Nucleic acid indexing techniques

Abstract

To provide methods for permitting high-throughput sequencing in a multi-sample preparation with reduced attribution errors.SOLUTION: Presented herein are techniques for indexing of nucleic acid for use in conjunction with sequencing. The techniques include generating indexed nucleic acid fragments from an individual sample, so that the index sequence incorporated into each index site of the nucleic acid fragments is selected from a plurality of distinguishable index sequences and so that a population of generated nucleic acid fragments presents each index sequence from the plurality of index sequences. In this manner, the generated indexed nucleic acid fragments from a single sample are indexed with a diverse mix of index sequences that reduce misassignment due to index read errors associated with low sequence diversity.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 582,175, entitled "NUCLEIC ACID INDEXING TECHNIQUES," filed November 6, 2017, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] The present disclosure relates generally to the field of data relating to biological samples, such as sequence data. More specifically, the present disclosure relates to techniques for indexing nucleic acids and resolving indexed sequences in acquired sequence data. [Background technology]

[0003] Gene sequencing is becoming an increasingly important area of ​​genetic research, and is expected to have future uses in diagnostics and other applications. In general, gene sequencing involves determining the order of nucleotides for nucleic acids, such as fragments of RNA or DNA. Next-generation sequencing technology facilitates higher throughput sequencing by using pooled sample preparations, such as multiple sample preparations. Within a pooled sample preparation, each individual sample can be tagged or otherwise marked so that each sequencing read from the pooled sample can be associated or attributed to an individual sample in the pool. However, the sequence data obtained may have errors and noise introduced from various sources, such as sample defects, sample preparation errors, and sequencing bias, which may reduce the accuracy of the sequence data associated with each individual sample. Therefore, it is desirable to develop a method that allows high-throughput sequencing in multiple sample preparations with reduced attribution errors. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Patent Application Publication No. 2007 / 0166705 [Patent Document 2] US Patent Application Publication No. 2006 / 0188901 [Patent Document 3] US Patent Application Publication No. 2006 / 0240439 [Patent Document 4] US Patent Application Publication No. 2006 / 0281109 [Patent Document 5] US Patent Application Publication No. 2005 / 0100900 [Patent Document 6] U.S. Patent No. 7,057,026 [Patent Document 7] WO 05 / 065814 [Patent Document 8] WO 06 / 064199; [Patent Document 9] WO 07 / 010,251 [Patent Document 10] U.S. Pat. No. 6,969,488 [Patent Document 11] U.S. Patent No. 6,172,218 [Patent Document 12] U.S. Patent No. 6,306,597 [Patent Document 13] U.S. Patent No. 7,001,792 [Patent Document 14] US 2009 / 0026082 A1 [Patent Document 15] US 2009 / 0127589 A1 [Patent Document 16] US 2010 / 0137143 A1 [Patent Document 17] US 2010 / 0282617 A1 [Patent Document 18] U.S. Patent No. 7,329,860 [Non-patent literature]

[0005] [Non-Patent Document 1] Soni & Meller, Clin. Chem. 53, 1996-2001 (2007) [Non-Patent Document 2] Healy, Nanomed. 2, 459-481 (2007) [Non-Patent Document 3] Cockroft et al. J. Am. Chem. Soc. 130, 818-820 (2008) [Non-Patent Document 4] Levene et al. Science 299, 682-686 (2003) [Non-Patent Document 5] Lundquist et al. Opt. Lett. 33, 1026-1028 (2008) [Non-Patent Document 6] Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008) Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides a novel approach for sample indexing of biological samples. As provided herein, sample indexing is a process by which acquired sequencing reads are associated with a particular sample. In particular, the disclosed indexing technique does not introduce cross-sample contamination, works robustly with an arbitrarily low number of samples (even as low as one for positive sample tracking), and even supports representation across multiplexed samples. The problems of index cross-contamination, low-plex sample indexing performance, and variable index performance are solved by using an indexing strategy that labels each nucleic acid fragment prepared from a sample with a potential pool of multiple indexes for the first and second indexes using dual indexing per fragment. The disclosed technique assigns multiple unique indexes to each sample in both the first and second indexes, in contrast to traditional combinatorial approaches. In one embodiment, the index sequence is an exogenous nucleic acid sequence that can be incorporated into nucleic acids from a sample for sample identification and / or sample association. For example, the index sequences provided herein can be incorporated into nucleic acid fragments derived from a sample of interest during sample preparation and / or during sequencing. Each index sequence has a sequence that is distinguishable from other index sequences used in the sequencing reaction. In certain embodiments, the index sequences are distinguishable from endogenous nucleic acids of one or more samples. The index sequences may be single-stranded or double-stranded and may be at least 5 bases, at least 6 bases, at least 7 bases, at least 8 bases, or more. In certain embodiments, the index sequences may be 5-8 bases, 5-10 bases, 5-15 bases, 5-25 bases, 8-10 bases, 8-12 bases, 8-15 bases, or 8-25 bases, etc. in length.

[0007] The disclosed technology provides indexed sequencing, where nucleic acid fragments from a particular sample are indexed using a predefined pool of index sequences at each index site. Although certain embodiments are disclosed in the context of two separate index sites per nucleic acid fragment (i.e., dual indexing), it should be understood that the disclosed technology can be used with nucleic acid fragments that have at least one index site, at least two index sites, and may incorporate three, four, or more index sites for each nucleic acid fragment. The indexed samples provided herein can be pooled with other indexed samples for sequencing, so long as the other indexed samples are indexed with different pools of index sequences at each index site. Thus, the technology of the present invention allows for pooled or multi-sample sequencing reactions, whereby each individual sample in a sequencing reaction is indexed with an assigned unique set of index sequences, and the sequencing reads obtained are associated with the appropriate sample of origin via the assigned unique index.

[0008] The techniques of the present invention improve sequencing accuracy compared to techniques that incorporate a single index sequence per sample fragment and / or at each index site. For example, sequencing analysis can be performed using rule-based assignment that discards any sequencing reads that do not contain a proper assigned index, e.g., at both the first and second index sites for dual-indexed fragments or at only one index site for single-indexed fragments. The assigned indexes selected for each sample are designed to be diverse and distinguishable using various sequencing techniques. For example, each index sequence has internal diversity, diversity within its assigned set, and diversity compared to other index sequences assigned to other samples in the pool. In this way, sequencing device errors in single base reads in the index are less likely to occur, because index diversity reduces misreads. In one example, if a sequencing run has low plex or a small number of samples, the index sequence data may still reflect a higher diversity than would be expected if the number of different index sequences at each index site had a 1:1 correspondence with the number of samples. That is, instead of using only a single index sequence per sample index site, whereby each sample is associated with only one index at each index site, resulting in such a 1:1 correspondence, each sample is associated with multiple possible index sequences at each index site. Thus, different or distinguishable index sequences are present for each index site in a ratio of at least 2:1, at least 3:1, or at least 4:1 for several samples. In one example, for sequencing techniques incorporating dedicated index reads (i.e., using index primers that target only upstream of the index sequence to sequence a limited number of bases corresponding to the length of the index sequence), the technique of the present invention and improved index diversity can result in more accurate base calling.

