Universal PCR to boost molecule recovery in cell free DNA library preparation and target enrichment workflows

Abstract

The present disclosure provides for methods of preparing nucleic acid libraries for sequencing. In particular, the present disclosure is directed to a workflow which employs a first round of amplification utilizing a universal polymerase chain reaction approach instead of a platform specific indexed polymerase chain reaction approach. Applicant has unexpectedly discovered that a workflow employing a first round of amplification which utilizes a universal polymerase chain reaction approach increases duplex recovery and / or genomic equivalent recovery as compared with a workflow employing a first round of amplification utilizing a platform specific indexed polymerase chain reaction.

Description

UNIVERSAL PCR TO BOOST MOLECULE RECOVERY IN CELL FREE DNA LIBRARY PREPARATION AND TARGET ENRICHMENT WORKFLOWSP38978-WO-1FIELD OF THE INVENTION

[0001] The present disclosure relates to methods of preparing nucleic acid libraries, such as nucleic acid libraries suitable for next-generation sequencing.BACKGROUND

[0002] Liquid biopsy, through the analysis of genetic and epigenetic changes in cell-free DNA(cfDNA), represents a promising non-invasive method for detecting and monitoring disease. This approach has shown significant potential in oncology and other tissue-specific conditions for early- stage disease detection, tracking disease progression, and identifying recurrence. By providing a realtime snapshot of tumor biology through a simple blood test, liquid biopsy offers advantages over traditional tissue biopsies, particularly in terms of patient comfort, accessibility, and the ability to monitor disease dynamics over time.

[0003] One of the primary challenges in liquid biopsy assays is the inherently low concentration of cfDNA molecules present in a typical sample. Compounding this issue is the fact that only a small subset of these cfDNA fragments originate from diseased tissue or tumor cells. This small fraction makes it difficult to reliably detect malignant DNA. Furthermore, the multi-step processes involved in cfDNA extraction, library preparation, and sequencing are prone to various technical errors and artifacts. These errors can produce false-positive signals, reducing the accuracy of the assay and hindering the detection of true genomic changes.

[0004] To overcome the challenge of low cfDNA availability, it is crucial to employ library preparation techniques that maximize the recovery of genomic equivalents. More specifically, Genomic equivalent Recovery (GER) is a metric used to indicate the proportion of input DNA that was actually sequenced. Since every molecule is a potential signal, with sufficient sequencing depth, higher GERs increase the likelihood of detecting very low variant molecules. It is believed that these methods enhance the sensitivity of the assay by ensuring that the highest possible number of cfDNA molecules, including circulating tumor (ctDNA), are captured and analyzed. Optimizing nucleic acid molecule recovery directly impacts the overall performance of the liquid biopsy, improving itscapacity to detect low-abundance mutations associated with early-stage disease or minimal residual disease (MRD).

[0005] Addressing error suppression is equally critical to improving the reliability of liquid biopsy assays. Several strategies can be employed to mitigate errors introduced during sample processing and sequencing, including the use of high-fidelity enzymes, unique molecular identifiers (UMIs), and duplex error correction. Duplex error correction is particularly effective, as it utilizes sequencing data from both the sense and antisense strands of the same DNA molecule to distinguish between true genomic alterations and low confidence artifacts. For duplex error correction to be successful, the library preparation method must be optimized for maximal duplex recovery, the metric which measures the percentage of sequenced reads that have both sense and antisense representation in sequencing data.BRIEF SUMMARY OF THE DISCLOSURE

[0006] It is desirable that for every nanogram of DNA that is obtained in a sample, the maximum number of nucleic acid molecules should be converted into library molecules for sequencing. Additionally, for each unique nucleic acid molecule that is converted into a library, both strands of a nucleic acid duplex should be converted.

