules with a plurality of first batch sequencing primers, a first plurality of sequencing polymerase and a plurality of detectably labeled multivalent molecules. In some embodiments, the first stage comprises binding detectably labeled multivalent molecules to complexed polymerases to form multivalent-complexed polymerases, and detecting the multivalent-complexed polymerases. In some embodiments, individual multivalent molecules comprise a core attached to multiple nucleotide arms and each nucleotide arm is attached to a nucleotide (e.g., a nucleotide unit) (e.g., FIGs. 1-5). In some embodiments, the multivalent molecules can be labeled with at least one detectable moiety that emits a signal. In some embodiments, the multivalent molecules can be labeled with at least one fluor ophore.

[0198] In some embodiments, individual complexed polymerases comprise a first sequencing polymerase bound to a nucleic acid duplex where the nucleic acid duplex comprises a template molecule hybridized to a sequencing primer. In some embodiments, the 59316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208detectably labeled multivalent molecules bind to the complexed polymerases to form a plurality of multivalent-complexed polymerases. In some embodiments, the detectably labeled multivalent molecules are bound to the complexed polymerases in the presence of a trapping reagent. In some embodiments, the trapping reagent can be formulated to promote binding of the detectably labeled multivalent molecules to the complexed polymerases. In some embodiments, the trapping reagent can be formulated to inhibit incorporation of the nucleotide unit of the multivalent molecules. In some embodiments, the trapping reagent comprises at least one solvent, at least one pH buffering agent, at least one non-catalytic cation, at least one viscosity agent, at least one chelating agent, at least one detergent, at least one monovalent cation, and at least one reducing agent. In some embodiments, the trapping reagent further comprises at least one chaotropic agent. In some embodiments, the trapping reagent further comprises an amino acid or a modified amino acid. In some embodiments, the trapping reagent further comprises a plurality of multivalent molecules. In some embodiments, the trapping reagent further comprises a first plurality of sequencing polymerases. In some embodiments, the at least one non-catalytic cation inhibits polymerase-catalyzed nucleotide incorporation.

[0199] In some embodiments, the multivalent-complexed polymerases can be exposed to excitation illumination to induce fluorescent signals from the multivalent-complexed polymerases. In some embodiments, the fluorescent signals from the multivalent-complexed polymerases can be imaged in the presence of an imaging reagent. In some embodiments, the imaging reagent can be formulated to reduce photo damage of the fluorescently-labeled multivalent-complexed polymerases upon exposure to the excitation illumination. In some embodiments, the imaging reagent can be formulated to inhibit polymerase-catalyzed nucleotide incorporation. In some embodiments, the imaging reagent comprises at least one solvent, at least one pH buffering agent, at least one chelating agent, at least one non-catalytic divalent cation, at least one compound for reducing photo-damage, at least one reducing agent, at least one detergent and at least one viscosity agent. In some embodiments, prior to conducting the second sequencing stage, the detectably labeled multivalent molecules can be dissociated from the complexed polymerases and removed (e.g., washing). In some embodiments, prior to conducting the sequencing second stage, the first plurality of sequencing polymerases can be dissociated from the first sub-population of template molecules. In some embodiments, the first sub-population of template molecules can remain immobilized to the support and the first batch sequencing primers can be retained and can remain hybridized to the first sub-population of template molecules.60316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0200] In some embodiments, the second stage of the two-stage sequencing method comprises contacting the first sub-population of template molecules and the retained first batch sequencing primers with a second plurality of sequencing polymerases and a plurality of nucleotides (e.g., non-conjugated free nucleotides). In some embodiments, the second stage comprises binding the plurality of nucleotides to the complexed polymerases to form nucleotide-complexed polymerases, and promoting nucleotide incorporation. In some embodiments, the second stage of the two-stage sequencing method comprises nucleotide incorporation and extension of the first batch sequencing primers.

[0201] In some embodiments, the plurality of nucleotides comprise fluorophore-labeled nucleotides. In some embodiments, the plurality of nucleotides are non-labeled. In some embodiments, when the nucleotides are fluorophore-labeled, detecting and imaging of the incorporated nucleotides can be performed. In some embodiments, when the nucleotides are non-labeled, detecting and imaging of the incorporated nucleotides can be omitted.

[0202] In some embodiments, the nucleotides comprise chain terminating nucleotides where individual nucleotides comprise a chain terminating moiety attached to the 3’ sugar position. In some embodiments, the nucleotides are not chain terminating nucleotides. In some embodiments, when the nucleotides comprise chain terminating nucleotides, the chain terminating moieties can be cleaved from the incorporated chain terminating nucleotides to generate an extendible 3 ’OH group.

[0203] In some embodiments, nucleotide incorporation can be conducted in the presence of a stepping reagent. In some embodiments, the stepping reagent can be formulated to promote polymerase-catalyzed nucleotide incorporation. In some embodiments, the stepping reagent comprises at least one solvent, at least one pH buffering agent, at least one monovalent cation, at least one catalytic cation, at least one viscosity agent, at least one chelating agent, at least one amino acid, at least one detergent. In some embodiments, the stepping reagent further comprises a plurality of nucleotides (e.g., non-conjugated free nucleotides). In some embodiments, the stepping reagent further comprises a second plurality of sequencing polymerases. In some embodiments, the at least one catalytic cation promotes polymerase-catalyzed nucleotide incorporation. In some embodiments, in the stepping reagent, the plurality of nucleotides comprises chain terminating nucleotides. In some embodiments, individual nucleotides comprise a chain terminating moiety attached to the 3’ sugar position. In some embodiments, in the stepping reagent, the plurality of nucleotides are not chain terminating nucleotides.61316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0204] In some embodiments, the sequencing of step (b) comprises conducting a two-stage sequencing method including repeating the first stage and second stage at least once thereby generating a plurality of first batch sequencing read products. In some embodiments, when conducting a two-stage sequencing method, one sequencing cycle comprises completion of a first and a second stage. In some embodiments, the sequencing of step (b) comprises conducting 4-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween. In some embodiments, the sequencing of step (b) comprises sequencing at least a portion of the first batch barcode and / or sequencing at least a portion of the first sample index. In some embodiments, the sequencing of step (b) comprises sequencing at least a portion of the first sequence of interest.

[0205] In some embodiments, prior to sequencing the second sub-population of template molecules, the plurality of first batch sequencing read products can be removed from the first sub-population of template molecules. In some embodiments, the first sub-population of template molecules can be retained on the support using a de-hybridization reagent. In some embodiments, the de-hybridization reagent comprises an SSC buffer (e.g., saline-sodium citrate) buffer, with or without formamide, at a temperature that promotes nucleic acid denaturation such as for example 50 - 90 °C. In some embodiments, the de-hybridization reagent comprises at least one solvent, at least one pH buffering agent, at least one reducing agent, at least one monovalent salt and at least one crowding agent. In some embodiments, the de-hybridization reagent further comprises at least one chaotropic agent. In some embodiments, the de-hybridization reagent further comprises at least one nucleic acid compaction agent. In some embodiments, the de-hybridization step can be conducted at a temperature that promotes nucleic acid denaturation such as for example 50 - 90 °C. In some embodiments, the first batch sequencing read products are not removed from the first subpopulation of template molecules.

[0206] In some embodiments, the sequencing reactions of the first sub-population of template molecules is stopped before initiating the sequencing reactions of the second subpopulation of template molecules.

[0207] In some embodiments, the methods for sequencing further comprises step (bl): conducting short read sequencing by performing up to 1000 sequencing cycles of the first sub-population of template molecules to generate a plurality of first batch sequencing read products. In some embodiments, the plurality of first batch sequencing read products62316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208comprises up to 1000 bases in length. In some embodiments, step (bl) comprises conducting 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween. In some embodiments, the first batch sequencing read products comprise a first batch barcode sequence. In some embodiments, the first batch sequencing read products comprise a first batch barcode sequence and a sample index sequence. In some embodiments, the first batch sequencing read products comprise a first batch barcode sequence and at least a portion of a first sequence of interest. In some embodiments, the first batch sequencing read products comprise a first batch barcode sequence, a sample index sequence, and at least a portion of a first sequence of interest. In some embodiments, the short read sequencing comprises hybridizing sequencing primers to sequencing primer binding sites on concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, 500 million - 1 billion of the first subpopulation of concatemer template molecules can be sequenced. In some embodiments, up to 1 billion, or up to 2 billion, or up to 3 billion, or up to 4 billion, or up to 5 billion of the first sub-population of concatemer template molecules can be sequenced. In some embodiments, up to 6 billion, or up to 7 billion, or up to 8 billion, or up to 9 billion, or up to 10 billion of the first sub-population of concatemer template molecules can be sequenced. In some embodiments, between about 500 million and about 10 billion, between about 1 billion and about 9 billion, between about 2 billion and about 8 billion, between about 3 billion and about 7 billion, between about 4 billion and about 6 billion, or any range therebetween of the first sub-population of concatemer template molecules can be sequenced.

[0208] In some embodiments, the sequencing of step (bl) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprise nucleotides, nucleotide analogs and / or multivalent molecules. In some embodiments, the reiterative sequencing of step (bl) comprises conducting a two-stage sequencing method described herein.

[0209] In some embodiments, the methods for sequencing further comprises step (b2): stopping and / or blocking the short read sequencing of step (bl). In some embodiments, the stopping / blocking comprises incorporating a chain terminating nucleotide to the 3’ terminal end of the first batch sequencing read products to inhibit further sequencing reactions.63316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208Exemplary chain terminating nucleotides include dideoxynucleotide or a nucleotide having a 2’ or 3’ chain terminating moiety.

[0210] In some embodiments, the methods for sequencing further comprise step (b3): removing the plurality of first batch sequencing read products from the template molecules of the first sub-population, and retaining the template molecules of the first sub-population. In some embodiments, the first batch sequencing read products can be removed from the template molecules by denaturation using heat and / or a de-hybridization reagent.

[0211] In some embodiments, the methods for sequencing further comprise step (b4): reiteratively sequencing the template molecules of the first sub-population by repeating steps (bl) - (b3) at least once. In some embodiments, the reiterative sequencing can be conducted 1-10 times, or 10-25 times, or 25-50 times, or any range therebetween, or more than 50 times. For example, the reiterative sequencing can be conducted up to 100 times.

[0212] In some embodiments, the sequences of all of the first batch sequencing read products can be determined and aligned with a first reference sequence to confirm the presence of the first sequence of interest. The first reference sequence can be the first batch barcode and / or the first sequence of interest.

[0213] In some embodiments, hybridizing the sequencing primers to the concatemer template molecules of step (bl) can be conducted with a hybridization reagent comprising an SSC buffer (e.g., 2X saline-sodium citrate) buffer with formamide (e.g., 10-20% formamide).

[0214] In some embodiments, in step (b3) the plurality of plurality of first batch sequencing read products can be removed from the template molecules and the plurality of template molecules can be retained using a de-hybridization reagent comprising an SSC buffer (e.g., saline-sodium citrate) buffer, with or without formamide, at a temperature that promotes nucleic acid denaturation such as for example 50 - 90 °C.

[0215] In some embodiments, in step (b3) the plurality of plurality of first batch sequencing read products can be removed from the template molecules and the plurality of template molecules can be retained using a de-hybridization reagent comprising at least one solvent, at least one pH buffering agent, at least one reducing agent, at least one monovalent salt and at least one crowding agent. In some embodiments, the de-hybridization reagent further comprises at least one chaotropic agent. In some embodiments, the de-hybridization reagent further comprises at least one nucleic acid compaction agent. In some embodiments, the de-hybridization of step (b3) can be conducted at a temperature that promotes nucleic acid denaturation such as for example 50 - 90 °C.64316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0216] In some embodiments, the methods for sequencing further comprise step (c): sequencing the second sub-population of template molecules using a plurality of second batch sequencing primers thereby generating a plurality of second batch sequencing read products and imaging the same region of the support to detect the sequencing reactions of the second sub-population of template molecules.

[0217] In some embodiments, the sequencing reactions of the first sub-population of template molecules is stopped before initiating the sequencing reactions of the second subpopulation of template molecules.

[0218] In some embodiments, the sequencing of step (c) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprise nucleotides, nucleotide analogs and / or multivalent molecules. Exemplary sequencing methods are described in WO2022266470, the contents of which are incorporated by reference in their entirety herein.

[0219] In some embodiments, the sequencing of step (c) comprises conducting a two-stage sequencing method. In some embodiments, the first stage generally comprises contacting the second sub-population of template molecules with a plurality of second batch sequencing primers, a first plurality of sequencing polymerase and a plurality of detectably labeled multivalent molecules. In some embodiments, the first stage comprises binding detectably labeled multivalent molecules to complexed polymerases to form multivalent-complexed polymerases, and detecting the multivalent-complexed polymerases. In some embodiments, individual multivalent molecules comprise a core attached to multiple nucleotide arms and each nucleotide arm is attached to a nucleotide (e.g., nucleotide unit) (e.g., FIGs. 1-5). In some embodiments, the multivalent molecules can be labeled with at least one detectable moiety that emits a signal. In some embodiments, the multivalent molecules can be labeled with at least one fluorophore.