[0009] By increasing the accuracy of the index sequence data, fewer sequencing reads are discarded following rule-based assignment, which also improves sequencing accuracy even for low concentration samples and low sample numbers. Furthermore, improved sample association through improved accuracy of index sequencing reduces the effects of index hopping (i.e., misassignment of sequencing reads to samples through index misidentification).

[0010] In one embodiment, a sample-indexed nucleic acid library preparation is provided, comprising: a first nucleic acid library prepared from a first sample, the first nucleic acid library comprising a first plurality of nucleic acid fragments, each of the first plurality of nucleic acid fragments comprising at least two different index sequences selected from a first set of index sequences; and a second nucleic acid library prepared from a second sample, the second nucleic acid library comprising a second plurality of nucleic acid fragments, each of the second plurality of nucleic acid fragments comprising at least two different index sequences selected from a second set of index sequences distinct from the first set of index sequences, wherein the index sequences are arranged in each nucleic acid fragment of the first plurality of nucleic acid fragments and the second plurality of nucleic acid fragments such that a first index sequence of the index sequences is located 5' of the target sequence and a second index sequence of the index sequences is located 3' of the target sequence.

[0011] In another embodiment, a method of sequencing a nucleic acid molecule is provided comprising: providing a plurality of dual-indexed nucleic acid fragments generated from a sample, wherein each individual nucleic acid fragment of the nucleic acid fragments comprises a 5' adaptor sequence, a 5' index sequence, a 3' adaptor sequence, and a 3' index sequence, wherein a plurality of different 5' index sequences selected from a first set of 5' index sequences associated with the sample and a plurality of different 3' index sequences selected from a second set of 3' index sequences associated with the sample are represented in the dual-indexed nucleic acid fragments, wherein the plurality of different 5' index sequences and the plurality of different 3' index sequences are distinguishable from one another; generating sequencing data representative of sequences of the dual-indexed nucleic acid fragments; and associating each individual one of the sequences with the sample only if the each individual sequence comprises both a 5' index sequence selected from the first set and a 3' index sequence selected from the second set.

[0012] In another embodiment, a multi-sample library preparation kit is provided that comprises a plurality of nucleic acid fragments, each fragment comprising an index sequence and an adaptor sequence. The plurality of nucleic acid fragments include a nucleic acid fragment set associated with a first sample, the nucleic acid fragment set having an index sequence selected from a first index set, the adapter sequence being the first adapter sequence, and the nucleic acid fragment set having an index sequence selected from a second index set, the adapter sequence being the second adapter sequence, each index sequence of the first index set and the second index set being represented in the nucleic acid fragment set associated with the first sample, and a nucleic acid fragment set associated with a second sample, the nucleic acid fragment set having an index sequence selected from a third index set, the adapter sequence being the first adapter sequence, and the nucleic acid fragment set having an index sequence selected from a fourth index set, the adapter sequence being the second adapter sequence, each index sequence of the third index set and the fourth index set being represented in the nucleic acid fragment set associated with the second sample, each of the first index set, the second index set, the third index set, and the fourth index set comprising a plurality of index sequences that are distinguishable from one another.

[0013] In another embodiment, a sequencing substrate is provided that includes a plurality of nucleic acid capture sequences immobilized on the substrate, each individual nucleic acid capture sequence including a first capture sequence that is complementary to a first adaptor sequence or a second capture sequence that is complementary to a second adaptor sequence. The sequencing substrate also includes a plurality of nucleic acid fragments linked to each nucleic acid capture sequence of the plurality of nucleic acid capture sequences, each individual fragment of the plurality of nucleic acid fragments including a first adaptor sequence and a second adaptor sequence, each individual fragment of the plurality of nucleic acid fragments including a first sequence that is complementary to one index sequence of the first set of unique index sequences and a second sequence that is complementary to one index sequence of the second set of unique index sequences, the first set of unique index sequences and the second set of unique index sequences being associated with only one sample from which the plurality of nucleic acid fragments originate, and each unique index sequence of the first set and the second set being present in at least one nucleic acid fragment of the plurality of nucleic acid fragments.

[0014] In another embodiment, a method of sequencing a nucleic acid molecule is provided comprising the steps of: providing a plurality of dual-indexed nucleic acid fragments generated from a sample, wherein each individual nucleic acid fragment of the dual-indexed nucleic acid fragments comprises a sequence of interest from the sample, a 5' adaptor sequence, a 5' index sequence, a 3' adaptor sequence, and a 3' index sequence, generating dual-indexed nucleic acid fragments, wherein a plurality of different 5' index sequences selected from a first set of 5' index sequences associated with the sample and a plurality of different 3' index sequences selected from a second set of 3' index sequences associated with the sample are represented in the dual-indexed nucleic acid fragments, wherein the plurality of different 5' index sequences and the plurality of different 3' index sequences are distinguishable from one another; generating sequencing data representative of the sequences of interest; generating sequencing data representative of the 5' index sequences and the 3' index sequences; and assigning each individual sequence of interest to a sample only if the each individual sequence of interest is associated with both a 5' index sequence selected from the first set and a 3' index sequence selected from the second set. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram of indexed nucleic acid fragments according to the present technology. [Diagram 2] FIG. 2 is a schematic diagram of an indexed nucleic acid fragment library according to the present technology. [Diagram 3] FIG. 3 is a flow diagram of a method for generating an indexed nucleic acid library of a sample according to the present technology. [Figure 4] FIG. 4 is a flow diagram of a method for sequencing a pooled, indexed nucleic acid library according to the technology of the present invention. [Diagram 5] FIG. 5 is a schematic diagram of a pooled, indexed nucleic acid fragment library according to the present technology. [Figure 6]FIG. 6 is a schematic diagram of sequencing of adaptors containing index sequences according to the technology of the present invention. [Figure 7] FIG. 7 is a flow diagram of a method for sequencing a nucleic acid library according to the present technology. [Figure 8] FIG. 8 is an example of an index set according to the technique of the present invention. [Figure 9] FIG. 9 is an example of an index set according to the technique of the present invention. [Figure 10] FIG. 10 is a nucleic acid sequencing kit according to the technology of the present invention. [Figure 11] FIG. 11 is a block diagram of a sequencing device configured to acquire sequencing data in accordance with the techniques of the present invention. [Figure 12] FIG. 12 is an example of a graphical user interface in accordance with the techniques of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The technology of the present invention is directed to indexed nucleic acids and sequencing and analysis using same. Sequencing of nucleic acids generates a large amount of raw data that is then analyzed and compiled to provide meaningful information about the sequenced samples. Sequencing technologies have evolved to process multiple samples simultaneously, which provides time and cost savings. However, such technologies present data processing challenges. Sequencing data acquired in a multi-sample sequencing run must be associated with its original sample before sequence assembly and analysis can be performed for each individual sample. However, in many cases, such sample association cannot be accurately completed using only endogenous nucleic acid sequences. Therefore, certain sequencing technologies incorporate unique exogenous barcode or index sequences into the nucleic acid prior to sequencing, and each sample is associated with a unique barcode or index. After the sequencing data is acquired, sequence reads with unique barcodes or indexes are assigned to the appropriate original sample.