[0007] In view of the foregoing, Applicant has developed a method of generating nucleic acid libraries, such as nucleic acid libraries suitable for sequencing, whereby universal primers are utilized in a first round of amplification ("a universal polymerase chain reaction (PCR) approach") instead of indexed, platform-specific primers. Applicant has unexpectedly discovered that a workflow employing a first round of amplification which utilizes a universal polymerase chain reaction approach increases genomic equivalent recovery (GER), and duplex recovery as compared with a workflow which employs a first round of amplification utilizing a platform specific indexed polymerase chain reaction (see FIGS. 4 and 5, herein). It is believed that the improved GER allows for greater sequencing depth at specific loci, which can directly enhance the assay's limit of detection for low-frequency variants. Additionally, the increased recovery of duplex molecules facilitates the implementation of duplex-based consensus error correction. This correction method provides more accurate variant detection by leveraging both DNA strands to reduce sequencing artifacts and minimize the overall error rate, thereby improving the precision of variant calls.

[0008] A first aspect of the present disclosure is a method of preparing a library comprising one or more target nucleic acid molecules, comprising: (a) obtaining a sample comprising one or morenucleic acid molecules; (b) ligating one or more universal adapters to each of the one or more nucleic acid molecules within the obtained sample to provide one or more universal adapter ligated nucleic acid molecules; (c) performing a first amplification, wherein the first amplification comprises amplifying the one or more universal adapter ligated nucleic acid molecules within the sample with one or more universal primers to provide one or more amplified nucleic acid molecules; (d) enriching the one or more amplified nucleic acid molecules in the sample for one or more target nucleic acid molecules to provide a target enriched sample; (e) performing a second amplification, wherein the second amplification comprises amplifying the one or more target nucleic acid molecules within the target enriched sample with one or more indexed, platform- specific primers to provide the library comprising the one or more target nucleic acid molecules.

[0009] In some embodiments, the method further comprises performing a third amplification. In some embodiments, the third amplification utilizes a third polymerase. In some embodiments, the third polymerase is the same or different than the first and / or second polymerases. In some embodiments, the third amplification utilizes universal primers.

[0010] In some embodiments, the one or more nucleic acid molecules comprise cfDNA. In some embodiments, the one or more nucleic acid molecules comprise ctDNA. In some embodiments, the obtained sample is derived from a liquid biopsy. In some embodiments, the obtained sample is derived from a tumor sample.

[0011] In some embodiments, the method further comprises sequencing the library comprising the one or more target nucleic acid molecules. In some embodiments, the sequencing comprises nextgenerations sequencing. In some embodiments, the next-generation sequencing comprises sequencing-by-synthesis.

[0012] In some embodiments, the one or more universal primers comprise one or more barcodes. In some embodiments, the one or more universal primers comprise a universal primer binding site.

[0013] In some embodiments, the first amplification utilizes a first polymerase. In some embodiments, the second amplification utilizes a second polymerase. In some embodiments, the first and second polymerases are the same. In some embodiments, the first and second polymerases are different.

[0014] In some embodiments, the enriching of the one or more amplified nucleic acid molecules comprises hybridization-based target enrichment. In some embodiments, the enriching of the one or more amplified nucleic acid molecules comprises primer extension target enrichment.

[0015] In some embodiments, the one or more indexed, platform-specific primers include a sample identifier or index sequence. In some embodiments, the one or more indexed, platformspecific primers include a platform-specific anchoring site. In some embodiments, the one or more indexed, platform-specific primers include (i) a sample identifier or index sequence; and (ii) a platform-specific anchoring site.

[0016] In some embodiments, the ligation of the one or more universal adapters to each of the one or more nucleic acid molecules utilizes a ligase. In some embodiments, the ligation of the one or more universal adapters to each of the one or more nucleic acid molecules utilizes a T4 ligase. In some embodiments, the T4 ligase is a modified T4 ligase, a mutated T4 ligase, or a variant of a T4 ligase.