[0220] In some embodiments, individual complexed polymerases comprise a first sequencing polymerase bound to a nucleic acid duplex. In some embodiments, the nucleic acid duplex comprises a template molecule hybridized to a sequencing primer. In some embodiments, the detectably labeled multivalent molecules bind to the complexed polymerases to form a plurality of multivalent-complexed polymerases. In some embodiments, the detectably labeled multivalent molecules are bound to the complexed polymerases in the presence of a trapping reagent. In some embodiments, the trapping reagent can be formulated to promote binding of the detectably labeled multivalent molecules 65316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208to the complexed polymerases. In some embodiments, the trapping reagent can be formulated to inhibit incorporation of the nucleotide unit of the multivalent molecules. In some embodiments, the trapping reagent comprises at least one solvent, at least one pH buffering agent, at least one non-catalytic cation, at least one viscosity agent, at least one chelating agent, at least one detergent, at least one monovalent cation, at least one reducing agent, and at least one chaotropic agent. In some embodiments, the trapping reagent further comprises a plurality of multivalent molecules. In some embodiments, the trapping reagent further comprises a first plurality of sequencing polymerases. In some embodiments, the at least one non-catalytic cation inhibits polymerase-catalyzed nucleotide incorporation.

[0221] In some embodiments, the multivalent-complexed polymerases can be exposed to excitation illumination to induce fluorescent signals from the multivalent-complexed polymerases. In some embodiments, the fluorescent signals from the multivalent-complexed polymerases can be imaged in the presence of an imaging reagent. In some embodiments, the imaging reagent can be formulated to reduce photo damage of the fluorescently-labeled multivalent-complexed polymerases upon exposure to the excitation illumination. In some embodiments, the imaging reagent can be formulated to inhibit polymerase-catalyzed nucleotide incorporation. In some embodiments, the imaging reagent comprises at least one solvent, at least one pH buffering agent, at least one chelating agent, at least one non-catalytic divalent cation, at least one compound for reducing photo-damage, at least one reducing agent, at least one detergent and at least one viscosity agent. In some embodiments, prior to conducting the second sequencing stage, the detectably labeled multivalent molecules can be dissociated from the complexed polymerases and removed (e.g., washing). In some embodiments, prior to conducting the second sequencing stage, the first plurality of sequencing polymerases can be dissociated from the second sub-population of template molecules. In some embodiments, the second sub-population of template molecules can remain immobilized to the support and the second batch sequencing primers can be retained and can remain hybridized to the second sub-population of template molecules.

[0222] In some embodiments, the second stage of the two-stage sequencing method generally comprises contacting the second sub-population of template molecules and the retained second batch sequencing primers with a second plurality of sequencing polymerases and a plurality of nucleotides (e.g., non-conjugated free nucleotides). In some embodiments, the second stage comprises binding the plurality of nucleotides to the complexed polymerases to form nucleotide-complexed polymerases, and promoting nucleotide incorporation. In some66316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208embodiments, the second stage of the two-stage sequencing method comprises nucleotide incorporation and extension of the second batch sequencing primer.

[0223] In some embodiments, the plurality of nucleotides comprise fluorophore-labeled nucleotides. In some embodiments, the plurality of nucleotides are non-labeled. In some embodiments, when the nucleotides are fluorophore-labeled, detecting and imaging of the incorporated nucleotides can be performed. In some embodiments, when the nucleotides are non-labeled, detecting and imaging of the incorporated nucleotides can be omitted.

[0224] In some embodiments, the nucleotides comprise chain terminating nucleotides where individual nucleotides comprise a chain terminating moiety attached to the 3’ sugar position. In some embodiments, the nucleotides are not chain terminating nucleotides. In some embodiments, when the nucleotides comprise chain terminating nucleotides, the chain terminating moieties can be cleaved from the incorporated chain terminating nucleotides to generate an extendible 3 ’OH group.

[0225] In some embodiments, nucleotide incorporation can be conducted in the presence of a stepping reagent. In some embodiments, the stepping reagent can be formulated to promote polymerase-catalyzed nucleotide incorporation. In some embodiments, the stepping reagent comprises at least one solvent, at least one pH buffering agent, at least one monovalent cation, at least one catalytic cation, at least one viscosity agent, at least one chelating agent, at least one amino acid, at least one detergent. In some embodiments, the stepping reagent further comprises a plurality of nucleotides (e.g., non-conjugated free nucleotides). In some embodiments, the stepping reagent further comprises a second plurality of sequencing polymerases. In some embodiments, the at least one catalytic cation promotes polymerase-catalyzed nucleotide incorporation. In some embodiments, in the stepping reagent, the plurality of nucleotides comprises chain terminating nucleotides where individual nucleotides comprise a chain terminating moiety attached to the 3’ sugar position. In some embodiments, in the stepping reagent, the plurality of nucleotides are not chain terminating nucleotides.

[0226] In some embodiments, the sequencing of step (c) comprises conducting a two-stage sequencing method including repeating the first stage and second stage at least once thereby generating a plurality of second batch sequencing read products. In some embodiments, when conducting a two-stage sequencing method, one sequencing cycle comprises completion of a first and a second stage. In some embodiments, the sequencing of step (c) comprises conducting 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-50067316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween. In some embodiments, the sequencing of step (c) comprises sequencing at least a portion of the second batch barcode and / or sequencing at least a portion of the second sample index. In some embodiments, the sequencing of step (c) comprises sequencing at least a portion of the second sequence of interest.

[0227] In some embodiments, prior to sequencing a subsequent sub-population of template molecules (e.g., after sequencing the second sub-population of template molecules), the plurality of second batch sequencing read products can be removed from the second subpopulation of template molecules and the second sub-population of template molecules can be retained on the support using a de-hybridization reagent. In some embodiments, the dehybridization reagent comprises an SSC buffer (e.g., saline-sodium citrate) buffer, with or without formamide, at a temperature that promotes nucleic acid denaturation such as for example 50 - 90 °C. In some embodiments, the de-hybridization reagent comprises at least one solvent, at least one pH buffering agent, at least one reducing agent, at least one monovalent salt and at least one crowding agent. In some embodiments, the de-hybridization reagent further comprises at least one chaotropic agent. In some embodiments, the de-hybridization reagent further comprises at least one nucleic acid compaction agent. In some embodiments, the de-hybridization step can be conducted at a temperature that promotes nucleic acid denaturation such as for example 50 - 90 °C. In some embodiments, the second batch sequencing read products are not removed from the second sub-population of template molecules.

[0228] In some embodiments, the sequencing reactions of the second sub-population of template molecules is stopped before initiating the sequencing reactions of the subsequent sub-population of template molecules.

[0229] In some embodiments, the methods for sequencing further comprise step (cl): conducting short read sequencing by performing up to 1000 sequencing cycles of the second sub-population of template molecules to generate a plurality of second batch sequencing read products that comprise up to 1000 bases in length. In some embodiments, step (cl) comprises conducting 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween. In some embodiments, the second batch sequencing read products comprise a second batch barcode sequence. In some embodiments, the second batch sequencing read products comprise a second batch barcode sequence and a sample index sequence. In some68316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208embodiments, the second batch sequencing read products comprise a second batch barcode sequence and at least a portion of a second sequence of interest. In some embodiments, the second batch sequencing read products comprise a second batch barcode sequence, a sample index sequence, and at least a portion of a second sequence of interest. In some embodiments, the short read sequencing comprises hybridizing sequencing primers to sequencing primer binding sites on concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, 500 million - 1 billion of the second sub-population of concatemer template molecules can be sequenced. In some embodiments, up to 1 billion, or up to 2 billion, or up to 3 billion, or up to 4 billion, or up to 5 billion of the second sub-population of concatemer template molecules can be sequenced. In some embodiments, up to 6 billion, or up to 7 billion, or up to 8 billion, or up to 9 billion, or up to 10 billion of the second sub-population of concatemer template molecules can be sequenced. In some embodiments, between about 500 million and about 10 billion, between about 1 billion and about 9 billion, between about 2 billion and about 8 billion, between about 3 billion and about 7 billion, between about 4 billion and about 6 billion, or any range therebetween of the second sub-population of concatemer template molecules can be sequenced.

[0230] In some embodiments, the sequencing of step (cl) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprise nucleotides, nucleotide analogs and / or multivalent molecules. In some embodiments, the reiterative sequencing of step (cl) comprises conducting a two-stage sequencing method described herein.

[0231] In some embodiments, the methods for sequencing further comprise step (c2): stopping and / or blocking the short read sequencing of step (cl). In some embodiments, the stopping / blocking comprises incorporating a chain terminating nucleotide to the 3’ terminal end of the first batch sequencing read products to inhibit further sequencing reactions.Exemplary chain terminating nucleotides include dideoxynucleotide or a nucleotide having a 2’ or 3’ chain terminating moiety.

[0232] In some embodiments, the methods for sequencing further comprise step (c3): removing the plurality of second batch sequencing read products from the template molecules of the second sub-population, and retaining the template molecules of the second subpopulation. In some embodiments, the second batch sequencing read products can be69316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208removed from the template molecules by denaturation using heat and / or a de-hybridization reagent.

[0233] In some embodiments, the methods for sequencing further comprise step (c4): reiteratively sequencing the template molecules of the second sub-population by repeating steps (cl) - (c3) at least once. In some embodiments, the reiterative sequencing can be conducted 1-10 times, or 10-25 times, or 25-50 times, or any range therebetween, or more than 50 times.

[0234] In some embodiments, the sequences of all of the second batch sequencing read products can be determined and aligned with a second reference sequence to confirm the presence of the second sequence of interest. The second reference sequence can be the second batch barcode and / or the second sequence of interest.

[0235] In some embodiments, hybridizing the sequencing primers to the concatemer template molecules of step (cl) can be conducted with a hybridization reagent comprising an SSC buffer (e.g., 2X saline-sodium citrate) buffer with formamide (e.g., 10-20% formamide).

[0236] In some embodiments, in step (c3) the plurality of plurality of second batch sequencing read products can be removed from the template molecules and the plurality of template molecules can be retained using a de-hybridization reagent comprising an SSC buffer (e.g., saline-sodium citrate) buffer, with or without formamide, at a temperature that promotes nucleic acid denaturation such as for example 50 - 90 °C.

[0237] In some embodiments, in step (c3) the plurality of plurality of second batch sequencing read products can be removed from the template molecules and the plurality of template molecules can be retained using a de-hybridization reagent comprising at least one solvent, at least one pH buffering agent, at least one reducing agent, at least one monovalent salt and at least one crowding agent. In some embodiments, the de-hybridization reagent further comprises at least one chaotropic agent. In some embodiments, the de-hybridization reagent further comprises at least one nucleic acid compaction agent. In some embodiments, the de-hybridization of step (b3) can be conducted at a temperature that promotes nucleic acid denaturation such as for example 50 - 90 °C.Re-Seeding a Support with Interrupted Sequencing

[0238] The present disclosure provides methods for re-seeding a support comprising step (a): providing a support comprising a plurality of surface capture primers immobilized to the support. In some embodiments, the plurality of capture primers have the same sequence. In some embodiments, the plurality of capture primers comprise at least two sub-populations of 70316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208capture primers including at least a first sub-population of capture primers having a first sequence and a second sub-population of capture primers having a second sequence. In some embodiments, the plurality of surface capture primers comprise single-stranded oligonucleotides. In some embodiments, the plurality of surface capture primers can be used to generate concatemer template molecules immobilized to the support. In some embodiments, the density of the plurality of surface capture primers is about 102- 1015per urn2, e.g. between about IO10and about 1015surface capture primers per mm2, between about 105and about 1015surface capture primers per mm2, between about 103and about 1014surface capture primers per mm2, between about 104and about 1013surface capture primers per mm2, between about 105and about 1012surface capture primers per mm2, between about 106and about 1011surface capture primers per mm2, between about 107and about IO10surface capture primers per mm2, or between about 108and about IO10surface capture primers per mm2, or any range therebetween.

[0239] In some embodiments, the plurality of surface capture primers can be immobilized to the support at random and non-pre-determined positions. In some embodiments, the plurality of surface capture primers can be immobilized to the support at pre-determined positions (e.g., a patterned support).

[0240] In some embodiments, the support is passivated with at least one polymer layer comprising a plurality of surface capture primers covalently tethered to the at least one polymer layer. In some embodiments, the plurality of surface capture primers are randomly distributed throughout and embedded within the at least one polymer layer.