[0017] Although such techniques facilitate the assignment of multi-sample sequencing data, errors in the sample assignment of sequencing reads still occur. Regardless of the source of sample assignment error introduced, sequence assignment errors can result in inaccuracies in subsequent genome assembly and / or data analysis performed on the misassigned data. If index sequence data is not accurately acquired by the sequencing device, sequencing reads of nucleic acid fragments (e.g., representative of nucleic acid fragments of 50-300 bp) may be assigned to the wrong sample based on the inaccurate data. Furthermore, certain sequencing techniques may be associated with a greater degree of index hopping or molecular recombination of indexes between samples. Index hopping may be caused by one end of the molecule (including the index region) moving between molecules in the template library. Although index hopping may occur at a low rate (~0.05%), even low levels of index hopping may be meaningful in clinical situations. In the case of single-index reads, migration results in incorrect assignment of the sequencing read to another sample, which leads to contamination in downstream analysis. Index hopping can also occur using dual indexing, which uses a combinatorial approach (e.g., assigning the same sequence to multiple samples in either index 1 or index 2, but ensuring that any particular combination of index 1 and index 2 is unique to a particular sample). In certain embodiments, the technology of the present invention is used in combination with unique indexes in the first and second index reads. In that case, even if swapping occurs, the observed index combination is not a member of the expected set of index pairs.

[0018] Provided herein is an indexing technique that results in reduced misassignment of sequencing reads. In contrast to techniques in which each sample is associated with a single barcode or index, the technique of the present invention provides multiple index sequences that are uniquely associated with each individual sample and are introduced during sample preparation. Multiple index sequences are introduced at the index site (or multiple index sites) such that each individual nucleic acid fragment prepared from a sample has at least one index sequence, and all of the different index sequences are present in the library of nucleic acid fragments of each sample. In this way, index sequence diversity per sample is introduced. This diversity can improve the accuracy of the index sequence data obtained. In particular, a sequencing device that acquires image data representing hundreds (or thousands) of nucleic acid fragments from two or more samples being sequenced simultaneously may have difficulty determining base calls when the samples lack nucleotide diversity, which can result in acquired image data that is difficult to interpret. For example, certain sequencing techniques evaluate differences in image signal intensity to make base calls. For samples with only a single index sequence per index site, the nucleotides in each sequencing cycle of the index sequence are as diverse as the total number of samples, which may be insufficiently diverse for runs with low sample numbers. Thus, in one embodiment, the indexing techniques provided herein result in improved acquisition of index sequence data through index diversity, allowing accurate sequencing even for low-plex sequencing runs.

[0019] To that end, FIG. 1 is a schematic diagram of an indexed nucleic acid fragment 12 of a sample according to the technology of the present invention. The indexed nucleic acid fragment 12 represents a fragment suitable for a sequencing run. The indexed nucleic acid fragment 12 includes an insert 14 of nucleic acid derived from the sample, i.e., an endogenous nucleic acid. The indexed nucleic acid fragment 12 also includes introduced or exogenous sequences that facilitate sequencing. Such sequences are located 5' and 3' of the insert 14 and may include one or more sequencing primer sequences 16, 18 that allow binding of a universal sequencing primer to one or both strands. The indexed nucleic acid fragment 12 also includes a first index site 20 and a second index site 22. In the illustrated embodiment, the indexed nucleic acid fragment 12 also includes a first adaptor sequence 26 and a second adaptor sequence 28. The one or more adapter sequences 26, 28 can be selected based on the desired sequencing platform and can be, for example, P7 and P5 adapters as shown, which facilitate attachment of the indexed nucleic acid fragments 12 to a flow cell or sequencing substrate.

[0020] The illustrated indexed nucleic acid fragment 12 may be a double-stranded fragment, where a first index sequence 20 may be located 5' of the insert 14 and a second index sequence 22 may be located 3' of the insert 14 in the forward strand, such that the index sequences 20, 22 flank the insert. The first index sequence 20 is selected from a first index set 30 that includes a plurality (e.g., two, three, four, or more) of index sequences, illustrated as index sequences 20a, 20b, 20c, 20d. The second index sequence 22, if present, is selected from a second index set 32 ​​that includes a plurality of index sequences, illustrated as index sequences 22a, 22b, 22c, 22d.

[0021] Within an index set (e.g., index set 30 or index set 32), the individual index sequences (e.g., index sequences 20a, 20b, 20c, 20d) are distinct from each other. That is, they have different sequences from each other as provided herein. Furthermore, the index sequences in the first index set 30 are also distinguishable from the index sequences in the second index set 32. In certain embodiments, the index sequences 20, 22 may be distinguishable from endogenous nucleic acids of one or more samples. However, in other embodiments, the index sequences may not necessarily be distinguishable from the insert 14.

[0022] The index sequences may be single-stranded or double-stranded and may be at least 5 bases, at least 6 bases, at least 7 bases, at least 8 bases, or more. In certain embodiments, the index sequences are 5-8 bases, 5-10 bases, 5-15 bases, 5-25 bases, 8-10 bases, 8-12 bases, 8-15 bases, or 8-25 bases, etc., in length. Further, in certain embodiments, the index sequences (e.g., index sequences 20, 22) are 30 bases or less, 25 bases or less, 20 bases or less, 15 bases or less in length. It should be understood that the lengths of the index sequences provided herein may refer to unique / distinguishable portions of the sequence within each index set (and relative to other index sets) and may exclude adjacent common or universal sequences of the indexed nucleic acid fragments 12 that may act as sequencing primers and are common among all of the indexed nucleic acid fragments 12 from a sample.

[0023] Although certain embodiments of the disclosed technology are discussed in the context of dual-indexed sequencing technology, it should be understood that the technology provided herein can also be used in the context of single-indexed sequencing. For example, nucleic acid fragment 12 can incorporate only one index sequence (e.g., index sequence 20 or 22) selected from an index set (e.g., index set 30). Furthermore, index sequences can be selected to be sequenced from one or both strands of double-stranded nucleic acid fragment 12, for example, in paired-end or single-read sequencing, depending on the desired sequencing technology.

[0024] 2 is a schematic diagram of a sequencing library 40 derived from a sample and including indexed nucleic acid fragments 12. As shown, the library 40 has a number of different configurations of index combinations. For example, a particular fragment 12a may be indexed using a combination of index 20a and index 22a, while another fragment 12b may be indexed using a combination of indexes 20b and 22a. During preparation of the library 40 from a sample, it is envisioned that the inserts 14 are modified in a generally random manner using an adaptor mixture that includes different index sequences 20, 22 in each index set (e.g., a first index set 30 and a second index set 32), such that any given insert 14 may be modified with any one of the individual index sequences 20a, 20b, 20c, or 20d of the first index set 30 and any one of the individual index sequences 22a, 22b, 22c, or 22d of the second index set 32. 2 shows some of the possible combinations of first index sequences 20 and second index sequences 22 present in a library. Furthermore, it should be understood that additional combinations are possible depending on the total number of index sequences in the particular index set used for indexing. It should also be understood that for a particular sample, multiple indexed nucleic acid fragments 12, each with a different insert 14, will have the same configuration or combination of index sequences 20, 22.