[0017] A second aspect of the present disclosure is a method of preparing a library comprising one or more target nucleic acid molecules, comprising: (a) obtaining a sample comprising one or more nucleic acid molecules; (b) ligating one or more universal adapters to each of the one or more nucleic acid molecules within the obtained sample to provide one or more universal adapter ligated nucleic acid molecules; (c) performing a first amplification, wherein the first amplification comprises amplifying the one or more universal adapter ligated nucleic acid molecules within the sample with one or more universal primers to provide one or more amplified nucleic acid molecules; (d) enriching the one or more amplified nucleic acid molecules in the sample for one or more target nucleic acid molecules to provide a target enriched sample; (e) performing a second amplification on the target enriched sample with the one or more universal primers to provide an amplified target enriched sample; and (f) performing a third amplification, wherein the third amplification comprises amplifying the one or more target nucleic acid molecules within the amplified target enriched sample with one or more indexed, platform-specific primers to provide the library comprising the one or more target nucleic acid molecules.

[0018] In some embodiments, the method further comprises sequencing the library comprising the one or more target nucleic acid molecules. In some embodiments, the sequencing comprises nextgenerations sequencing. In some embodiments, the next-generation sequencing comprises sequencing-by-synthesis.

[0019] In some embodiments, the one or more universal primers comprise one or more barcodes. In some embodiments, the one or more universal primers comprise a universal primer binding site. In some embodiments, the first amplification utilizes a first polymerase. In someembodiments, the second amplification utilizes a second polymerase. In some embodiments, the first, second, and third polymerases are the same.

[0020] In some embodiments, the enriching of the one or more amplified nucleic acid molecules comprises hybridization-based target enrichment. In some embodiments, the enriching of the one or more amplified nucleic acid molecules comprises primer extension target enrichment.

[0021] In some embodiments, the one or more indexed, platform-specific primers include a sample identifier or index sequence. In some embodiments, the one or more indexed, platformspecific primers include a platform-specific anchoring site. In some embodiments, the one or more indexed, platform-specific primers include (i) a sample identifier or index sequence; and (ii) a platform-specific anchoring site.

[0022] In some embodiments, the ligation of the one or more universal adapters to each of the one or more nucleic acid molecules utilizes a ligase. In some embodiments, the ligation of the one or more universal adapters to each of the one or more nucleic acid molecules utilizes a T4 ligase. In some embodiments, the T4 ligase is a modified T4 ligase, a mutated T4 ligase, or a variant of a T4 ligase. In some embodiments, the one or more nucleic acid molecules comprise cfDNA. In some embodiments, the obtained sample comprises a liquid biopsy. In some embodiments, the obtained is derived from a tumor sample.

[0023] A third aspect of the present disclosure is a kit comprising: (a) one or more universal primers; and (b) one or more indexed, platform-specific primers. In some embodiments, the one or more universal primers comprise one or more barcodes and a universal primer binding site. In some embodiments, the one or more indexed, platform-specific primers include a sample identifier or index sequence. In some embodiments, one or more indexed, platform-specific primers include a platformspecific anchoring site. In some embodiments, the kit further comprises a polymerase. In some embodiments, the polymerase is an archaeal DNA polymerase. In some embodiments, the kit further comprises a ligase. In some embodiments, the ligase is a T4 ligase. In some embodiments, the T4 ligase is a modified T4 ligase, a mutated T4 ligase, or a variant of a T4 ligase. In some embodiments, the fit further comprises a next-generation sequencing instrument.BRIEF DESCRIPTION OF THE FIGURES

[0024] For a general understanding of the features of the disclosure, reference is made to the drawings. In the drawings, like reference numerals have been used throughout to identify identical elements.

[0025] FIG. 1 depicts a workflow utilizing a universal polymerase chain reaction approach in accordance with one embodiment of the present disclosure. In particular, universal primers are used in PCR1 ; and amplification with platform specific indexed primers is performed at PCR2.

[0026] FIG. 2 depicts a workflow which utilizes index, platform-specific primers during a first round of amplification. This represents a "standard workflow" employed for library preparation and target enrichment of nucleic acid molecules, e.g., cfDNA. Platform specific indexed primers are used in PCR1 ; and amplification with platform specific primers is performed at PCR2.