[0241] In some embodiments, the support lacks any contours (e.g., wells, protrusions, and the like) arranged in a pre-determined pattern where the contours have features that are sites for attachment (e.g., immobilization) of the nucleic acid template molecules.

[0242] In some embodiments, the support lacks partitions and / or barriers that would create separate regions of the support.

[0243] In some embodiments, the plurality of surface capture primers are located at predetermined positions on the at least one polymer layer and / or the plurality of surface capture primers are embedded within the at least one polymer layer at pre-determined locations.

[0244] In some embodiments, the support includes contours (e.g., wells, protrusions, and the like) arranged in a pre-determined pattern where the contours have features that are sites for attachment of the nucleic acid template molecules. In some embodiments, the support includes interstitial regions arranged in a pre-determined pattern where the interstitial regions are sites designed to have no attached template molecules.71316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0245] In some embodiments, the methods for re-seeding a support further comprise step (b): distributing on the support a first plurality of circularized nucleic acid library molecules under a condition suitable for hybridizing individual circularized library molecules to individual surface capture primers and conducting a rolling circle amplification reaction in a template-dependent manner using individual circularized library molecules in the first plurality as templates, thereby generating a first plurality of nucleic acid concatemer template molecules immobilized to the support. In some embodiments, a subset of the surface capture primers hybridize to individual circularized library molecules to generate a first plurality of concatemer template molecules. In some embodiments, the number of surface capture primers immobilized to the support exceeds the number of first plurality of circularized nucleic acid library molecules distributed onto the support. In some embodiments, the support comprises up to 500 million of a first plurality of concatemer template molecules immobilized thereon, or up to 1 billion a first plurality of concatemer template molecules immobilized thereon, or up to 2 billion a first plurality of concatemer template molecules immobilized thereon, or up to 3 billion a first plurality of concatemer template molecules immobilized thereon, or up to 4 billion a first plurality of concatemer template molecules immobilized thereon, or up to 5 billion a first plurality of concatemer template molecules immobilized thereon, or up to 6 billion a first plurality of concatemer template molecules immobilized thereon. In some embodiments, the support comprises up to 7 billion concatemer template molecules immobilized thereon, or up to 8 billion concatemer template molecules immobilized thereon, or up to 9 billion concatemer template molecules immobilized thereon, or up to 10 billion concatemer template molecules immobilized thereon, or up to 20 billion concatemer template molecules immobilized thereon. In some embodiments, the support comprises between about 500 million and about 20 billion concatemer template molecules immobilized thereon, between about 1 billion and about 10 billion concatemer template molecules immobilized thereon, between about 2 billion and about 9 billion concatemer template molecules immobilized thereon, between about 3 billion and about 8 billion concatemer template molecules immobilized thereon, between about 4 billion and about 7 billion concatemer template molecules immobilized thereon, or between about 5 billion and about 6 billion concatemer template molecules immobilized thereon, or any range therebetween. In some embodiments, individual concatemer template molecules in the first plurality comprise a plurality of tandem copies of a polynucleotide unit. In some embodiments, each polynucleotide unit comprises a sequence of interest and a batch seeding sequencing primer binding site sequence. In some embodiments, the first plurality of72316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208circularized library molecules can be generated using padlock probes, single-stranded splint strands, or double-stranded adaptors. In some embodiments, the first plurality of circularized library molecules comprise a mixture of any combination of circularized padlock probes, linear library molecules circularized using single-stranded splint strands, and / or linear library molecules circularized using double-stranded adaptors. Methods for generating circularized library molecules are described herein.

[0246] In some embodiments, in the methods for re-seeding a support of step (b), individual circularized library molecules in the first plurality comprise a sequence of interest, a seeding batch sequencing primer binding site sequence which corresponds to the sequence of interest, and a surface capture primer binding site. In some embodiments, a pre-determined first seeding batch sequencing primer binding site sequence can be linked to a given sequence of interest in the first plurality of circularized library molecules, thus the pre-determined first seeding batch sequencing primer binding site sequence corresponds to a given sequence of interest in the first plurality of circularized library molecules. In some embodiments, a predetermined first seeding batch sequencing primer binding site sequence can be linked to different sequences of interest in a first plurality of circularized library molecules.

[0247] In some embodiments, individual circularized library molecules in the first plurality further comprise a seeding batch barcode sequence which corresponds to the sequence of interest. In some embodiments, a pre-determined first seeding batch barcode sequence can be linked to a given sequence of interest in the first plurality of circularized library molecules, thus the pre-determined first seeding batch barcode sequence corresponds to a given sequence of interest in the first plurality of circularized library molecules. In some embodiments, a pre-determined first seeding batch barcode sequence can be linked to different sequences of interest in a first plurality of circularized library molecules.

[0248] In some embodiments, individual circularized library molecules in the first plurality comprise a sequence of interest and an identical seeding batch sequencing primer binding site sequence which corresponds to the sequence of interest. In some embodiments, individual circularized library molecules further comprise a surface capture primer binding site and a first seeding batch barcode sequence which corresponds to the sequence of interest.

[0249] In some embodiments, the sequences of interest in the first plurality of circularized nucleic acid library molecules are about 50-250 bases in length, or about 250-500 bases in length, or about 500-800 bases in length, or about 800-1200 bases in length, or any range therebetween, or up to 2000 bases in length.73316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0250] In some embodiments, the concentration of the first plurality of circularized nucleic acid library molecules that are distributed onto the support can be about 1-5 pM, or about 5-10 pM, or about 10-50 pM, or any range therebetween.

[0251] In some embodiments, in the methods for re-seeding a support of step (b), the first plurality of circularized nucleic acid library molecules comprise a plurality of subpopulations of circularized library molecules including at least a first and second subpopulation of circularized library molecules.

[0252] In some embodiments, individual circularized library molecules in the first subpopulation comprise the same first sub-population seeding batch sequencing primer binding site sequences. In some embodiments, individual circularized library molecules in the first sub-population have the same sequence of interest or different sequences of interest. In some embodiments, the first sub-population seeding batch sequencing primer binding site sequence corresponds to the first sequence of interest, or the first sub-population seeding batch sequencing primer binding site sequence corresponds to one of the sequences of interest in the first sub-population. In some embodiments, a pre-determined first sub-population seeding batch sequencing primer binding site sequence can be linked to a given sequence of interest in the first sub-population of circularized library molecules, thus the pre-determined first subpopulation seeding batch sequencing primer binding site sequence corresponds to a given sequence of interest in the first sub-population of circularized library molecules. In some embodiments, a pre-determined first sub-population seeding batch sequencing primer binding site sequence can be linked to different sequences of interest in a first sub-population of circularized library molecules.

[0253] In some embodiments, individual circularized library molecules in the first subpopulation further comprise a first sub-population seeding batch barcode sequence which corresponds to the first sequence of interest. In some embodiments, the first sub-population seeding batch barcode sequence corresponds to one of the sequences of interest in the first sub-population. In some embodiments, a pre-determined first sub-population seeding batch barcode sequence can be linked to a given sequence of interest in the first sub-population of circularized library molecules, thus the pre-determined first sub-population seeding batch barcode sequence corresponds to a given sequence of interest in the first sub-population of circularized library molecules. In some embodiments, a pre-determined first sub-population seeding batch barcode sequence can be linked to different sequences of interest in a first subpopulation of circularized library molecules.74316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0254] In some embodiments, individual circularized library molecules in the first subpopulation further comprise a sample index sequence that can be used in a multiplex assay to distinguish sequences of interest obtained from different sample sources. In some embodiments, individual circularized library molecules in the first sub-population further comprise a surface capture primer binding site. In some embodiments, individual circularized library molecules in the first sub-population further comprise a surface pinning primer binding site. In some embodiments, individual circularized library molecules in the first subpopulation further comprise a compaction oligonucleotide binding site.

[0255] In some embodiments, the sequences of interest in the first sub-population of circularized nucleic acid library molecules are about 50-250 bases in length, or about 250-500 bases in length, or about 500-800 bases in length, or about 800-1200 bases in length, any range therebetween, or up to 2000 bases in length.

[0256] In some embodiments, in the methods for re-seeding a support of step (b), the method comprises conducting a rolling circle amplification reaction, in a template-dependent manner, using individual circularized library molecules in the first sub-population, thereby generating a first sub-population concatemer template molecules immobilized to the support. In some embodiments, a subset of the surface capture primers hybridize to individual circularized library molecules to generate the plurality of first sub-population concatemer template molecules.

[0257] In some embodiments, the first sub-population concatemer template molecules can be immobilized to the support at random and non-predetermined positions on the support, or at pre-determined positions on the support (e.g., patterned support).

[0258] In some embodiments, in the methods for re-seeding a support of step (b), individual circularized library molecules in the second sub-population comprise the same second sub-population seeding batch sequencing primer binding site sequence and have the same sequence of interest or different sequences of interest. In some embodiments, the second sub-population seeding batch sequencing primer binding site sequence corresponds to the second sequence of interest. In some embodiments, the second sub-population seeding batch sequencing primer binding site sequence corresponds to one of the sequences of interest in the second sub-population. In some embodiments, a pre-determined second subpopulation seeding batch sequencing primer binding site sequence can be linked to a given sequence of interest in the second sub-population of circularized library molecules, thus the pre-determined second sub-population seeding batch sequencing primer binding site sequence corresponds to a given sequence of interest in the second sub-population of75316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208circularized library molecules. In some embodiments, a pre-determined second subpopulation seeding batch sequencing primer binding site sequence can be linked to different sequences of interest in a second sub-population of circularized library molecules.

[0259] In some embodiments, individual circularized library molecules in the second subpopulation further comprise a second sub-population seeding batch barcode sequence which corresponds to the second sequence of interest, or the second sub-population seeding batch barcode sequence corresponds to one of the sequences of interest in the second subpopulation. In some embodiments, a pre-determined second sub-population seeding batch barcode sequence can be linked to a given sequence of interest in the second sub-population of circularized library molecules, thus the pre-determined second subs-population seeding batch barcode sequence corresponds to a given sequence of interest in the second subpopulation of circularized library molecules. In some embodiments, a pre-determined second sub-population seeding batch barcode sequence can be linked to different sequences of interest in a second sub-population of circularized library molecules.

[0260] In some embodiments, individual circularized library molecules in the second subpopulation further comprise a sample index sequence that can be used in a multiplex assay to distinguish sequences of interest obtained from different sample sources. In some embodiments, individual circularized library molecules in the second sub-population further comprise a surface capture primer binding site. In some embodiments, individual circularized library molecules in the second sub-population further comprise a surface pinning primer binding site. In some embodiments, individual circularized library molecules in the second sub-population further comprise a compaction oligonucleotide binding site.

[0261] In some embodiments, the sequences of interest in the second sub-population of circularized nucleic acid library molecules are about 50-250 bases in length, or about 250-500 bases in length, or about 500-800 bases in length, or about 800-1200 bases in length, or any range therebetween, or up to 2000 bases in length.

[0262] In some embodiments, the first sub-population seeding batch sequencing primer binding site sequence and second sub-population seeding batch sequencing primer binding site sequence have different sequences.

[0263] In some embodiments, in the methods for re-seeding a support of step (b), the method comprises conducting a rolling circle amplification reaction, in a template-dependent manner, using individual circularized library molecules in the second sub-population, thereby generating a plurality of second sub-population concatemer template molecules immobilized to the support. In some embodiments, a subset of the surface capture primers hybridize to 76316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208individual circularized library molecules to generate the plurality of second sub-population concatemer template molecules.

[0264] In some embodiments, the second sub-population concatemer template molecules can be immobilized to the support at random and non-predetermined positions on the support, or at pre-determined positions on the support (e.g., patterned support).

[0265] In some embodiments, in the methods for re-seeding a support of step (b), the rolling circle amplification reaction comprises contacting the primed circularized library molecules with a plurality of a strand displacing polymerase, and a plurality of nucleotides which include dATP, dCTP, dGTP, dTTP.

[0266] In some embodiments, the plurality of nucleotide further comprises a plurality of a nucleotide having a scissile moiety (e.g., uracil).

[0267] In some embodiments, the rolling circle amplification reaction of step (b) can be conducted in the presence of a plurality of compaction oligonucleotides. In some embodiments, the rolling circle amplification reaction of step (b) can be conducted in the absence of a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides can hybridize to two different locations on the same the template molecule to pull together distal portions of the template molecule causing compaction of the template molecule to form a DNA nanoball.

[0268] In some embodiments, the methods for re-seeding a support further comprise step (c): sequencing at least a subset of the first plurality of concatemer template molecules, thereby generating a first plurality of sequencing read products. In some embodiments, the sequencing of step (c) comprises imaging a region of the support to detect the sequencing reactions of the first plurality of concatemer template molecules.