[0025] FIG. 3 is a flow diagram of an embodiment of a method 50 for preparing an indexed library 40 of FIG. 2. After obtaining a sample of interest (step 52), the nucleic acids in the sample are fragmented (step 54). The fragmented nucleic acids are contacted with a plurality of diverse index sequences of an index set. In particular, at the beginning of library preparation, one or more index sets can be associated with or assigned to a particular sample. A library preparation can then be prepared using only the one or more index sets assigned to the sample (step 56). In certain embodiments, the plurality of index sequences assigned to a sample, e.g., the first index set 30 or the second index set 32, comprises equal concentrations of each individual index sequence in the set, e.g., index sequences 20, 22. That is, when the index set 30 has three different index sequences 20a, 20b, 20c, they are provided in a 1:1:1 ratio relative to each other, and in one embodiment, they are provided as a mixture with equal concentrations (or approximately equal concentrations within a range of 0.9 to 1.1 relative to each other) of each index sequence 20.

[0026] The fragmented nucleic acids are modified with adaptors (step 58) that contain index sequences assigned to the sample (e.g., as shown in FIG. 4). In other embodiments, index sequences may be added to the fragmented nucleic acids in a separate step from the addition of the adaptors. As a result of the modification, indexed nucleic acid fragments are generated. Providing substantially equal concentrations of the individual index sequences of each index set may promote relatively equal incorporation of each individual index sequence within the indexed nucleic acid fragments 12.

[0027] FIG. 4 is a schematic diagram of an embodiment of library preparation of a library of indexed nucleic acid fragments 12 from a sample 60. However, it should be understood that the illustrated method is an example and that the indexed nucleic acids 12 provided herein can be prepared using other library preparation techniques, such as tagmentation. In certain embodiments, the library is used to derive single-stranded template molecules that can be used in a sequencing reaction. The library can be formed from nucleic acid fragments that have common sequences at the 5' and 3' ends, but have diversity at one or more index and insert sites. As described in more detail below, the nucleic acid fragments in the library can contain regions of common sequence at (or proximal to) the 5' and 3' ends. In certain embodiments, the nucleic acid fragments of the library are "branched" such that the common sequence at the 5' end of each individual template in the library is not identical and is not fully complementary to the common sequence at the 3' end of the template. However, in other embodiments, the adaptors are not branched, e.g., fully complementary.

[0028] Sample 60 is fragmented to produce fragmented nucleic acids 62 with overhanging ends that are made blunt by a number of methods known to those of skill in the art. In one method, the ends of the fragmented DNA are end-repaired using T4 DNA polymerase and Klenow polymerase, and then phosphorylated using polynucleotide kinase enzyme to produce phosphorylated fragmented nucleic acids 64. A single "A" deoxynucleotide is then added to both 3' ends of the DNA molecules using Taq polymerase enzyme, resulting in 1-base 3' overhang fragmented nucleic acids 68 that are complementary to the 1-base 3' "T" overhangs on the double-stranded ends of the branched adapters.

[0029] A ligation reaction is then carried out between the branched adapters 70 and the 1 base 3' overhang fragmented nucleic acids 68 using a suitable ligase enzyme (e.g., T4 DNA ligase) that joins two copies of the adapters, one at each end, to each DNA fragment to form the adapter-target constructs 72. The products of this reaction can be purified from the unligated adapters by a number of techniques, such as size inclusion chromatography, preferably by electrophoresis through an agarose gel slab followed by excision of portions of the agarose containing DNA that are larger in size than the adapters.

[0030] In a particular depicted embodiment, the branched adapters 70 implemented in library preparation include a diverse mixture of indexes in the first index sequence 20 and the second index sequence 22. In contrast to other techniques where the adapters ligated to the sample or insert nucleic acid are generally identical, the branched adapters 70 are prepared using a mixture of index sequences, for example from index set 30 and index set 32. Thus, the branched adapters 70 are not all identical to each other, but are diverse based on the particular combination of index sequences in the first index sequence 20 and the second index sequence 22. However, the sequencing primers 16, 18 and adapter sequences are shared or common between the branched adapters 70. The adapter-target constructs 72 can be amplified to generate indexed nucleic acid fragments 12, which can then be denatured to separate the double-stranded structures into single-stranded molecules prior to sequencing.

[0031] Depending on the desired sequencing platform, the library 40 of indexed nucleic acid fragments 12 can be prepared from a single sample and pooled with other libraries from each of the other samples and prepared in a similar manner. Figure 5 is a schematic diagram of libraries with dedicated or assigned distinct index sets 30, 32 that can be pooled and distinguished after sequencing via the distinguishable index sequences present in the first index set 30a, 30b, 30c and / or the distinguishable index sequences present in the second index set 32a, 32b, 32c. The first index set 30a used in the creation of library 40a includes a separate set of index sequences that are not present in the other libraries 40b, 40c and do not overlap with the other index sets (30b, 32b, 30c, 32c) present in the other libraries 40b, 40c. It should also be understood that the first index set 30a is also separate and non-overlapping from the second index set 32a used in the same library 40a.

[0032] FIG. 6 is a flow diagram of a method 80 for sequencing pooled libraries (e.g., the library of FIG. 5) from different samples and assigning sequencing data to the correct sample using index sequences provided herein. In step 82, individual indexed sample fragments (e.g., indexed nucleic acid fragments 12) are prepared from individual samples using at least one index set that includes multiple index sequences. The individual indexed sample fragments are pooled in step 84 with other indexed sample fragments from different samples prepared using different (i.e., distinguishable from the indexes referred to in step 82) index sets to generate combined sample fragments. The pooling or combining can be performed during loading of the samples onto the sequencing substrate. In one embodiment, the combined samples are loaded into different lanes of a flow cell. Because the lanes of the flow cell are separated from each other during sequencing, the index set used in the sample in the first lane of the flow cell can be used to index other samples in different lanes, as long as the samples in the same lane are not indexed using the same index set.

[0033] In step 86, sequencing data representing the sequencing of the combined sample fragments is obtained, and in step 88, the sequencing reads are associated with individual samples only if they contain an index sequence of the assigned index set. In certain embodiments, when each of the indexed sample fragments has a first index sequence and a second index sequence, the rule-based assignment requires that both the first index sequence and the second index sequence are members of the assigned index set for a given sample. A failure at only one index site is sufficient to remove the read with index hopping, resulting in removal of the sequencing read from downstream analysis (e.g., genome assembly). In certain embodiments, the failed sequencing reads can be saved for quality assessment. That is, certain potential index sequences can be associated with larger index failures, and these can be tracked for redesign.