[0027] FIG. 3 depicts a workflow utilizing a universal polymerase chain reaction approach in accordance with one embodiment of the present disclosure.

[0028] FIG. 4 illustrates duplex recovery rates for libraries prepared using universal primers during a first round of amplification as compared with libraries prepared using indexed, platform specific primers during a first round of amplification.

[0029] FIG. 5 illustrates GER rates for libraries prepared using universal primers during a first round of amplification as compared with libraries prepared using indexed, platform specific primers during a first round of amplification.DETAILED DESCRIPTION

[0030] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0031] As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "includes" is defined inclusively, such that "includes A or B" means including A, B, or A and B.

[0032] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only termsclearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of' or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0033] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.

[0034] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0035] As used herein, the term "adapter" refers to a nucleotide sequence that may be added to another sequence to import additional properties to that sequence. An adapter can be single- ordouble-stranded or may have both a single-stranded portion and a double-stranded portion. The ligation of an adapter to a target polynucleotide or a target polynucleotide strand of interest enables the generation of amplification-ready products of the target polynucleotide or the target polynucleotide strand of interest. The target polynucleotide molecules may be fragmented or not prior to the addition of adaptors.

[0036] As used herein, the term "amplicon" refers to the product of a polynucleotide amplification reaction; that is, a clonal population of polynucleotides, which may be single stranded or double stranded, which are replicated from one or more starting sequences. The one or more starting sequences may be one or more copies of the same sequence, or they may be a mixture of different sequences. In some embodiments, amplicons are formed by the amplification of a single starting sequence. In some embodiments, amplicons may be produced by a variety of amplification reactions whose products comprise replicates of the one or more starting, or target, nucleic acids. In some embodiments, amplification reactions producing amplicons are "template- driven" in that base pairing of reactants, either nucleotides or oligonucleotides, have complements in a template polynucleotide that are required for the creation of reaction products.

[0037] As used herein "amplification" refers to a process in which a copy number increases. Amplification may be a process in which replication occurs repeatedly over time to form multiple copies of a template. Amplification can produce an exponential or linear increase in the number of copies as amplification proceeds. Exemplary amplification strategies include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), rolling circle replication (RCA), cascade-RCA, nucleic acid-based amplification (NASBA), and the like. Also, amplification can utilize a linear or circular template. Amplification can be performed under any suitable temperature conditions, such as with thermal cycling or isothermally. Furthermore, amplification can be performed in an amplification mixture (or reagent mixture), which is any composition capable of amplifying a nucleic acid target, if any, in the mixture. PCR amplification relies on repeated cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication. PCR can be performed by thermal cycling between two or more temperature setpoints, such as a higher denaturation temperature and a lower annealing / extension temperature, or among three or more temperature setpoints, such as a higher denaturation temperature, a lower annealing temperature, and an intermediate extension temperature, among others. PCR can be performed with a thermostable polymerase, such as Taq DNA polymerase. PCR produces an exponential increase in the amount of a product amplicon over successive cycles. PCR is described, for example, in U.S. Pat. No. 4,683,202;U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,000,159; U.S. Pat. No. 4,965,188; U.S. Pat. No. 5,176,995), the disclosures of each are hereby incorporated by reference herein in their entirety.

[0038] As used herein, the term "biological sample," "tissue sample," "specimen" or the like refers to any sample including a biomolecule (such as a protein, a peptide, a nucleic acid, a lipid, a carbohydrate, or a combination thereof) that is obtained from any organism including viruses. Other examples of organisms include mammals (such as humans; veterinary animals like cats, dogs, horses, cattle, and swine; and laboratory animals like mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections and needle biopsies of tissue), cell samples (such as cytological smears such as Pap smears or blood smears or samples of cells obtained by microdissection), or cell fractions, fragments, or organelles (such as obtained by lysing cells and separating their components by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (for example, obtained by a surgical biopsy or a needle biopsy), nipple aspirates, cerumen, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample (such as a homogenized or liquefied sample) prepared from a tumor or a portion thereof obtained from a subject.