[0269] In some embodiments, the concatemer template molecules immobilized to the support in the first plurality are sequenced. For example, at least 30-50%, or at least 50-70%, or at least 70-90% of the concatemer template molecules in the first plurality are sequenced. In some embodiments, 500 million - 1 billion of the first plurality of concatemer template molecules can be sequenced. In some embodiments, up to 1 billion, or up to 2 billion, or up to 3 billion, or up to 4 billion, or up to 5 billion of the first plurality of concatemer template molecules can be sequenced. In some embodiments, up to 6 billion, or up to 7 billion, or up to 8 billion, or up to 9 billion, or up to 10 billion of the first plurality of concatemer template molecules can be sequenced. In some embodiments, between about 500 million and about 10 billion concatemer template molecules, between about 1 billion and about 9 billion concatemer template molecules, between about 2 billion and about 8 billion concatemer 77316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208template molecules, between about 3 billion and about 7 billion concatemer template molecules, between about 4 billion and about 5 billion concatemer template molecules, or any range therebetween of concatemer template molecules of the first plurality of concatemer template molecules can be sequenced.

[0270] In some embodiments, the full length of the concatemer template molecules in the first plurality are sequenced. In some embodiments, a partial length of the concatemer template molecules in the first plurality are sequenced.

[0271] In some embodiments, the sequencing of step (c) comprises hybridizing sequencing primers to sequencing primers binding sites on the first plurality of concatemer template molecules immobilized to the support and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, the concatemer template molecules in the first plurality can be subjected to 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.

[0272] In some embodiments, a partial length of the concatemer template molecules in the first plurality are reiteratively sequenced.

[0273] In some embodiments, in the methods for re-seeding a support of step (c), a first sub-population of the concatemer template molecules in the first plurality are sequenced using the first batch sequencing primer binding sites in the first sub-population of concatemer template molecules.

[0274] In some embodiments, the full length of the concatemer template molecules in the first sub-population are sequenced. In some embodiments, a partial length of the concatemer template molecules in the first sub-population are sequenced.

[0275] In some embodiments, the sequencing of step (c) comprises hybridizing sequencing primers to sequencing primers binding sites on the first sub-population of the first plurality of concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, the concatemer template molecules in the first sub-population can be subjected to 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.

[0276] In some embodiments, a partial length of the concatemer template molecules in the first sub-population are reiteratively sequenced.78316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0277] In some embodiments, the sequencing of step (c) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprise nucleotides, nucleotide analogs and / or multivalent molecules.

[0278] In some embodiments, the sequencing of step (c) comprises conducting a two-stage sequencing method. In some embodiments, the first stage generally comprises contacting the first sub-population of template molecules in the first plurality with a plurality of first batch sequencing primers, a first plurality of sequencing polymerase and a plurality of detectably labeled multivalent molecules. In some embodiments, the first stage comprises binding detectably labeled multivalent molecules to complexed polymerases to form multivalent-complexed polymerases, and detecting the multivalent-complexed polymerases. In some embodiments, individual multivalent molecules comprise a core attached to multiple nucleotide arms and each nucleotide arm is attached to a nucleotide (e.g., nucleotide unit) (e.g., FIGs. 1-5). In some embodiments, the multivalent molecules can be labeled with at least one detectable moiety that emits a signal. In some embodiments, the multivalent molecules can be labeled with at least one fluorophore.

[0279] In some embodiments, individual complexed polymerases comprise a first sequencing polymerase bound to a nucleic acid duplex where the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a sequencing primer. In some embodiments, the detectably labeled multivalent molecules bind to the complexed polymerases to form a plurality of multivalent-complexed polymerases. In some embodiments, the detectably labeled multivalent molecules are bound to the complexed polymerases in the presence of a trapping reagent. In some embodiments, the trapping reagent can be formulated to promote binding of the detectably labeled multivalent molecules to the complexed polymerases. In some embodiments, the trapping reagent can be formulated to inhibit incorporation of the nucleotide unit of the multivalent molecules. In some embodiments, the trapping reagent comprises at least one solvent, at least one pH buffering agent, at least one non-catalytic cation, at least one viscosity agent, at least one chelating agent, at least one detergent, at least one monovalent cation, at least one reducing agent, and at least one chaotropic agent. In some embodiments, the trapping reagent further comprises a plurality of multivalent molecules. In some embodiments, the trapping reagent further comprises a first plurality of sequencing polymerases. In some embodiments, the at least one non-catalytic cation inhibits polymerase-catalyzed nucleotide incorporation.79316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0280] In some embodiments, the multivalent-complexed polymerases can be exposed to excitation illumination to induce fluorescent signals from the multivalent-complexed polymerases. In some embodiments, the fluorescent signals from the multivalent-complexed polymerases can be imaged in the presence of an imaging reagent. In some embodiments, the imaging reagent can be formulated to reduce photo damage of the fluorescently-labeled multivalent-complexed polymerases upon exposure to the excitation illumination. In some embodiments, the imaging reagent can be formulated to inhibit polymerase-catalyzed nucleotide incorporation. In some embodiments, the imaging reagent comprises at least one solvent, at least one pH buffering agent, at least one chelating agent, at least one non-catalytic divalent cation, at least one compound for reducing photo-damage, at least one reducing agent, at least one detergent and at least one viscosity agent. In some embodiments, prior to conducting the second sequencing stage, the detectably labeled multivalent molecules can be dissociated from the complexed polymerases and removed (e.g., washing). In some embodiments, prior to conducting the second sequencing stage, the first plurality of sequencing polymerases can be dissociated from the first sub-population of template molecules in the first plurality. In some embodiments, the first sub-population of template molecules in the first plurality can remain immobilized to the support and the first batch sequencing primers can be retained and can remain hybridized to the first sub-population of template molecules in the first plurality.

[0281] In some embodiments, the second stage of the two-stage sequencing method comprises contacting the first sub-population of template molecules in the first plurality and the retained first batch sequencing primers with a second plurality of sequencing polymerases and a plurality of nucleotides (e.g., non-conjugated free nucleotides). In some embodiments, the second stage comprises binding the plurality of nucleotides to the complexed polymerases to form nucleotide-complexed polymerases, and promoting nucleotide incorporation. In some embodiments, the second stage of the two-stage sequencing method comprises nucleotide incorporation and extension of the first batch sequencing primer.

[0282] In some embodiments, the plurality of nucleotides comprise fluorophore-labeled nucleotides. In some embodiments, the plurality of nucleotides are non-labeled. In some embodiments, when the nucleotides are fluorophore-labeled, then detecting and imaging of the incorporated nucleotides can be performed. In some embodiments, when the nucleotides are non-labeled, detecting and imaging of the incorporated nucleotides can be omitted.

[0283] In some embodiments, the nucleotides comprises chain terminating nucleotides where individual nucleotides comprise a chain terminating moiety attached to the 3’ sugar 80316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208position. In some embodiments, the nucleotides are not chain terminating nucleotides. In some embodiments, when the nucleotides comprise chain terminating nucleotides, the chain terminating moieties can be cleaved from the incorporated chain terminating nucleotides to generate an extendible 3 ’OH group.

[0284] In some embodiments, nucleotide incorporation can be conducted in the presence of a stepping reagent. In some embodiments, the stepping reagent can be formulated to promote polymerase-catalyzed nucleotide incorporation. In some embodiments, the stepping reagent comprises at least one solvent, at least one pH buffering agent, at least one monovalent cation, at least one catalytic cation, at least one viscosity agent, at least one chelating agent, at least one amino acid, at least one detergent. In some embodiments, the stepping reagent further comprises a plurality of nucleotides (e.g., non-conjugated free nucleotides). In some embodiments, the stepping reagent further comprises a second plurality of sequencing polymerases. In some embodiments, the at least one catalytic cation promotes polymerase-catalyzed nucleotide incorporation. In some embodiments, in the stepping reagent, the plurality of nucleotides comprises chain terminating nucleotides where individual nucleotides comprise a chain terminating moiety attached to the 3’ sugar position. In some embodiments, in the stepping reagent, the plurality of nucleotides are not chain terminating nucleotides.

[0285] In some embodiments, the sequencing of step (c) comprises conducting a two-stage sequencing method including repeating the first stage and second stage at least once thereby generating a plurality of first batch sequencing read products. In some embodiments, when conducting a two-stage sequencing method, one sequencing cycle comprises completion of a first and a second stage. In some embodiments, the sequencing of step (c) comprises conducting 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.

[0286] In some embodiments, in the methods for re-seeding a support of step (c), a second sub-population of concatemer template molecules in the first plurality are sequenced using the second batch sequencing primer binding sites in the second sub-population of concatemer template molecules.

[0287] In some embodiments, the full length of the concatemer template molecules in the second sub-population are sequenced. In some embodiments, a partial length of the concatemer template molecules in the second sub-population are sequenced.81316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0288] In some embodiments, the sequencing of step (c) comprises hybridizing sequencing primers to sequencing primers binding sites on the second sub-population of the first plurality of concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, the concatemer template molecules in the second sub-population plurality can be subjected to 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.

[0289] In some embodiments, a partial length of the concatemer template molecules in the second sub-population are reiteratively sequenced.

[0290] In some embodiments, the sequencing of step (c) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprise nucleotides, nucleotide analogs and / or multivalent molecules.

[0291] In some embodiments, the sequencing of step (c) comprises conducting a two-stage sequencing method. In some embodiments, the first stage comprises contacting the second sub-population of template molecules in the first plurality with a plurality of second batch sequencing primers, a first plurality of sequencing polymerase and a plurality of detectably labeled multivalent molecules. In some embodiments, the first stage comprises binding detectably labeled multivalent molecules to complexed polymerases to form multivalent-complexed polymerases, and detecting the multivalent-complexed polymerases. In some embodiments, individual multivalent molecules comprise a core attached to multiple nucleotide arms and each nucleotide arm is attached to a nucleotide (e.g., nucleotide unit) (e.g., FIGs. 1-5). In some embodiments, the multivalent molecules can be labeled with at least one detectable moiety that emits a signal. In some embodiments, the multivalent molecules can be labeled with at least one fluorophore.

[0292] In some embodiments, individual complexed polymerases comprise a first sequencing polymerase bound to a nucleic acid duplex where the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a sequencing primer. In some embodiments, the detectably labeled multivalent molecules bind to the complexed polymerases to form a plurality of multivalent-complexed polymerases. In some embodiments, the detectably labeled multivalent molecules are bound to the complexed polymerases in the presence of a trapping reagent. In some embodiments, the trapping reagent can be formulated to promote binding of the detectably labeled multivalent molecules 82316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208to the complexed polymerases. In some embodiments, the trapping reagent can be formulated to inhibit incorporation of the nucleotide unit of the multivalent molecules. In some embodiments, the trapping reagent comprises at least one solvent, at least one pH buffering agent, at least one non-catalytic cation, at least one viscosity agent, at least one chelating agent, at least one detergent, at least one monovalent cation, at least one reducing agent, and at least one chaotropic agent. In some embodiments, the trapping reagent further comprises a plurality of multivalent molecules. In some embodiments, the trapping reagent further comprises a first plurality of sequencing polymerases. In some embodiments, the at least one non-catalytic cation inhibits polymerase-catalyzed nucleotide incorporation.

[0293] In some embodiments, the multivalent-complexed polymerases can be exposed to excitation illumination to induce fluorescent signals from the multivalent-complexed polymerases. In some embodiments, the fluorescent signals from the multivalent-complexed polymerases can be imaged in the presence of an imaging reagent. In some embodiments, the imaging reagent can be formulated to reduce photo damage of the fluorescently-labeled multivalent-complexed polymerases upon exposure to the excitation illumination. In some embodiments, the imaging reagent can be formulated to inhibit polymerase-catalyzed nucleotide incorporation. In some embodiments, the imaging reagent comprises at least one solvent, at least one pH buffering agent, at least one chelating agent, at least one non-catalytic divalent cation, at least one compound for reducing photo-damage, at least one reducing agent, at least one detergent and at least one viscosity agent. In some embodiments, prior to conducting the second sequencing stage, the detectably labeled multivalent molecules can be dissociated from the complexed polymerases and removed (e.g., washing). In some embodiments, prior to conducting the second sequencing stage, the first plurality of sequencing polymerases can be dissociated from the second sub-population of template molecules in the first plurality. In some embodiments, the second sub-population of template molecules in the first plurality can remain immobilized to the support and the second batch sequencing primers can be retained and can remain hybridized to the second sub-population of template molecules in the first plurality.