[0034] 7 is a schematic diagram of an embodiment of a sequencing technique 90 for obtaining sequencing data provided herein. As shown, the nucleic acid to be sequenced is immobilized on a substrate 92 via a capture probe 94 that is complementary to that used in combination with a template strand 96 derived from denaturation of an indexed nucleic acid fragment 12. The first sequencing read, read 1, is the sequence of the insert 14 obtained via contact with a read 1 primer 98 that targets (i.e., is complementary to) one of the sequencing primer sequences 16, 18. For example, if the template strand 96 is captured using a capture probe 94 complementary to the p7 (or other 5') adaptor, then the read 1 primer 98 can be complementary to the sequencing primer sequence 18.

[0035] After removal of the read 1 product, the technique 90 may also obtain a first index read of the index sequence 20 at the p7 side of the insert using a first index primer 100 that may be targeted to the sequencing primer 16. In the illustrated embodiment, the first index read is on the same strand as read 1. While the read 1 read may be 100-150 bases, the index read may be relatively shorter, for example 8-12 bases, or may be of a length comparable to the known length of the index sequences 20, 22. In this way, sequencing resources are conserved. To obtain a second index read from the same strand after removal of the first index product, the template strand captured on the 3' end via the capture probe 104 may be used. For example, the second index read may be obtained using a second index read primer 106 that targets a portion of the p5 (or other 3') adaptor or adjacent sequence. However, in other embodiments, the second index read may be obtained from the resynthesized complementary strand. After removal of the second index read product, a complementary strand 110 to the template strand 96 is synthesized and the original template strand 96 is removed. The synthesized strand is then contacted with a read 2 primer 112 to obtain a read 2 sequence that is the reverse complement of the read 1 sequence. It is envisioned that the first index primer 100, the second index primer 106, and the read 1 and read 2 primers 98, 112 are universal for all template strands 96, regardless of the origin of the template sample and the index sequence. Based on the obtained first index and second index reads, the sequencing data of read 1 and read 2 can be associated with a particular sample.

[0036] As provided herein, index sets provide additional diversity for a given sample in the index reads. When assigning a single index sequence to each sample, there is a risk that not all nucleotides are represented in any given cycle of index reads when the number of samples is low. If that happens, the sequencing run may fail to produce usable data because the intensity correction and base calling algorithms do not work as designed in the index cycle, which results in failure to assign reads to samples. The most extreme example of this scenario is the sequencing of a single sample, which may be done, for example, for positive sample tracking and / or to remove non-indexed Phi X from the reads. A solution to this problem is to label each sample with multiple index sequences so that there is sufficient nucleotide representation for the primary analysis pipeline to function as designed. Figure 8 is a representation of an index set (e.g., index set 30) with nucleotides N1, N2, etc. The index set may be selected such that for any given position, the nucleotides are diverse within the sequence of the index set. For example, the nucleotides may vary such that at position 120, three or more of A, C, T, and G are represented across the index set. In FIG. 9, which depicts a particular embodiment of index set 30, three different nucleotides are represented at position 120 and four different nucleotides are represented at position 122. Thus, the index set may be designed such that at each nucleotide position, at least three different nucleotides are represented across the index set. In other embodiments, four different nucleotides are represented across the index set at at least half of the nucleotide positions in the index sequence. Furthermore, in certain embodiments, the index sequences are internally diverse; that is, at least a portion of adjacent nucleotides of individual index sequences differ from one another.

[0037] Furthermore, certain indexes may not perform as well as others, resulting in certain samples being under-represented even when the concentration of sample input is the same. The benefit of placing multiple index sequences in each sample is that the overall impact of poor performance of one index sequence is limited. In this strategy, high and low performing index sequences can also be grouped together to further improve the uniformity of representation across different samples.

[0038] Tables 1 and 2 are examples of index sets (e.g., index sets 30, 32) and individual index arrays (e.g., index arrays 20, 22) that make up the index sets according to the techniques of the present invention. For example, the index arrays (e.g., index arrays 20, 22) under each individual group # are assumed to be a single index set. That is, group #0 includes four different index arrays F7-001, F7-002, F7-003, and F7-004 that together form a single index set. Group #1 includes an additional four different index arrays F7-005, F7-006, F7-007, and F7-008 that together form a single index set, and so on.

[0039] In the index sets shown below, there are four indexes per index set. However, it should be understood that the size of the index set may vary and may include three, four, or more individual distinct index sequences. The index sets shown were selected to ensure that within any group, there is a balanced representation of nucleotides. Specifically, within any group, the second least frequent nucleotide in each cycle must be present in at least 25% of the index sequences.

[0040] Table 1 shows index sets that may be the P7 side index set 30. However, in certain embodiments, the sequences in Table 1 may be used as the index set 32 ​​on the P5 side.

[0041] [Table 1A]

[0042] [Table 1B]

[0043] [Table 1C]

[0044] [Table 1D]

[0045] Table 2 shows index sets that may be P5 index sets 32. In certain embodiments, the index sets are designed to be used in combination with P7 index sets 30 having the same group number to generate indexed nucleic acid fragments for a sample. For example, certain paired sets may have completed quality assessments when used together and may be associated with low levels of index hopping or generally evenly distributed amplification yields. In other embodiments, any given P7 (or 5') index set 30 may be used with any other P5 index set 32.

[0046] [Table 2A]

[0047] [Table 2B]

[0048] [Table 2C]

[0049] [Table 2D]

[0050] FIG. 10 is an example of a sample preparation kit 150 for sequencing that may be used in conjunction with the techniques of the present invention, i.e., for preparing indexed nucleic acid fragments 12 from individual samples, and in certain embodiments, for sequencing the indexed nucleic acid fragments 12. The sample preparation kit 150 may include a first index set 30 and, if used, a second index set 32. In certain embodiments, the first index set 30 and / or the second index set 32 ​​may be provided in the form of an adapter nucleic acid that includes additional elements such as primer sequences, adapter sequences, etc. The first index set 30 and / or the second index set 32 ​​may be provided in each individual container in premixed amounts such that each individual index sequence is present in approximately equal concentration, and index diversity for a given sample is less susceptible to user error. In embodiments where the adapters are branched dual-indexed adapters, both index sets 30, 32 may be present in a single adapter nucleic acid that may be provided in a single container. The sample preparation kit 150 may also include appropriate primers 152 for use in combination with a desired sequencing platform. The sample preparation kit 150 may also include one or more sample preparation enzymes, buffers, and / or reagents 154. The sample preparation kit 150 may be provided as a pre-packaged kit for preparing a library from a single sample, or in certain embodiments, may be provided as a multi-sample kit having multiple different index sets 30, 32.