[0039] As used herein, the term "ligation" refers to a condensation reaction joining two nucleic acid strands wherein a 5 '-phosphate group of one molecule reacts with the 3 '-hydroxyl group of another molecule. Ligation is typically an enzymatic reaction catalyzed by a ligase (e.g., a T4 ligase, a modified T4 ligase, or a variant of a T4 ligase, as described further herein) or a topoisomerase. Ligation may join two single strands to create one single-stranded molecule. Ligation may also join two strands each belonging to a double-stranded molecule thus joining two double-stranded molecules. Ligation may also join both strands of a double-stranded molecule to both strands of another double-stranded molecule thus joining two double-stranded molecules. Ligation may also join two ends of a strand within a double-stranded molecule thus repairing a nick in the double-stranded molecule.

[0040] As used herein, the term "nanopore" refers to a pore, channel, or passage formed or otherwise provided in a membrane or other barrier material that has a characteristic width or diameter of about 0.1 nm to about 1000 nm. A nanopore can be made of a naturally occurring pore-forming protein, such as a-hemolysin from S. aureus, or a mutant or variant of a wild-type pore-formingprotein, either non- naturally occurring (i.e., engineered) such as a-HL-C46, or naturally occurring. A membrane may be an organic membrane, such as a lipid bilayer, or a synthetic membrane made of a non-naturally occurring polymeric material. The nanopore may be disposed adjacent or in proximity to a sensor, a sensing circuit, or an electrode coupled to a sensing circuit, such as, for example, a complementary metal-oxide semiconductor (CMOS) or field effect transistor (FET) circuit.

[0041] As used herein, the term "next generation sequencing" refers to sequencing technologies having high-throughput sequencing as compared to traditional Sanger- and capillary electrophoresis-based approaches, wherein the sequencing process is performed in parallel, for example producing thousands or millions of relatively small sequence reads at a time. Some examples of next generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. These technologies produce shorter reads (anywhere from about 25 - about 500 bp) but many hundreds of thousands or millions of reads in a relatively short time. Examples of such sequencing devices available from Illumina (San Diego, CA) include, but are not limited to iSEQ, MiniSEQ, MiSEQ, NextSEQ, NoveSEQ.

[0042] It is believed that the Illumina next-generation sequencing technology uses clonal amplification and sequencing by synthesis (SBS) chemistry to enable rapid sequencing. The process simultaneously identifies DNA bases while incorporating them into a nucleic acid chain. Each base emits a unique fluorescent signal as it is added to the growing strand, which is used to determine the order of the DNA sequence. A non-limiting example of a sequencing device available from ThermoFisher Scientific (Waltham, MA) includes the Ion Personal Genome Machine™ (PGM™) System.

[0043] It is believed that Ion Torrent sequencing measures the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase. A non-limiting example of a sequencing device available from Pacific Biosciences (Menlo Park, CA) includes the PacBio Sequel Systems. A non-limiting example of a sequencing device available from Roche (Pleasanton, CA) is the Roche 454. Next-generation sequencing methods may also include nanopore sequencing methods. In general, three nanopore sequencing approaches have been pursued: strand sequencing in which the bases of DNA are identified as they pass sequentially through a nanopore, exonuclease-based nanopore sequencing in which nucleotides are enzymatically cleaved one-by-one from a DNA molecule and monitored as they are captured by and pass through the nanopore, and a nanopore sequencing by synthesis (SBS) approach in which identifiable polymer tags are attached to nucleotidesand registered in nanopores during enzyme-catalyzed DNA synthesis. Common to all these methods is the need for precise control of the reaction rates so that each base is determined in order.