[0294] In some embodiments, the second stage of the two-stage sequencing method generally comprises contacting the second sub-population of template molecules in the first plurality and the retained second batch sequencing primers with a second plurality of sequencing polymerases and a plurality of nucleotides (e.g., non-conjugated free nucleotides). In some embodiments, the second stage comprises binding the plurality of nucleotides to the complexed polymerases to form nucleotide-complexed polymerases, and promoting83316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208nucleotide incorporation. In some embodiments, the second stage of the two-stage sequencing method comprises nucleotide incorporation and extension of the second batch sequencing primer.

[0295] In some embodiments, the plurality of nucleotides comprise fluorophore-labeled nucleotides, or the nucleotides are non-labeled. In some embodiments, when the nucleotides are fluorophore-labeled, then detecting and imaging of the incorporated nucleotides can be performed. In some embodiments, when the nucleotides are non-labeled, detecting and imaging of the incorporated nucleotides can be omitted.

[0296] In some embodiments, the nucleotides comprises chain terminating nucleotides where individual nucleotides comprise a chain terminating moiety attached to the 3’ sugar position. In some embodiments, the nucleotides are not chain terminating nucleotides. In some embodiments, when the nucleotides comprise chain terminating nucleotides, then the chain terminating moieties can be cleaved from the incorporated chain terminating nucleotides to generate an extendible 3 ’OH group.

[0297] In some embodiments, nucleotide incorporation can be conducted in the presence of a stepping reagent. In some embodiments, the stepping reagent can be formulated to promote polymerase-catalyzed nucleotide incorporation. In some embodiments, the stepping reagent comprises at least one solvent, at least one pH buffering agent, at least one monovalent cation, at least one catalytic cation, at least one viscosity agent, at least one chelating agent, at least one amino acid, at least one detergent. In some embodiments, the stepping reagent further comprises a plurality of nucleotides (e.g., non-conjugated free nucleotides). In some embodiments, the stepping reagent further comprises a second plurality of sequencing polymerases. In some embodiments, the at least one catalytic cation promotes polymerase-catalyzed nucleotide incorporation. In some embodiments, in the stepping reagent, the plurality of nucleotides comprises chain terminating nucleotides where individual nucleotides comprise a chain terminating moiety attached to the 3’ sugar position. In some embodiments, in the stepping reagent, the plurality of nucleotides are not chain terminating nucleotides.

[0298] In some embodiments, the sequencing of step (c) comprises conducting a two-stage sequencing method including repeating the first stage and second stage at least once thereby generating a plurality of second batch sequencing read products. In some embodiments, when conducting a two-stage sequencing method, one sequencing cycle comprises completion of a first and a second stage. In some embodiments, the sequencing of step (c) comprises conducting 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-7584316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.

[0299] In some embodiments, the methods for re-seeding a support further comprise reiteratively sequencing the first sub-population of the first plurality of concatemer template molecules, which comprises step (cl): conducting short read sequencing by performing up to 1000 sequencing cycles of the first sub-population of concatemer template molecules to generate a plurality of first sub-population batch sequencing read products that comprise up to 1000 bases in length. In some embodiments, step (cl) comprises conducting 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.

[0300] In some embodiments, the first sub-population batch sequencing read products comprise the first sub-population seeding batch barcode sequence.

[0301] In some embodiments, the first sub-population batch sequencing read products comprise the first sub-population seeding batch barcode sequence and the sample index sequence.

[0302] In some embodiments, the first sub-population batch sequencing read products comprise the first sub-population seeding batch barcode sequence and at least a portion of the first sequence of interest.

[0303] In some embodiments, the first sub-population batch sequencing read products comprise the first sub-population seeding batch barcode sequence, the sample index sequence, and at least a portion of the first sequence of interest.

[0304] In some embodiments, in step (cl), the short read sequencing comprises hybridizing sequencing primers to sequencing primer binding sites on the first sub-population of concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, 500 million - 1 billion of the first sub-population of concatemer template molecules can be sequenced. In some embodiments, up to 1 billion, or up to 2 billion, or up to 3 billion, or up to 4 billion, or up to 5 billion of the first sub-population of concatemer template molecules can be sequenced. In some embodiments, up to 6 billion, or up to 7 billion, or up to 8 billion, or up to 9 billion, or up to 10 billion of the first sub-population of concatemer template molecules can be sequenced. In some embodiments, between about 500 million and about 10 billion concatemer template molecules, between about 1 billion and about 9 billion concatemer 85316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208template molecules, between about 2 billion and about 8 billion concatemer template molecules, between about 3 billion and about 7 billion concatemer template molecules, between about 4 billion and about 5 billion concatemer template molecules, or any range therebetween of the first sub-population of concatemer template molecules can be sequenced.

[0305] In some embodiments, the sequencing of step (cl) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprise nucleotides, nucleotide analogs and / or multivalent molecules. In some embodiments, the reiterative sequencing of step (cl) comprises conducting a two-stage sequencing method described herein.

[0306] In some embodiments, the methods for re-seeding a support further comprise step (c2): stopping and / or blocking the short read sequencing of step (cl). In some embodiments, the stopping and / or blocking comprises incorporating a chain terminating nucleotide to the 3’ terminal end of the first sub-population batch sequencing read products to inhibit further sequencing reactions. Exemplary chain terminating nucleotides include dideoxynucleotide or a nucleotide having a 2’ or 3’ chain terminating moiety.

[0307] In some embodiments, the methods for re-seeding a support further comprise step (c3): removing the plurality of first sub-population batch sequencing read products and retaining the concatemer template molecules of the first sub -population. In some embodiments, step (c3) is optional. In some embodiments, the first sub-population batch sequencing read products can be removed from the concatemer template molecules by denaturation using heat and / or a de-hybridization reagent.

[0308] In some embodiments, the methods for re-seeding a support further comprise step (c4): reiteratively sequencing the concatemer template molecules of the first sub-population by repeating steps (cl) - (c3) at least once. In some embodiments, the reiterative sequencing can be conducted 1-10 times, or 10-25 times, or 25-50 times or more.

[0309] In some embodiments, the sequences of the first sub-population batch sequencing read products can be determined and aligned with a first reference sequence to confirm the presence of the first sequence of interest. The first reference sequence can be the first subpopulation seeding batch barcode and / or the first sequence of interest.

[0310] In some embodiments, the methods for re-seeding a support further comprise reiteratively sequencing the second sub-population of concatemer template molecules in a manner similar to steps (cl) - (c4) as described above for the first sub-population of concatemer template molecules.86316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0311] In some embodiments, hybridizing the sequencing primers to the concatemer template molecules of any of steps (cl) can be conducted with a hybridization reagent comprising an SSC buffer (e.g., 2X saline-sodium citrate) buffer with formamide (e.g., 10-20% formamide).

[0312] In some embodiments, in step (c3) the plurality of first sub-population batch sequencing read products can be removed from the template molecules and the plurality of template molecules can be retained using a de-hybridization reagent comprising an SSC buffer (e.g., saline-sodium citrate) buffer, with or without formamide, at a temperature that promotes nucleic acid denaturation such as for example 50 - 90 °C.

[0313] In some embodiments, in step (c3) the plurality of first sub-population batch sequencing read products can be removed from the template molecules and the plurality of template molecules can be retained using a de-hybridization reagent comprising at least one solvent, at least one pH buffering agent, at least one reducing agent, at least one monovalent salt and at least one crowding agent. In some embodiments, the de-hybridization reagent further comprises at least one chaotropic agent. In some embodiments, the de-hybridization reagent further comprises at least one nucleic acid compaction agent. In some embodiments, the de-hybridization of step (c3) can be conducted at a temperature that promotes nucleic acid denaturation such as for example 50 - 90 °C.

[0314] In some embodiments, the methods for re-seeding a support further comprise step (d): distributing on the support a second plurality of circularized nucleic acid library molecules under a condition suitable for hybridizing individual circularized library molecules o to individual surface capture primers and conducting a second rolling circle amplification reaction, in a template-dependent manner using individual circularized library molecules in the second plurality as templates, thereby generating a second plurality of nucleic acid concatemer template molecules immobilized to the support. In some embodiments, the support comprises up to 500 million of a second plurality of concatemer template molecules immobilized thereon, or up to 1 billion a second plurality of concatemer template molecules immobilized thereon, or up to 2 billion a second plurality of concatemer template molecules immobilized thereon, or up to 3 billion a second plurality of concatemer template molecules immobilized thereon, or up to 4 billion a second plurality of concatemer template molecules immobilized thereon, or up to 5 billion a second plurality of concatemer template molecules immobilized thereon, or up to 6 billion a second plurality of concatemer template molecules immobilized thereon. In some embodiments, the support comprises up to 7 billion concatemer template molecules immobilized thereon, or up to 8 billion concatemer template 87316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208molecules immobilized thereon, or up to 9 billion concatemer template molecules immobilized thereon, or up to 10 billion concatemer template molecules immobilized thereon, or up to 20 billion concatemer template molecules immobilized thereon. In some embodiments, the support comprises between about 500 million and about 20 billion concatemer template molecules immobilized thereon, between about 1 billion and about 10 billion concatemer template molecules immobilized thereon, between about 2 billion and about 9 billion concatemer template molecules immobilized thereon, between about 3 billion and about 8 billion concatemer template molecules immobilized thereon, between about 4 billion and about 7 billion concatemer template molecules immobilized thereon, or between about 5 billion and about 6 concatemer billion template molecules immobilized thereon, or any range therebetween. In some embodiments, individual concatemer template molecules in the second plurality comprise a plurality of tandem copies of a polynucleotide unit, where each polynucleotide unit comprises a sequence of interest and a batch seeding sequencing primer binding site sequence. In some embodiments, the first plurality of concatemer template molecules of step (c) can be completely sequenced or the sequencing can be interrupted at any time prior to distributing the second plurality of circularized nucleic acid library molecules onto the support of step (d). In some embodiments, the second plurality of circularized library molecules can be generated using padlock probes, single-stranded splint strands, or double-stranded adaptors. In some embodiments, the second plurality of circularized library molecules comprise a mixture of any combination of circularized padlock probes, linear library molecules circularized using single-stranded splint strands, and / or linear library molecules circularized using double-stranded adaptors. Methods for generating circularized library molecules are described herein.

[0315] In some embodiments, in the methods for re-seeding the support of step (d), individual circularized library molecules in the second plurality comprise a sequence of interest, a seeding batch sequencing primer binding site sequence which corresponds to the sequence of interest, and a surface capture primer binding site. In some embodiments, a predetermined second seeding batch sequencing primer binding site sequence can be linked to a given sequence of interest in the second plurality of circularized library molecules. In some embodiments, a pre-determined second seeding batch sequencing primer binding site sequence can be linked to different sequences of interest in a second plurality of circularized library molecules), thus the pre-determined second seeding batch sequencing primer binding site sequence corresponds to a given sequence of interest in the second plurality of circularized library molecules.88316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0316] In some embodiments, individual circularized library molecules in the second plurality further comprise a seeding batch barcode sequence which corresponds to the sequence of interest.

[0317] In some embodiments, a pre-determined second seeding batch barcode sequence can be linked to a given sequence of interest in the second plurality of circularized library molecules, thus the pre-determined second seeding batch barcode sequence corresponds to a given sequence of interest in the second plurality of circularized library molecules. In some embodiments, a pre-determined second seeding batch barcode sequence can be linked to different sequences of interest in a second plurality of circularized library molecules.

[0318] In some embodiments, individual circularized library molecules in the second plurality comprise a sequence of interest, the same seeding batch sequencing primer binding site sequence which corresponds to the sequence of interest, and individual circularized library molecules further comprise a surface capture primer binding site, and a second seeding batch barcode sequence which corresponds to the sequence of interest.

[0319] In some embodiments, the sequences of interest in the second plurality of circularized nucleic acid library molecules are about 50-250 bases in length, or about 250-500 bases in length, or about 500-800 bases in length, or about 800-1200 bases in length, or any range therebetween, or up to 2000 bases in length.

[0320] In some embodiments, the concentration of the second plurality of circularized nucleic acid library molecules that are distributed onto the support can be about 1-5 pM, or about 5-10 pM, or about 10-50 pM, or any range therebetween.

[0321] In some embodiments, in the methods for re-seeding a support of step (d), the second plurality of circularized nucleic acid library molecules comprise a plurality of subpopulations of circularized library molecules including at least a third and fourth subpopulation of circularized library molecules.

[0322] In some embodiments, individual circularized library molecules in the third subpopulation comprise the same third sub-population seeding batch sequencing primer binding site sequence and have the same sequence of interest. In some embodiments, individual circularized library molecules in the third sub-population comprise the same third subpopulation seeding batch sequencing primer binding site sequence and have different sequences of interest. In some embodiments, the third sub-population seeding batch sequencing primer binding site sequence corresponds to the third sequence of interest, or the third sub-population seeding batch sequencing primer binding site sequence corresponds to one of the sequences of interest in the third sub-population. In some embodiments, a pre- 89316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208determined third sub-population seeding batch sequencing primer binding site sequence can be linked to a given sequence of interest in the third sub-population of circularized library molecules, thus the pre-determined third sub-population seeding batch sequencing primer binding site sequence corresponds to a given sequence of interest in the third sub-population of circularized library molecules. In some embodiments, a pre-determined third subpopulation seeding batch sequencing primer binding site sequence can be linked to different sequences of interest in a third sub-population of circularized library molecules.