[0051] FIG. 11 is an illustration of a configuration of a sequencing device 160 that may be used in combination with the disclosed embodiments to obtain sequencing data from indexed nucleic acids (e.g., sequencing reads, read 1, read 2, index reads, index read 1, index read 2, multi-sample sequencing data) that have been assigned to individual samples using the indexing techniques provided herein. The sequencing device 160 may be implemented according to any sequencing technology, such as those incorporating the sequencing bi-synthesis method described in U.S. Patent Application Publication Nos. 2007 / 0166705; 2006 / 0188901; 2006 / 0240439; 2006 / 0281109; 2005 / 0100900; U.S. Patent No. 7,057,026; WO 05 / 065814; WO 06 / 064199; WO 07 / 010,251 (the disclosures of which are incorporated herein by reference in their entirety). Alternatively, a sequencing by ligation technology may be used in the sequencing device 160. Such techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides and are described in U.S. Patent Nos. 6,969,488; 6,172,218; and 6,306,597, the disclosures of which are incorporated by reference in their entireties. Some embodiments may utilize nanopore sequencing, in which a target nucleic acid strand, or nucleotides exonuclease removed from a target nucleic acid, are passed through a nanopore.As the target nucleic acid or nucleotide passes through the nanopore, each type of base can be identified by measuring the change in the electrical conductance of the pore (U.S. Pat. No. 7,001,792; Soni & Meller, Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2, 459-481 (2007); and Cockroft et al. J. Am. Chem. Soc. 130, 818-820 (2008); the disclosures of which are incorporated herein by reference in their entireties). Yet other embodiments include detection of protons released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use electrical detectors and related technology available from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies), or the sequencing methods and systems described in US 2009 / 0026082 A1; US ​​2009 / 0127589 A1; US ​​2010 / 0137143 A1; or US 2010 / 0282617 A1, each of which is incorporated herein by reference in its entirety. Certain embodiments can utilize methods involving real-time monitoring of DNA polymerase activity. Nucleotide incorporation can be detected through fluorescence resonance energy transfer (FRET) interactions between fluorophore-bearing polymerases and gamma-phosphate-labeled nucleotides, or using zero-mode waveguides, for example, as described in Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entireties. Other suitable alternative techniques include, for example, fluorescence in situ sequencing (FISSEQ), and massively parallel signature sequencing (MPSS).In certain embodiments, the sequencing device 160 can be an Illumina (La Jolla, Calif.) HiSeq, MiSeq, or HiScanSQ. In other embodiments, the sequencing device 160 can be configured to function using a CMOS sensor with nanowells fabricated over photodiodes such that DNA precipitates are aligned one-to-one with each photodiode.

[0052] The sequencing device 160 may be a "one channel" detection device in which only two of the four nucleotides are labeled and detectable for any given image. For example, thymine may have a permanent fluorescent label, while adenine uses the same fluorescent label in a removable form. Guanine may be permanently dark, and cytosine may be initially dark but have a label added during the cycle. Thus, each cycle may involve an initial image and a second image, where a dye is cleaved from any adenine and added to any cytosine, such that only thymine and adenine are detectable in the initial image, but only thymine and cytosine are detectable in the second image. Any base that is dark throughout both images is a guanine, and any base that is detectable throughout both images is a thymine. A base that is detectable in the first image but not in the second image is an adenine, and a base that is not detectable in the first image but is detectable in the second image is a cytosine. By combining the information from the initial and second images, one channel can be used to distinguish all four bases.

[0053] In the illustrated embodiment, the sequencing device 160 includes a separate sample processing device 162 and associated computer 164. However, as described, these may be implemented as a single device. Furthermore, the associated computer 164 may be local to or networked with the sample processing device 162. In the illustrated embodiment, a biological sample may be loaded onto a sample substrate 170, e.g., a flow cell or slide, of the sample processing device 162, and the sample substrate is imaged to generate sequence data. For example, reagents that interact with the biological sample may fluoresce at a particular wavelength in response to an excitation beam generated by the imaging module 172, thereby returning radiation for imaging. For example, fluorescent moieties may be generated by fluorescently tagged nucleic acids that hybridize to their complementary molecules or fluorescently tagged nucleotides that have been incorporated into oligonucleotides using a polymerase. As will be appreciated by those skilled in the art, the wavelengths at which sample dyes are excited and at which they fluoresce depend on the absorption and emission spectra of the particular dyes. Such returned radiation may back-propagate through light-directing optics. This retrograde beam may generally be directed towards the detection optics of the imaging module 172 .

[0054] The detection optics of the imaging module can be based on any suitable technology, for example, a charge-coupled device (CCD) sensor that generates pixelated image data based on photons impinging on a location in the device. However, it will be understood that any of a variety of other detectors can also be used, including, but not limited to, a detector array configured for time-delay integration (TDI) operation, a complementary metal-oxide semiconductor (CMOS) detector, an avalanche photodiode (APD) detector, a Geiger mode photon counter, or any other suitable detector. TDI mode detection can be coupled with line scanning as described in U.S. Pat. No. 7,329,860 (hereby incorporated by reference). Other useful detectors are described, for example, in the references provided herein above in the context of various nucleic acid sequencing methodologies.

[0055] The imaging module 172 may be under the control of the processor, for example, via a processor 174, and the sample processing device 162 may also include an I / O controller 176, an internal bus 178, a non-volatile memory 180, a RAM 182 and any other memory structures capable of storing executable instructions, and other suitable hardware components that may be similar to those described with respect to FIG. 11. Additionally, the associated computer 164 may also include a processor 184, an I / O controller 186, a communication module, and a memory architecture such as a RAM 188 and a non-volatile memory 190 capable of storing executable instructions 192. The hardware components may be coupled by an internal bus 194, which may also be coupled to a display 196. In embodiments in which the sequencing device 160 is implemented as an all-in-one device, certain extraneous hardware elements may be eliminated.

[0056] The processor 184 may be programmed to assign individual sequencing reads to samples based on one or more associated index sequences according to the techniques provided herein. In certain embodiments, based on image data acquired by the imaging module 172, the sequencing device 160 may be configured to generate sequencing data including base calls for each base of the sequencing read. Furthermore, based on the image data, even for sequential sequencing reads, individual reads may be linked to the same position, and thus the same template strand, via the image data. In this manner, the index sequencing reads may be associated with the sequencing reads of the insert sequence before being assigned to the original sample. The processor 184 may also be programmed to perform downstream analysis on the sequence corresponding to the insert for a particular sample after the assignment of the sequencing reads to the samples.

[0057] FIG. 12 is an example of a graphical user interface screen 200 that may be generated by the sequencing device 160 for user input of information regarding a sequencing reaction using indexed nucleic acid fragments provided herein. For example, a user may provide input regarding the name or identification of each sample in a sequencing run, the number of index sites, and the particular index set or sets to be used for each sample. In one embodiment, the index sets are commercially available, and the user interface screen 200 provides a drop-down menu of commercially available index sets. Each individual sample may then be associated with the selected commercially available index set or sets. Based on the selection, a processor (e.g., processor 184) of the sequencing device 160 accesses stored index sequence information corresponding to the selected index set from memory, and uses the accessed index sequence information to assign index sequence reads acquired by the sequencing device 160 to a particular sample. Once assigned to a particular sample based on one or more index sequence reads, sequencing reads that represent inserts and are associated with the imaged positions of the index sequence reads are co-assigned to a particular sample.

[0058] The technical effects of the disclosed embodiments include improved and more accurate indexing of nucleic acid sequences. Improved indexing can reduce incorrectly assigned sequencing reads from multiplexes (e.g., multiple samples) to more meaningful information for clinicians. Furthermore, the improved accuracy of sequencing read assignment associated with the technology of the present invention facilitates commercially and time-saving high-throughput sequencing strategies. The index sequences provided herein address biases introduced in sequencing data that affect sequencing coverage counts.