[0044] Strand sequencing requires a method for slowing down the passage of the DNA through the nanopore and decoding a plurality of bases within the channel; ratcheting approaches, taking advantage of molecular motors, have been developed for this purpose. Exonuclease-based sequencing requires the release of each nucleotide close enough to the pore to guarantee its capture and its transit through the pore at a rate slow enough to obtain a valid ionic current signal. In addition, both methods rely on distinctions among the four natural bases, two relatively similar purines and two similar pyrimidines.

[0045] The nanopore SBS approach utilizes synthetic polymer tags attached to the nucleotides that are designed specifically to produce unique and readily distinguishable ionic current blockade signatures for sequence determination. In some embodiments, sequencing of nucleic acid molecules via nanopore sequencing comprises preparing nanopore sequencing complexes and determining polynucleotide sequences. Methods of preparing nanopores and nanopore sequencing are described in U.S. Patent Application Publication No. 2017 / 0268052, and PCT Publication Nos. WO20 14 / 074727, W02006 / 028508, WO2012 / 083249, and WO / 2014 / 074727, the disclosures of which are hereby incorporated by reference herein in their entireties. In some embodiments, tagged nucleotides may be used in the determination of the polynucleotide sequences (see, e.g., PCT Publication No. WO / 2020 / 131759, WO / 2013 / 191793, and WO / 2015 / 148402, the disclosures of which are hereby incorporated by reference herein in their entireties).

[0046] Analysis of the data generated by sequencing is generally performed using software and / or statistical algorithms that perform various data conversions, e.g., conversion of signal emissions into base calls, conversion of base calls into consensus sequences for a nucleic acid template, etc. Such software, statistical algorithms, and the use of such are described in detail, in U.S. Patent Application Publication Nos. 2009 / 0024331 2017 / 0044606 and in PCT Publication No. WO / 2018 / 034745, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0047] As used herein, the terms "nucleic acid" or "nucleic acid molecule" as used herein, refer to a high-molecular-weight biochemical macromolecule composed of nucleotide chains that convey genetic information. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The monomers from which nucleic acids are constructed are called nucleotides. Each nucleotide consists of three components: a nitrogenous heterocyclic base, either apurine or a pyrimidine (also known as a nucleobase); and a pentose sugar. Different nucleic acid types differ in the structure of the sugar in their nucleotides; DNA contains 2-deoxyribose while RNA contains ribose.

[0048] As used herein, the term "nucleotide" refers to a nucleoside-5'-oligophosphate compound, or structural analog of a nucleoside-5'-oligophosphate, which can act as a substrate or inhibitor of a nucleic acid polymerase. Exemplary nucleotides include, but are not limited to, nucleoside-5'-triphosphates (e.g., dATP, dCTP, dGTP, dTTP, and dUTP); nucleosides (e.g., dA, dC, dG, dT, and dU) with 5'-oligophosphate chains of 4 or more phosphates in length (e.g., 5'- tetraphosphosphate, 5'-pentaphosphosphate, 5'-hexaphosphosphate, 5'-heptaphosphosphate, 5'- octaphosphosphate); and structural analogs of nucleoside-5 '-triphosphates that can have a modified base moiety (e.g., a substituted purine or pyrimidine base), a modified sugar moiety (e.g., an Ci- alkylated sugar), and / or a modified oligophosphate moiety (e.g., an oligophosphate comprising a thiophosphate, a methylene, and / or other bridges between phosphates).

[0049] As used herein, the "polymerase" as used herein, refers to an enzyme that catalyzes the process of replication of nucleic acids. More specifically, DNA polymerase catalyzes the polymerization of deoxyribonucleotides alongside a DNA strand, which the DNA polymerase "reads" and uses as a template. The newly polymerized molecule is complementary to the template strand and identical to the template's partner strand.

[0050] As used herein, the term "primer" refers to an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. Extension of a primer is usually carried out with a nucleic acid polymerase, such as a DNA or RNA polymerase. The sequence of nucleotides added in the extension process is determined by the sequence of the template polynucleotide. Usually, primers are extended by a DNA polymerase. Primers usually have a length in the range of from 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides. Primers are employed in a variety of nucleic amplification reactions, for example, linear amplification reactions using a single primer, or polymerase chain reactions, employing two or more primers. Guidance for selecting the lengths and sequences of primers for particular applications is well known to those of ordinary skill in the art, as evidenced by the following reference that is incorporated by reference herein in its entirety: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2ndEdition (Cold Spring Harbor Press, New York, 2003).