[0323] In some embodiments, individual circularized library molecules in the third subpopulation further comprise a third sub-population seeding batch barcode sequence which corresponds to the third sequence of interest, or the third sub-population seeding batch barcode sequence corresponds to one of the sequences of interest in the third sub-population. In some embodiments, a pre-determined third sub-population seeding batch barcode sequence can be linked to a given sequence of interest in the third sub-population of circularized library molecules, thus the pre-determined third sub-population seeding batch barcode sequence corresponds to a given sequence of interest in the third sub-population of circularized library molecules. In some embodiments, a pre-determined third sub-population seeding batch barcode sequence can be linked to different sequences of interest in a third sub-population of circularized library molecules.

[0324] In some embodiments, individual circularized library molecules in the third subpopulation further comprise a sample index sequence that can be used in a multiplex assay to distinguish sequences of interest obtained from different sample sources. In some embodiments, individual circularized library molecules in the third sub-population further comprise a surface capture primer binding site. In some embodiments, individual circularized library molecules in the third sub-population further comprise a surface pinning primer binding site. In some embodiments, individual circularized library molecules in the third subpopulation further comprise a compaction oligonucleotide binding site.

[0325] In some embodiments, the sequences of interest in the third sub-population of circularized nucleic acid library molecules are about 50-250 bases in length, or about 250-500 bases in length, or about 500-800 bases in length, or about 800-1200 bases in length, or any range therebetween, or up to 2000 bases in length.

[0326] In some embodiments, in the methods for re-seeding a support of step (d), the method comprises conducting a rolling circle amplification reaction, in a template-dependent manner using individual circularized library molecules in the third sub-population, thereby generating a plurality of third sub-population concatemer template molecules immobilized to 90316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208the support. In some embodiments, a subset of the surface capture primers hybridize to individual circularized library molecules to generate the plurality of third sub-population concatemer template molecules.

[0327] In some embodiments, the third sub-population concatemer template molecules can be immobilized to the support at random and non-predetermined positions, or at predetermined positions (e.g., patterned support).

[0328] In some embodiments, in the methods for re-seeding a support of step (d), individual circularized library molecules in the fourth sub-population comprise the same fourth sub-population seeding batch sequencing primer binding site sequence and have the same sequence of interest or different sequences of interest. In some embodiments, the fourth sub-population seeding batch sequencing primer binding site sequence corresponds to the fourth sequence of interest, or the fourth sub-population seeding batch sequencing primer binding site sequence corresponds to one of the sequences of interest in the fourth subpopulation. In some embodiments, a pre-determined fourth sub-population seeding batch sequencing primer binding site sequence can be linked to a given sequence of interest in the fourth sub-population of circularized library molecules, thus the pre-determined fourth subpopulation seeding batch sequencing primer binding site sequence corresponds to a given sequence of interest in the fourth sub-population of circularized library molecules. In some embodiments, a pre-determined fourth sub-population seeding batch sequencing primer binding site sequence can be linked to different sequences of interest in a fourth subpopulation of circularized library molecules.

[0329] In some embodiments, individual circularized library molecules in the fourth subpopulation further comprise a fourth sub-population seeding batch barcode sequence which corresponds to the fourth sequence of interest, or the fourth sub-population seeding batch barcode sequence corresponds to one of the sequences of interest in the fourth subpopulation. In some embodiments, a pre-determined fourth sub-population seeding batch barcode sequence can be linked to a given sequence of interest in the fourth sub-population of circularized library molecules, thus the pre-determined fourth subs-population seeding batch barcode sequence corresponds to a given sequence of interest in the fourth sub-population of circularized library molecules. In some embodiments, a pre-determined fourth sub-population seeding batch barcode sequence can be linked to different sequences of interest in a fourth sub-population of circularized library molecules

[0330] In some embodiments, individual circularized library molecules in the fourth subpopulation further comprise a sample index sequence that can be used in a multiplex assay to 91316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208distinguish sequences of interest obtained from different sample sources. In some embodiments, individual circularized library molecules in the fourth sub-population further comprise a surface capture primer binding site. In some embodiments, individual circularized library molecules in the fourth sub-population further comprise a surface pinning primer binding site. In some embodiments, individual circularized library molecules in the fourth sub-population further comprise a compaction oligonucleotide binding site.

[0331] In some embodiments, the sequences of interest in the fourth sub-population of circularized nucleic acid library molecules are about 50-250 bases in length, or about 250-500 bases in length, or about 500-800 bases in length, or about 800-1200 bases in length, or any range therebetween, or up to 2000 bases in length.

[0332] In some embodiments, the third sub-population seeding batch sequencing primer binding site sequence and fourth sub-population seeding batch sequencing primer binding site sequence have different sequences.

[0333] In some embodiments, in the methods for re-seeding a support of step (d), the method comprises conducting a rolling circle amplification reaction, in a template-dependent manner using individual circularized library molecules in the fourth sub-population, thereby generating a fourth sub-population concatemer template molecules immobilized to the support. In some embodiments, a subset of the surface capture primers hybridize to individual circularized library molecules to generate the fourth sub-population concatemer template molecules.

[0334] In some embodiments, the fourth sub-population concatemer template molecules can be immobilized to the support at random and non-predetermined positions, or at predetermined positions (e.g., patterned support).

[0335] In some embodiments, in the methods for re-seeding a support of step (d), the rolling circle amplification reaction comprises contacting the primed circularized library molecules with a plurality of a strand displacing polymerase, and a plurality of nucleotides which include dATP, dCTP, dGTP, dTTP.

[0336] In some embodiments, the plurality of nucleotide further comprises a plurality of a nucleotide having a scissile moiety (e.g., uracil).

[0337] In some embodiments, the rolling circle amplification reaction of step (d) can be conducted in the presence, or in the absence, of a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides can hybridize to two different locations on the same the template molecule to pull together distal portions of the template molecule causing compaction of the template molecule to form a DNA nanoball.92316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0338] In some embodiments, the methods for re-seeding a support further comprise step (e): sequencing at least a subset of the second plurality of immobilized concatemer template molecules thereby generating a second plurality of sequencing read products. In some embodiments, the sequencing of step (e) comprises imaging a region of the support to detect the sequencing reactions of the second plurality of template molecules. In some embodiments, the same region of the support is sequenced in steps (c) and (e). In some embodiments, different regions of the support are sequenced in steps (c) and (e).

[0339] In some embodiments, the concatemer template molecules in the second plurality are sequenced. For example, at least 30-50%, or at least 50-70%, or at least 70-90% of the concatemer template molecules in the second plurality are sequenced. In some embodiments, 500 million - 1 billion of the second plurality of concatemer template molecules can be sequenced. In some embodiments, up to 1 billion, or up to 2 billion, or up to 3 billion, or up to 4 billion, or up to 5 billion of the second plurality of concatemer template molecules can be sequenced. In some embodiments, up to 6 billion, or up to 7 billion, or up to 8 billion, or up to 9 billion, or up to 10 billion of the second plurality of concatemer template molecules can be sequenced. In some embodiments, between about 500 million and about 10 billion concatemer template molecules, between about 1 billion and about 9 billion concatemer template molecules, between about 2 billion and about 8 billion concatemer template molecules, between about 3 billion and about 7 billion concatemer template molecules, between about 4 billion and about 5 billion concatemer template molecules, or any range therebetween of concatemer template molecules of the second plurality of concatemer template molecules can be sequenced.

[0340] In some embodiments, the full length of the concatemer template molecules in the second plurality are sequenced. In some embodiments, a partial length of the concatemer template molecules in the second plurality are sequenced.

[0341] In some embodiments, the sequencing of step (e) comprises hybridizing sequencing primers to sequencing primers binding sites on the second plurality of concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, the concatemer template molecules in the second plurality can be subjected to 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.93316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0342] In some embodiments, a partial length of the concatemer template molecules in the second plurality are reiteratively sequenced.

[0343] In some embodiments, in the methods for re-seeding a support of step (e), the third sub-population of the concatemer template molecules in the second plurality are sequenced using the third batch sequencing primer binding sites in the third sub-population of concatemer template molecules.

[0344] In some embodiments, the full length of the concatemer template molecules in the third sub-population are sequenced. In some embodiments, a partial length of the concatemer template molecules in the third sub-population are sequenced.

[0345] In some embodiments, the sequencing of step (e) comprises hybridizing sequencing primers to sequencing primers binding sites on the third sub-population of the second plurality of concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, the immobilized concatemer template molecules in the third sub-population can be subjected to 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.

[0346] In some embodiments, a partial length of the concatemer template molecules in the third sub-population are reiteratively sequenced.

[0347] In some embodiments, the sequencing of step (e) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprise nucleotides, nucleotide analogs and / or multivalent molecules.

[0348] In some embodiments, the sequencing of step (e) comprises conducting a two-stage sequencing method. In some embodiments, the first stage generally comprises contacting the third sub-population of template molecules in the second plurality with a plurality of third batch sequencing primers, a first plurality of sequencing polymerase and a plurality of detectably labeled multivalent molecules. In some embodiments, the first stage comprises binding detectably labeled multivalent molecules to complexed polymerases to form multivalent-complexed polymerases, and detecting the multivalent-complexed polymerases. In some embodiments, individual multivalent molecules comprise a core attached to multiple nucleotide arms and each nucleotide arm is attached to a nucleotide (e.g., nucleotide unit) (e.g., FIGs. 1-5). In some embodiments, the multivalent molecules can be94316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208labeled with at least one detectable moiety that emits a signal. In some embodiments, the multivalent molecules can be labeled with at least one fluorophore.

[0349] In some embodiments, individual complexed polymerases comprise a first sequencing polymerase bound to a nucleic acid duplex where the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a sequencing primer. In some embodiments, the detectably labeled multivalent molecules bind to the complexed polymerases to form a plurality of multivalent-complexed polymerases. In some embodiments, the detectably labeled multivalent molecules are bound to the complexed polymerases in the presence of a trapping reagent. In some embodiments, the trapping reagent can be formulated to promote binding of the detectably labeled multivalent molecules to the complexed polymerases. In some embodiments, the trapping reagent can be formulated to inhibit incorporation of the nucleotide unit of the multivalent molecules. In some embodiments, the trapping reagent comprises at least one solvent, at least one pH buffering agent, at least one non-catalytic cation, at least one viscosity agent, at least one chelating agent, at least one detergent, at least one monovalent cation, at least one reducing agent, and at least one chaotropic agent. In some embodiments, the trapping reagent further comprises a plurality of multivalent molecules. In some embodiments, the trapping reagent further comprises a first plurality of sequencing polymerases. In some embodiments, the at least one non-catalytic cation inhibits polymerase-catalyzed nucleotide incorporation.

[0350] In some embodiments, the multivalent-complexed polymerases can be exposed to excitation illumination to induce fluorescent signals from the multivalent-complexed polymerases. In some embodiments, the fluorescent signals from the multivalent-complexed polymerases can be imaged in the presence of an imaging reagent. In some embodiments, the imaging reagent can be formulated to reduce photo damage of the fluorescently-labeled multivalent-complexed polymerases upon exposure to the excitation illumination. In some embodiments, the imaging reagent can be formulated to inhibit polymerase-catalyzed nucleotide incorporation. In some embodiments, the imaging reagent comprises at least one solvent, at least one pH buffering agent, at least one chelating agent, at least one non-catalytic divalent cation, at least one compound for reducing photo-damage, at least one reducing agent, at least one detergent and at least one viscosity agent. In some embodiments, prior to conducting the second sequencing stage, the detectably labeled multivalent molecules can be dissociated from the complexed polymerases and removed (e.g., washing). In some embodiments, prior to conducting the second sequencing stage, the first plurality of sequencing polymerases can be dissociated from the third sub-population of template95316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208molecules in the second plurality. In some embodiments, the third sub-population of template molecules in the second plurality can remain immobilized to the support and the third batch sequencing primers can be retained and can remain hybridized to the third sub-population of template molecules in the second plurality.

[0351] In some embodiments, the second stage of the two-stage sequencing method comprises contacting the third sub-population of template molecules in the second plurality and the retained third batch sequencing primers with a second plurality of sequencing polymerases and a plurality of nucleotides (e.g., non-conjugated free nucleotides). In some embodiments, the second stage comprises binding the plurality of nucleotides to the complexed polymerases to form nucleotide-complexed polymerases, and promoting nucleotide incorporation. In some embodiments, the second stage of the two-stage sequencing method comprises nucleotide incorporation and extension of the third batch sequencing primer.