[0059] While only certain features of the present disclosure have been illustrated and described herein, many modifications and changes will occur to those skilled in the art, and it is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the present disclosure. [Explanation of symbols]

[0060] 12 Indexed Nucleic Acid Fragments 14 Insert 16 Sequencing primer sequences 18 Sequencing primer sequence 20 First index site 22 Second index site 26 First adapter sequence 28 Second Adapter Sequence 30 First Index Set 32 Second set of indexes 40 Sequencing Library 60 samples 62 Fragmented Nucleic Acids 64 Phosphorylated Fragmented Nucleic Acids 68 1-base 3' overhang fragmented nucleic acid 70 Branch adapter 72 Adapter-Target Constructs 92 Substrate 94 Capture Probes 96 Template strand 98 Primer for lead 1 100 First Index Primer 104 Capture Probe 106 Second Index Read Primer 110 Complementary strand 112 Primer for lead 2 120 positions 122 positions 150 Sample Preparation Kit 152 Primer 154 Reagents 160 Sequencing Device 162 Sample processing device 164 Associated Computers 170 Sample Substrate 172 Imaging Module 174 processors 176 I / O control section 178 Internal Bus 180 Non-volatile memory 182 RAM 184 processors 186 I / O control unit 188 RAM 190 Non-volatile memory 192 Executable Instructions 194 Internal Bus 196 displays 200 Graphical User Interface Screens

Claims

1. 1. A sample-indexed nucleic acid library preparation comprising: a first nucleic acid library prepared from a first sample, said first nucleic acid library comprising a first plurality of nucleic acid fragments, each of said first plurality of nucleic acid fragments comprising at least two different index sequences selected from a first set of index sequences; A second nucleic acid library prepared from a second sample, the second nucleic acid library comprising a second plurality of nucleic acid fragments, each of the second plurality of nucleic acid fragments comprising at least two different index sequences selected from a second set of index sequences distinct from the first set of index sequences. Including, the index sequences are arranged in each nucleic acid fragment of the first plurality of nucleic acid fragments and the second plurality of nucleic acid fragments such that a first index sequence of the index sequence is located 5' of the target sequence and a second index sequence of the index sequence is located 3' of the target sequence; Library preparation.

2. (i) the first plurality of nucleic acid fragments comprises individual nucleic acid fragments that have different combinations of two different index sequences from the first set of index sequences relative to other individual nucleic acid fragments within the first plurality of nucleic acid fragments; and / or 2. The library preparation of claim 1, wherein (ii) the second plurality of nucleic acid fragments comprises individual nucleic acid fragments having different combinations of two different index sequences from the second set of index sequences relative to other individual nucleic acid fragments in the second plurality of nucleic acid fragments.

3. 3. The library preparation of claim 1 or claim 2, wherein each index sequence of the first set of index sequences is present in the first plurality of nucleic acid fragments in a ratio of between 0.9 and 1.1 relative to other index sequences in the first set of index sequences, and optionally each index sequence of the first set of index sequences is present in approximately equal proportions in the first plurality of nucleic acid fragments.

4. 4. The library preparation of any one of claims 1 to 3, wherein a portion of an index sequence from the first set of index sequences is located only 5' to the target sequence in the first plurality of nucleic acid fragments and not 3' to the target sequence.

5. 5. The method of claim 4, wherein another portion of index sequences from said first set of index sequences is located only 3' to said target sequences in said first plurality of nucleic acid fragments and not 5' to said target sequences.

6. 6. The library preparation of any one of claims 1 to 5, wherein each index sequence of the second set of index sequences is present in the second plurality of nucleic acid fragments in a ratio of between 0.9 and 1.1 relative to other index sequences in the second set of index sequences, and optionally, each index sequence of the second set of index sequences is present in approximately equal proportions in the second plurality of nucleic acid fragments.

7. 7. The library preparation of any one of claims 1 to 6, wherein a portion of index sequences from the second set of index sequences are located only 5' to the target sequences in the second plurality of nucleic acid fragments and not 3' to the target sequences.

8. 8. The library preparation of claim 7, wherein another portion of index sequences from said second set of index sequences are located only 3' to said target sequences in said second plurality of nucleic acid fragments and not 5' to said target sequences.

9. 9. The library preparation of any one of claims 1 to 8, wherein the first sample and the second sample are nucleic acid samples from different individuals.

10. 1. A method for sequencing a nucleic acid molecule, comprising: providing a plurality of dual-indexed nucleic acid fragments generated from a sample, each individual nucleic acid fragment of the nucleic acid fragments comprising a 5' adaptor sequence, a 5' index sequence, a 3' adaptor sequence, and a 3' index sequence, wherein a plurality of different 5' index sequences selected from a first set of 5' index sequences associated with the sample and a plurality of different 3' index sequences selected from a second set of 3' index sequences associated with the sample are represented in the dual-indexed nucleic acid fragments, wherein the plurality of different 5' index sequences and the plurality of different 3' index sequences are distinguishable from one another; generating sequencing data representative of the sequences of the dual-indexed nucleic acid fragments; and associating each of said sequences with said sample only if said each of said sequences comprises both said 5' index sequence selected from said first set and said 3' index sequence selected from said second set. A method comprising:

11. 11. The method of claim 10, comprising contacting the dual-indexed nucleic acid fragments with a sequencing substrate comprising immobilized capture molecules, the capture molecules configured to hybridize to the 5' adaptor and the 3' adaptor sequences of the nucleic acid fragments.

12. 12. The method of claim 10 or claim 11, comprising removing individual sequences in the sequencing data that contain only one of the 5' index sequence or the 3' index sequence.

13. 13. The method of any one of claims 10 to 12, comprising providing dual-indexed nucleic acid fragments from a second sample, wherein the dual-indexed nucleic acid fragments from the second sample are modified with the 5' adaptor sequence and the 3' adaptor sequence, and wherein a second plurality of different 5' indexing sequences and a second plurality of different 3' indexing sequences are represented in the dual-indexed nucleic acid fragments from the second sample, and wherein the second plurality of different 5' indexing sequences and the second plurality of different 3' indexing sequences are distinguishable from each other and from the plurality of different 5' indexing sequences and the plurality of different 3' indexing sequences.

14. 14. The method of claim 13, comprising simultaneously contacting the dual-indexed nucleic acid fragments from the sample and the second sample with a sequencing substrate comprising immobilized capture molecules, the capture molecules configured to hybridize to the 5' adaptor and the 3' adaptor sequences of the nucleic acid fragments.