[0051] As used herein, the term "sequence," when used in reference to a nucleic acid molecule, refers to the order of nucleotides (or bases) in the nucleic acid molecules. In cases, where different species of nucleotides are present in the nucleic acid molecule, the sequence includes an identification of the species of nucleotide (or base) at respective positions in the nucleic acid molecule. A sequence is a property of all or part of a nucleic acid molecule. The term can be used similarly to describe the order and positional identity of monomeric units in other polymers such as amino acid monomeric units of protein polymers.

[0052] As used herein, the term "sequencing" refers to the determination of the order and position of bases in a nucleic acid molecule. More particularly, the term "sequencing" refers to biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. Sequencing, as the term is used herein, can include without limitation parallel sequencing or any other sequencing method known of those skilled in the art, for example, chain-termination methods, rapid DNA sequencing methods, wandering-spot analysis, Maxam-Gilbert sequencing, dye- terminator sequencing, or using any other modern automated DNA sequencing instruments.

[0053] As used herein, the term "universal" refers to a nucleic acid molecule (e.g., primer or other oligonucleotide) that can be added to any nucleic acid molecule and perform its function irrespectively of the sequence of the nucleic acid molecule. The universal molecule may perform its function by hybridizing to the complement, e.g., a universal primer to a universal primer binding site in a universal primer.

[0054] As used herein, the phrase "universal adapter" refers to a DNA molecule containing a universal primer binding site or a universal primer binding sequence that may be covalently linked to the 5' and 3' ends of one or more nucleic acid molecules, such as double stranded nucleic acid molecules. The addition of the universal adapters provides universal priming sequences to the 5' and 3' ends of the one or more nucleic acid molecules from which PCR amplification may take place, amplifying each of the one or more nucleic acid molecules using a single pair of amplification primers.

[0055] As used herein, the phrase "universal primer binding site" refers to a region or site including universal sequence that can be used for amplifying and / or sequencing a nucleic acid molecule (or a fragment thereof) ligated to a universal adapter.

[0056] As used herein, the phrase "universal primer binding sequence" refers to a DNA sequence that may be appended to one or more nucleic acid molecules, for example by ligation, PCR,or ligation mediated PCR. Once added to the one or more nucleic acid molecules, primers specific to the universal primer binding sequence can be used to amplify the one or more nucleic acid molecules using a single pair of amplification primers. Universal priming sequences are typically not related to target sequences within one or more nucleic acid molecules.

[0057] OVERVIEW

[0058] The present disclosure provides for methods of preparing nucleic acid libraries for sequencing. In particular, the present disclosure is directed to a workflow which employs a first round of amplification utilizing a universal polymerase chain reaction approach (FIG. 1) instead of a platform specific indexed polymerase chain reaction approach (FIG. 2). For instance, and with reference to FIG. 1, the present disclosure employs a universal workflow for library preparation and target enrichment of nucleic acid molecules, where universal primers are utilized in a first round of amplification (e.g., polymerase chain reaction); and where a second round of amplification utilizes platform specific indexed primers, i.e., indexes and sequencer-specific sequences are added by polymerase chain reaction in a second round of PCR (see FIG. 1). Alternatively, two initial rounds of amplification may be performed where the first and second rounds of amplification utilize universal primers; and where a third round of amplification utilizes platform specific primers. For instance, a first round of amplification may be performed with universal primers, a second round of amplification may be performed with universal primers (such as after target enrichment), and a third round of amplification may be performed with platform specific index primers.