[0352] In some embodiments, the plurality of nucleotides comprise fluorophore-labeled nucleotides, or the nucleotides are non-labeled. In some embodiments, when the nucleotides are fluorophore-labeled, detecting and imaging of the incorporated nucleotides can be performed. In some embodiments, when the nucleotides are non-labeled, detecting and imaging of the incorporated nucleotides can be omitted.

[0353] In some embodiments, the nucleotides comprises chain terminating nucleotides where individual nucleotides comprise a chain terminating moiety attached to the 3’ sugar position. In some embodiments, the nucleotides are not chain terminating nucleotides. In some embodiments, when the nucleotides comprise chain terminating nucleotides, the chain terminating moieties can be cleaved from the incorporated chain terminating nucleotides to generate an extendible 3 ’OH group.

[0354] In some embodiments, nucleotide incorporation can be conducted in the presence of a stepping reagent. In some embodiments, the stepping reagent can be formulated to promote polymerase-catalyzed nucleotide incorporation. In some embodiments, the stepping reagent comprises at least one solvent, at least one pH buffering agent, at least one monovalent cation, at least one catalytic cation, at least one viscosity agent, at least one chelating agent, at least one amino acid, at least one detergent. In some embodiments, the stepping reagent further comprises a plurality of nucleotides (e.g., non-conjugated free nucleotides). In some embodiments, the stepping reagent further comprises a second plurality of sequencing polymerases. In some embodiments, the at least one catalytic cation promotes polymerase-catalyzed nucleotide incorporation. In some embodiments, in the stepping 96316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208reagent, the plurality of nucleotides comprises chain terminating nucleotides. In some embodiments, individual nucleotides comprise a chain terminating moiety attached to the 3’ sugar position. In some embodiments, in the stepping reagent, the plurality of nucleotides are not chain terminating nucleotides.

[0355] In some embodiments, the sequencing of step (e) comprises conducting a two-stage sequencing method including repeating the first stage and second stage at least once thereby generating a plurality of third batch sequencing read products. In some embodiments, when conducting a two-stage sequencing method, one sequencing cycle comprises completion of a first and a second stage. In some embodiments, the sequencing of step (e) comprises conducting 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.

[0356] In some embodiments, in the methods for re-seeding a support of step (e), the fourth sub-population of the concatemer template molecules in the second plurality are sequenced using the fourth batch sequencing primer binding sites in the fourth sub-population of concatemer template molecules.

[0357] In some embodiments, the full length of the concatemer template molecules in the fourth sub-population are sequenced. In some embodiments, a partial length of the concatemer template molecules in the fourth sub-population are sequenced.

[0358] In some embodiments, the sequencing of step (e) comprises hybridizing sequencing primers to sequencing primers binding sites on the fourth sub-population of the second plurality of concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, the concatemer template molecules in the fourth sub-population can be subjected to 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.

[0359] In some embodiments, a partial length of the concatemer template molecules in the fourth sub-population are reiteratively sequenced.

[0360] In some embodiments, the sequencing of step (e) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprise nucleotides, nucleotide analogs and / or multivalent molecules.97316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0361] In some embodiments, the sequencing of step (e) comprises conducting a two-stage sequencing method. In some embodiments, the first stage comprises contacting the fourth sub-population of template molecules in the second plurality with a plurality of fourth batch sequencing primers, a first plurality of sequencing polymerase and a plurality of detectably labeled multivalent molecules. In some embodiments, the first stage comprises binding detectably labeled multivalent molecules to complexed polymerases to form multivalent-complexed polymerases, and detecting the multivalent-complexed polymerases. In some embodiments, individual multivalent molecules comprise a core attached to multiple nucleotide arms and each nucleotide arm is attached to a nucleotide (e.g., nucleotide unit) (e.g., FIGs. 1-5). In some embodiments, the multivalent molecules can be labeled with at least one detectable moiety that emits a signal. In some embodiments, the multivalent molecules can be labeled with at least one fluorophore.

[0362] In some embodiments, individual complexed polymerases comprise a first sequencing polymerase bound to a nucleic acid duplex where the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a sequencing primer. In some embodiments, the detectably labeled multivalent molecules bind to the complexed polymerases to form a plurality of multivalent-complexed polymerases. In some embodiments, the detectably labeled multivalent molecules are bound to the complexed polymerases in the presence of a trapping reagent. In some embodiments, the trapping reagent can be formulated to promote binding of the detectably labeled multivalent molecules to the complexed polymerases. In some embodiments, the trapping reagent can be formulated to inhibit incorporation of the nucleotide unit of the multivalent molecules. In some embodiments, the trapping reagent comprises at least one solvent, at least one pH buffering agent, at least one non-catalytic cation, at least one viscosity agent, at least one chelating agent, at least one detergent, at least one monovalent cation, at least one reducing agent, and at least one chaotropic agent. In some embodiments, the trapping reagent further comprises a plurality of multivalent molecules. In some embodiments, the trapping reagent further comprises a first plurality of sequencing polymerases. In some embodiments, the at least one non-catalytic cation inhibits polymerase-catalyzed nucleotide incorporation.

[0363] In some embodiments, the multivalent-complexed polymerases can be exposed to excitation illumination to induce fluorescent signals from the multivalent-complexed polymerases. In some embodiments, the fluorescent signals from the multivalent-complexed polymerases can be imaged in the presence of an imaging reagent. In some embodiments, the imaging reagent can be formulated to reduce photo damage of the fluorescently-labeled 98316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208multivalent-complexed polymerases upon exposure to the excitation illumination. In some embodiments, the imaging reagent can be formulated to inhibit polymerase-catalyzed nucleotide incorporation. In some embodiments, the imaging reagent comprises at least one solvent, at least one pH buffering agent, at least one chelating agent, at least one non-catalytic divalent cation, at least one compound for reducing photo-damage, at least one reducing agent, at least one detergent and at least one viscosity agent. In some embodiments, prior to conducting the second sequencing stage, the detectably labeled multivalent molecules can be dissociated from the complexed polymerases and removed (e.g., washing). In some embodiments, prior to conducting the second sequencing stage, the first plurality of sequencing polymerases can be dissociated from the fourth sub-population of template molecules in the second plurality. In some embodiments, the fourth sub-population of template molecules in the second plurality can remain immobilized to the support and the fourth batch sequencing primers can be retained and can remain hybridized to the fourth subpopulation of template molecules in the second plurality.

[0364] In some embodiments, the second stage of the two-stage sequencing method comprises contacting the fourth sub-population of template molecules in the second plurality and the retained fourth batch sequencing primers with a second plurality of sequencing polymerases and a plurality of nucleotides (e.g., non-conjugated free nucleotides). In some embodiments, the second stage comprises binding the plurality of nucleotides to the complexed polymerases to form nucleotide-complexed polymerases, and promoting nucleotide incorporation. In some embodiments, the second stage of the two-stage sequencing method comprises nucleotide incorporation and extension of the fourth batch sequencing primer.

[0365] In some embodiments, the plurality of nucleotides comprise fluorophore-labeled nucleotides, or the nucleotides are non-labeled. In some embodiments, when the nucleotides are fluorophore-labeled, then detecting and imaging of the incorporated nucleotides can be performed. In some embodiments, when the nucleotides are non-labeled, detecting and imaging of the incorporated nucleotides can be omitted.

[0366] In some embodiments, the nucleotides comprises chain terminating nucleotides where individual nucleotides comprise a chain terminating moiety attached to the 3’ sugar position. In some embodiments, the nucleotides are not chain terminating nucleotides. In some embodiments, when the nucleotides comprise chain terminating nucleotides, then the chain terminating moieties can be cleaved from the incorporated chain terminating nucleotides to generate an extendible 3 ’OH group.99316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0367] In some embodiments, nucleotide incorporation can be conducted in the presence of a stepping reagent. In some embodiments, the stepping reagent can be formulated to promote polymerase-catalyzed nucleotide incorporation. In some embodiments, the stepping reagent comprises at least one solvent, at least one pH buffering agent, at least one monovalent cation, at least one catalytic cation, at least one viscosity agent, at least one chelating agent, at least one amino acid, at least one detergent. In some embodiments, the stepping reagent further comprises a plurality of nucleotides (e.g., non-conjugated free nucleotides). In some embodiments, the stepping reagent further comprises a second plurality of sequencing polymerases. In some embodiments, the at least one catalytic cation promotes polymerase-catalyzed nucleotide incorporation. In some embodiments, in the stepping reagent, the plurality of nucleotides comprises chain terminating nucleotides where individual nucleotides comprise a chain terminating moiety attached to the 3’ sugar position. In some embodiments, in the stepping reagent, the plurality of nucleotides are not chain terminating nucleotides.

[0368] In some embodiments, the sequencing of step (e) comprises conducting a two-stage sequencing method including repeating the first stage and second stage at least once thereby generating a plurality of fourth batch sequencing read products. In some embodiments, when conducting a two-stage sequencing method, one sequencing cycle comprises completion of a first and a second stage. In some embodiments, the sequencing of step (e) comprises conducting 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.

[0369] In some embodiments, the methods for re-seeding a support further comprise reiteratively sequencing the third sub-population of concatemer template molecules, which comprises step (el): conducting short read sequencing by performing up to 1000 sequencing cycles of the third sub-population of the second plurality of concatemer template molecules to generate a plurality of second sub-population batch sequencing read products that comprise up to 1000 bases in length. In some embodiments, step (el) comprises conducting 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.

[0370] In some embodiments, the third sub-population batch sequencing read products comprise the third sub-population seeding batch barcode sequence.100316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0371] In some embodiments, the third sub-population batch sequencing read products comprise the third sub-population seeding batch barcode sequence and the sample index sequence.

[0372] In some embodiments, the third sub-population batch sequencing read products comprise the third sub-population seeding batch barcode sequence and at least a portion of the second sequence of interest.

[0373] In some embodiments, the third sub-population batch sequencing read products comprise the third sub-population seeding batch barcode sequence, the sample index sequence, and at least a portion of the second sequence of interest.

[0374] In some embodiments, in step (el), the short read sequencing comprises hybridizing sequencing primers to sequencing primer binding sites on the third subpopulation of concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, 500 million - 1 billion of the third sub-population of concatemer template molecules can be sequenced. In some embodiments, up to 1 billion, or up to 2 billion, or up to 3 billion, or up to 4 billion, or up to 5 billion of the third sub-population of concatemer template molecules can be sequenced. In some embodiments, up to 6 billion, or up to 7 billion, or up to 8 billion, or up to 9 billion, or up to 10 billion of the third sub-population of concatemer template molecules can be sequenced. In some embodiments, between about 500 million and about 10 billion concatemer template molecules, between about 1 billion and about 9 billion concatemer template molecules, between about 2 billion and about 8 billion concatemer template molecules, between about 3 billion and about 7 billion concatemer template molecules, between about 4 billion and about 5 billion concatemer template molecules, or any range therebetween of the third sub-population of concatemer template molecules can be sequenced.

[0375] In some embodiments, the sequencing of step (el) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprise nucleotides, nucleotide analogs and / or multivalent molecules. In some embodiments, the reiterative sequencing of step (el) comprises conducting a two-stage sequencing method described herein.

[0376] In some embodiments, the methods for re-seeding a support further comprise step (e2): stopping and / or blocking the short read sequencing of step (el). In some embodiments, the stopping / blocking comprises incorporating a chain terminating nucleotide to the 3’101316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208terminal end of the second sub-population batch sequencing read products to inhibit further sequencing reactions. Exemplary chain terminating nucleotides include dideoxynucleotide or a nucleotide having a 2’ or 3’ chain terminating moiety.

[0377] In some embodiments, the methods for re-seeding a support further comprise step (e3): removing the plurality of second sub-population batch sequencing read products and retaining the concatemer template molecules of the second sub-population. In some embodiments, step (e3) is optional. In some embodiments, the third sub-population batch sequencing read products can be removed from the concatemer template molecules by denaturation using heat and / or a de-hybridization reagent.

[0378] In some embodiments, the methods for re-seeding a support further comprise step (e4): reiteratively sequencing the concatemer template molecules of the third sub-population by repeating steps (el) - (e3) at least once. In some embodiments, the reiterative sequencing can be conducted 1-10 times, or 10-25 times, or 25-50 times, or any range therebetween or more than 50 times.

[0379] In some embodiments, the sequences of the third sub-population batch sequencing read products can be determined and aligned with a second reference sequence to confirm the presence of the second sequence of interest. The second reference sequence can be the third sub-population seeding batch barcode and / or the second sequence of interest.