15. 1. A multi-sample library preparation kit comprising a plurality of nucleic acid fragments, each fragment comprising an index sequence and an adaptor sequence, the plurality of nucleic acid fragments comprising: a nucleic acid fragment set associated with a first sample, the nucleic acid fragment set comprising nucleic acid fragments having the index sequence selected from a first index set, the adapter sequence being a first adapter sequence, and nucleic acid fragments having the index sequence selected from a second index set, the adapter sequence being a second adapter sequence, each index sequence of the first index set and the second index set being represented in the nucleic acid fragment set associated with the first sample; and A set of nucleic acid fragments associated with a second sample, the set of nucleic acid fragments comprising nucleic acid fragments having the index sequence selected from a third index set, the adapter sequence being a first adapter sequence, and nucleic acid fragments having the index sequence selected from a fourth index set, the adapter sequence being a second adapter sequence, each index sequence of the third index set and the fourth index set being represented in the set of nucleic acid fragments associated with the second sample. Including, each of the first index set, the second index set, the third index set, and the fourth index set includes a plurality of index arrays that are distinguishable from one another; Multi-sample library preparation kit.

16. The first set of indexes: CCATACTA, TGTGCGCT, CACATTGC, and ATCCGGAG a first plurality of unique index sequences including: each of the first plurality of unique index sequences is represented in a nucleic acid fragment set associated with the first sample; 16. The multi-sample library preparation kit of claim 15.

17. The second set of indexes: T.C.G.C.T.T.C. ATTGGAGG, AACTAGAC, and CGGACTAT a second plurality of unique index sequences comprising: each of the second plurality of unique index sequences is represented in a set of nucleic acid fragments associated with the first sample; 17. The multi-sample library preparation kit of claim 16.

18. 18. The multiple sample library preparation kit of any one of claims 15 to 17, wherein the first adapter sequence is a p5 adapter sequence and / or the second adapter sequence is a p7 adapter sequence.

19. 19. The multiple sample library preparation kit of any one of Claims 15 to 18, wherein the index sequence is located between the adapter sequence and a sequencing primer in each nucleic acid fragment.

20. (i) the first index set comprises a first plurality of unique index sequences that are at least 5 bases in length and differ from one another such that at least three of the individual unique index sequences have a different base at a first base; (ii) the first index set comprises a first plurality of unique index sequences that are at least 5 bases in length and that differ from one another such that at each base position of the plurality of unique index sequences, at least three different nucleotides are represented in the plurality of unique index sequences; and / or (iii) the first index set comprises a first plurality of unique index sequences that are at least 5 bases in length and that differ from one another such that at least three different nucleotides are represented in the plurality of unique index sequences at a majority of base positions of the plurality of unique index sequences; 20. A multiple sample library preparation kit according to any one of claims 15 to 19.

21. A plurality of nucleic acid capture sequences immobilized on a substrate, each individual nucleic acid capture sequence comprising: A first capture sequence that is complementary to the first adapter sequence or a second capture sequence that is complementary to the second adapter sequence. A sequencing substrate comprising: a plurality of nucleic acid fragments linked to each nucleic acid capture sequence of the plurality of nucleic acid capture sequences, each individual fragment of the plurality of nucleic acid fragments comprising the first adaptor sequence and the second adaptor sequence, each individual fragment of the plurality of nucleic acid fragments comprising a first sequence complementary to one index sequence of a first set of unique index sequences and a second sequence complementary to one index sequence of a second set of unique index sequences, the first set of unique index sequences and the second set of unique index sequences being associated with only one sample from which the plurality of nucleic acid fragments are derived, and each unique index sequence of the first set and the second set is present in at least one nucleic acid fragment of the plurality of nucleic acid fragments; Sequencing substrate.

22. (i) the plurality of nucleic acid capture sequences are single-stranded; (ii) each unique index sequence of the first set is present in the plurality of nucleic acid fragments in a ratio of about 0.9 to about 1.1 relative to any other unique index sequence of the first set; and / or (iii) each unique index sequence is at least 6 bases in length; 22. The sequencing substrate of claim 21.

23. 23. The sequencing substrate of claim 21 or claim 22, comprising an additional plurality of nucleic acid fragments derived from each of a plurality of additional samples, wherein the additional plurality of nucleic acid fragments from each individual sample of the additional samples does not comprise sequences complementary to the first set of unique index sequences and the second set of unique index sequences.

24. 1. A method for sequencing a nucleic acid molecule, comprising: providing a plurality of dual-indexed nucleic acid fragments generated from a sample, each individual nucleic acid fragment of the dual-indexed nucleic acid fragments comprising a sequence of interest derived from the sample, a 5' adaptor sequence, a 5' index sequence, a 3' adaptor sequence, and a 3' index sequence to generate dual-indexed nucleic acid fragments, wherein a plurality of different 5' index sequences selected from a first set of 5' index sequences associated with the sample and a plurality of different 3' index sequences selected from a second set of 3' index sequences associated with the sample are represented in the dual-indexed nucleic acid fragments, wherein the plurality of different 5' index sequences and the plurality of different 3' index sequences are distinguishable from one another; generating sequencing data representative of said sequence of interest; generating sequencing data representing the 5' index sequence and the 3' index sequence; and assigning each sequence of interest to the sample only if the each sequence of interest is associated with both the 5' index sequence selected from the first set and the 3' index sequence selected from the second set. A method comprising:

25. 25. The method of claim 24, wherein each sequence of interest is associated with both the 5' index sequence selected from the first set and the 3' index sequence selected from the second set based on a common position of the sequencing data representing the 5' index sequence and the 3' index sequence with the sequencing data representing the sequence of interest.

26. 25. The method of claim 24, comprising assigning sequence data of another individual sequence of interest to the sample based on complementarity with the individual sequence of interest.

27. 27. The method of any one of Claims 24 to 26, wherein the sequencing data representing the sequence of interest as well as the 5' index sequence and the 3' index sequence are generated from the same single strand of the dual-indexed nucleic acid fragment.

28. 28. The method of any one of Claims 24 to 27, wherein the sequencing data representing the 5' index sequence and the 3' index sequence are generated from different single strands of the dual-indexed nucleic acid fragment.

29. 1. A computer-implemented method for assigning sequencing data representing a sequence of interest to a sample, comprising: providing sequencing data representative of a sequence of interest and sequencing data representative of a 5' index sequence and a 3' index sequence, said sequencing data being obtained from a step of sequencing a plurality of dual-indexed nucleic acid fragments generated from a sample, wherein each individual nucleic acid fragment of said dual-indexed nucleic acid fragments comprises a sequence of interest, a 5' adaptor sequence, a 5' index sequence, a 3' adaptor sequence, and a 3' index sequence derived from said sample to generate dual-indexed nucleic acid fragments, wherein a plurality of different 5' index sequences selected from a first set of 5' index sequences associated with said sample and a plurality of different 3' index sequences selected from a second set of 3' index sequences associated with said sample are represented in said dual-indexed nucleic acid fragments, wherein the plurality of different 5' index sequences and the plurality of different 3' index sequences are distinguishable from one another; processing the sequencing data to assign each sequence of interest to the sample only if the each sequence of interest is associated with both the 5' index sequence selected from the first set and the 3' index sequence selected from the second set. The method includes:

30. 30. The method of claim 29, wherein each sequence of interest is associated with both the 5' index sequence selected from the first set and the 3' index sequence selected from the second set based on a common position of the sequencing data representing the 5' index sequence and the 3' index sequence with the sequencing data representing the sequence of interest.

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