[0380] In some embodiments, the methods for re-seeding a support further comprise reiteratively sequencing the fourth sub-population of concatemer template molecules in a manner similar to steps (el) - (e4) as described above for the third sub-population of concatemer template molecules.

[0381] In some embodiments, hybridizing the sequencing primers to the concatemer template molecules of any of steps (el) can be conducted with a hybridization reagent comprising an SSC buffer (e.g., 2X saline-sodium citrate) buffer with formamide (e.g., 10-20% formamide).

[0382] In some embodiments, in step (e3) the plurality of third sub-population batch sequencing read products can be removed from the template molecules and the plurality of template molecules can be retained using a de-hybridization reagent comprising an SSC buffer (e.g., saline-sodium citrate) buffer, with or without formamide, at a temperature that promotes nucleic acid denaturation such as for example 50 - 90 °C.

[0383] In some embodiments, in step (e3) the plurality of third sub-population batch sequencing read products can be removed from the template molecules and the plurality of template molecules can be retained using a de-hybridization reagent comprising at least one 102316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208solvent, at least one pH buffering agent, at least one reducing agent, at least one monovalent salt and at least one crowding agent. In some embodiments, the de-hybridization reagent further comprises at least one chaotropic agent. In some embodiments, the de-hybridization reagent further comprises at least one nucleic acid compaction agent. In some embodiments, the de-hybridization of step (e3) can be conducted at a temperature that promotes nucleic acid denaturation such as for example 50 - 90 °C.Methods for Determining Nucleic Acid Template Density on a Support

[0384] Conventional methods for achieving a desired density of immobilized nucleic acid template molecules for massively parallel sequencing include determining the concentration of library molecules in-solution prior to immobilizing the library molecules on the support. The conventional methods typically employ qPCR and / or a fluorometer with a fluorescentbased assay (e.g., Qubit). Even when the desired in-solution library concentration is achieved, these convention methods can yield immobilized template densities that are too high or too low.

[0385] The present disclosure provides methods for determining the density of nucleic acid template molecules that are already immobilized to a support, thus providing more accurate density information of the template molecules that are immobilized to the support. In some embodiments, the density determining methods can be used to determine the density of a mixture of template molecules including at least a first and second sub-population of template molecules immobilized to a support. In some embodiments, when the density of any given sub-population of immobilized template molecules is determined to be too low, then the support can be re-seeded with that particular sub-population of library molecule, which can then be amplified to increase the density.

[0386] The present disclosure provides methods for determining nucleic acid template density comprising step (a): providing a support comprising a plurality of nucleic acid template molecules immobilized to the support. In some embodiments, the plurality of template molecules comprises a plurality of sub-populations of template molecules, including at least a first and a second sub-population of template molecules. In some embodiments, the plurality of template molecules comprises 1-50 sub-populations, or 50-100 sub-populations, or 100-150 sub-populations, or 150-200 sub-populations, or any range therebetween, or more than 200 sub-populations of template molecules.103316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208

[0387] In some embodiments, individual template molecules of the first sub-population comprise (i) a first batch sequencing primer binding site, (ii) a first sequence of interest, and (iii) optionally a first batch barcode sequence and / or a first batch sample index sequence.

[0388] In some embodiments, individual template molecules within the first subpopulation comprise the same first batch sequencing primer binding site. In some embodiments, individual template molecules within the first sub-population comprise the same sequence of interest, or comprise different sequences of interest. In some embodiments, the sequence of the first batch sequencing primer binding site sequence corresponds to the first sequence of interest, or the first batch sequencing primer binding site sequence corresponds to one of the first sequences of interest in the first sub-population. In some embodiments, a pre-determined first batch sequencing primer binding site sequence can be linked to, i.e. can be used to selectively sequence in a batch sequencing workflow, a given sequence of interest in the first sub-population. In some embodiments, a pre-determined first batch sequencing primer binding site sequence can be linked to different sequences of interest in the first sub-population. Thus, the pre-determined first batch sequencing primer binding site sequence corresponds to a given sequence of interest or sequences of interest in the first sub-population.

[0389] In some embodiments, the sequences of interest in the first sub-population are about 50-250 bases in length, or about 250-500 bases in length, or about 500-800 bases in length, or about 800-1200 bases in length, or any range therebetween, or up to 2000 bases in length.[039...

Claims

Attorney Docket No. ELEM-025 / 001WO 340101-2208What is claimed:

1. A method for nucleic acid sequencing comprising:a) providing a support comprising a plurality of nucleic acid template molecules immobilized to the support, wherein the plurality of nucleic acid template molecules comprises at least a first and a second sub-population of template molecules,• wherein individual template molecules in the first sub-population of template molecules comprises a first batch sequencing primer binding site, a first batch barcode sequence and at least one first sequence-of- interest,• wherein the individual template molecules in the second sub-population of template molecules comprises a second batch sequencing primer binding site, a second batch barcode sequence and at least one second sequence-of-interest,b) sequencing the first sub-population of template molecules using a plurality of first batch sequencing primers, thereby generating a plurality of first batch sequencing read products and imaging a region of the support to detect the first batch sequencing read products; andc) sequencing the second sub-population of template molecules using a plurality of second batch sequencing primers, thereby generating a plurality of second batch sequencing read products and imaging the same region of the support to detect the second batch sequencing read products.

2. The method of claim 1, wherein the first batch sequencing primer binding site and the second batch sequencing primer binding site have different sequences.

3. The method of claim 1 or 2, wherein the first batch barcode sequence and the second batch barcode sequence are different.

4. The method of any one of claims 1-3, wherein sequencing the first sub-population of template molecules of step (b) comprises:• Step (bl): conducting short read sequencing by performing up to 1000 sequencing cycles of the first sub-population of template molecules to generate a 255316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208plurality of first batch sequencing read products that comprise up to 1000 bases in length;• Step (b2): stopping and / or blocking the short read sequencing of step (bl); • Step (b3): removing the plurality of first batch sequencing read products and retaining the first sub-population of template molecules; and optionally• Step (b4): repeating steps (bl) - (b3) at least once.

5. The method of any one of claims 1-4, wherein sequencing the second sub-population of template molecules of step (c) comprises:• Step (cl): conducting short read sequencing by performing up to 1000 sequencing cycles of the second sub-population of template molecules to generate a plurality of second batch sequencing read products that comprise up to 1000 bases in length;• Step (c2): stopping and / or blocking the short read sequencing of step (cl); • Step (c3): removing the plurality of second batch sequencing read products and retaining the second sub-population of template molecules; and optionally • Step (c4): repeating steps (cl) - (c3) at least once.

6. The method of any one of claims 1-5, wherein the first sub-population of template molecules have the same first batch sequencing primer binding site, and have the same sequence of interest or different sequences of interest.

7. The method of any one of claims 1-6, wherein the individual template molecules of the second sub-population of template molecules have the same second batch sequencing primer binding site, and have the same sequence of interest or different sequences of interest.

8. The method of any one of claims 1-7, wherein the plurality of nucleic acid template molecules immobilized to the support are at a density of about 102- 1015template molecules 2per mm .

9. The method of any one of claims 1-7, wherein the plurality of nucleic acid template molecules are immobilized to the support at a high density.256316574825Attorney Docket No. ELEM-025 / 001WO 340101-220810. The method of any one of claims 1-9, wherein at least some individual template molecules of the first and second sub-populations of template molecules comprise nearest neighbor template molecules that touch each other and / or overlap each other when viewed from any angle of the support including above, below or side views of the support.

11. The method of any one of claims 1-10, wherein the support lacks partitions and / or barriers that separate regions of the support.

12. The method of any one of claims 1-11, wherein the plurality of template molecules are immobilized to the support at random and non-determined positions on the support.

13. The method of any one of claims 1-11, wherein the plurality of template molecules are immobilized to the support at pre-determined positions on the support (e.g., a patterned support).

14. The method of any one of claims 1-13, wherein the plurality of nucleic acid template molecules comprises concatemer template molecules comprising at least a first and second sub-population of concatemer template molecules.

15. The method of claim 14, wherein individual concatemer template molecules in the first sub-population of concatemer template molecules comprise a plurality of tandem polynucleotide units comprising a first sequence of interest, a first batch sequencing primer binding site sequence which corresponds to the first sequence of interest, and a first batch barcode sequence which corresponds to the first sequence of interest.

16. The method of claim 14 or 15, wherein individual concatemer template molecules in the second sub-population of concatemer template molecules comprise a plurality of tandem polynucleotide units comprising a second sequence of interest, a second batch sequencing primer binding site sequence which corresponds to the second sequence of interest, and a second batch barcode sequence which corresponds to the second sequence of interest.

17. The method of any one of claims 1-16, wherein the first batch sequencing read products comprise:257316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208• the first batch barcode sequence; or• the first batch barcode sequence and the first sequence of interest.

18. The method of any one of claims 1-17, wherein the second batch sequencing read products comprise:• the second batch barcode sequence; or• the second batch barcode sequence and the second sequence of interest.

19. A method for re-seeding a support, comprising:a) providing a support comprising a plurality of surface capture primers immobilized to the support;b) distributing on the support a first plurality of circularized library molecules under a condition suitable for hybridizing individual circularized library molecules to individual surface capture primers, and conducting a first rolling circle amplification reaction thereby generating a first plurality of concatemer template molecules immobilized to the support;c) sequencing at least a subset of the first plurality of concatemer template molecules, thereby generating a first plurality of sequencing read products; d) distributing on the support a second plurality of circularized library molecules under a condition suitable for hybridizing individual circularized library molecules of the second plurality to individual surface capture primers, and conducting a second rolling circle amplification reaction thereby generating a second plurality of concatemer template molecules immobilized to the support; ande) sequencing at least a subset of the second plurality of concatemer template molecules, thereby generating a second plurality of sequencing read products.

20. The method of claim 19, wherein the first plurality of circularized library molecules comprises:• circularized padlock probes;• linear library molecules circularized using single-stranded splint strands;• linear library molecules circularized using double-stranded adaptors; or258316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208• a mixture of any combination of circularized padlock probes, linear library molecules circularized using single-stranded splint strands and / or linear library molecules circularized using double-stranded adaptors.

21. The method of claim 19 or 20, wherein the plurality of surface capture primers are immobilized to the support at random and non-pre-determined positions.

22. The method of claim 19 or 20, wherein the plurality of surface capture primers are immobilized to the support at pre-determined positions.

23. The method of any one of claims 19-22, wherein individual circularized library molecules in the first plurality of circularized library molecules comprise a first seeding batch sequencing primer binding site, a first seeding batch barcode sequence, and a first sequence of interest.

24. The method of any one of claims 19-23, wherein the first plurality of sequencing read products of step (c) comprises:• a first seeding batch barcode sequence; or• a first seeding batch barcode sequence and a first sequence of interest.

25. The method of any one of claims 19-24, wherein second individual circularized library molecules in the second plurality of circularized library molecules comprise a second seeding batch sequencing primer binding site, a second seeding batch barcode sequence, and a second sequence of interest.

26. The method of any one of claims 19-25, wherein the second plurality of sequencing read products of step (e) comprises:• a second seeding batch barcode sequence; or• a second seeding batch barcode sequence and a second sequence of interest.

27. The method of any one of claims 19-26, wherein sequencing at least the subset of the first plurality of concatemer template molecules of step (c) comprises:259316574825Attorney Docket No. ELEM-025 / 001WO 340101-2208• Step (cl): conducting short read sequencing by performing up to 1000 sequencing cycles of the first plurality of concatemer template molecules to generate a first plurality of sequencing read products that comprise up to 1000 bases in length;• Step (c2): stopping and / or blocking the short read sequencing of step (cl);• Step (c3): removing the first plurality of sequencing read products and retaining the first plurality of immobilized concatemer template molecules; and optionally • Step (c4): repeating steps (cl) - (c3) at least once.

28. The method of any one of claims 19-27, wherein the sequencing at least the subset of the second plurality of concatemer template molecules of step (e) comprises:• Step (el): conducting short read sequencing by performing up to 1000 sequencing cycles of the second plurality of concatemer template molecules to generate a second plurality of sequencing read products that comprise up to 1000 bases in length;• Step (e2): stopping and / or blocking the short read sequencing of step (el);• Step (e3): removing the second plurality of sequencing read products and retaining the second plurality of immobilized concatemer template molecules; and optionally• Step (e4): repeating steps (el) - (e3) at least once.

29. The method of any one of claims 19-28, wherein the plurality of surface capture primers immobilized to the support are at a density of about 102- 1015capture primers per 2mm .

30. The method of any one of claims 19-28, wherein at least some of the surface capture primers comprise nearest neighbor surface capture primers that touch each other and / or overlap each other when viewed from any angle of the support including above, below or side views of the support.

31. The method of any one of claims 19-30, wherein the support lacks partitions and / or barriers that separate regions of the support.260316574825

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