[0059] The universal polymerase chain reaction approach differs from the approach set forth in FIG. 2 (a platform specific indexed polymerase chain reaction approach) where indexes and sequencer-specific sequences are utilized in the first round of polymerase chain reaction. Applicant has surprisingly discovered that the presently disclosed workflow (FIG. 1) increases duplex recovery and / or genomic equivalent recovery as compared with standard workflows (FIG. 2).

[0060] The universal workflow of the present disclosure is further illustrated in FIG. 3. In some embodiments, a sample comprising one or more nucleic acid molecules is obtained (step 101); and where one or more universal adapters are ligated to one or more nucleic acid molecules within the obtained sample to provide one or more adapter ligated nucleic acid molecules (step 102). Following the ligation of the one or more universal adapters to the one or more nucleic acid molecules (step 102), the one or more universal adapter ligated nucleic acid molecules are amplified (step 103) with a set of universal primers specific for the universal primer binding sites within the ligated universal adapters to provide a sample including one or more amplified nucleic acid molecules. Next,the sample including the one or more amplified nucleic acid molecules is enriched for one or more target nucleic acid molecules (step 104). During the step of enrichment, non- target nucleic acid molecules are removed from the sample to provide for a sample enriched with amplified target nucleic acid molecules. Finally, a second round of amplification is performed utilized indexed, platformspecific primers to provide a nucleic acid library for sequencing (step 105), such as sequencing using next-generation sequencing (step 106). In some embodiments, the method may comprise one or more additional rounds of amplification. For instance, an additional round of amplification may be performed between steps 103 and 105, where the additional round of amplification may utilize universal primers. In some embodiments, the additional round of amplification is performed after target enrichment.

[0061] These and other aspects are described in further detail herein.

[0062] SAMPLE PREPARATION

[0063] In some embodiments, a sample comprising one or more nucleic acid molecules is obtained (step 101) and prepared for downstream processing. In some embodiments, samples may be obtained from any source including a target nucleic acid molecule having one or more modified nucleotides, e.g., tissue (including tumor tissue or formalin-fixed paraffin- embedded (FFPE) tissue), blood, skin, swab (e.g., buccal, vaginal), urine, saliva, etc. In some embodiments, the sample is a liquid biopsy sample.

[0064] In some embodiments, the sample is derived from a subject or a patient, such as a subject or a patient diagnosed with a disease or suspected of having a disease. In some embodiments, the sample may include a fragment of a solid tissue, or a tumor sample derived from the subject or the patient, e.g., by biopsy. As used herein, the term "tumor sample" encompasses samples prepared from a tumor or from a sample potentially including or suspected of comprising cancer cells, or to be tested for the potential presence of cancer cells, such as a lymph node. As used herein, the term "tumor" refers to a mass or a neoplasm, which itself is defined as an abnormal new growth of cells that usually grow more rapidly than normal cells and will continue to grow if not treated sometimes resulting in damage to adjacent structures. Tumor sizes can vary widely. A tumor may be solid, or fluid filled. A tumor can refer to benign (not malignant, generally harmless), or malignant (capable of metastasis) growths. Some tumors can include neoplastic cells that are benign (such as carcinoma in situ) and, simultaneously, contain malignant cancer cells (such as adenocarcinoma). This should be understood to include neoplasms found in multiple locations throughout the body. Therefore, for purposes of thepresent disclosure, tumors include primary tumors, lymph nodes, lymphatic tissue, and metastatic tumors.

[0065] Methods for isolating nucleic acid molecules from obtained samples and / or purifying the obtained samples are known (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001) and several kits are commercially available (e.g., High Pure RNA Isolation Kit, High Pure Viral Nucleic Acid Kit, and MagNA Pure LC Total Nucleic Acid Isolation Kit, DNA Isolation Kit for Cells and Tissues, DNA Isolation Kit for Mammalian Blood, High Pure FFPET DNA Isolation Kit, available from Roche). In the context of the presently disclosed methods, nucleic acid molecules, including genomic DNA, can be collected, purified, and / or isolated.

[0066] It will be appreciated that nucleic acid molecules may be isolated from obtained samples using any of a variety of procedures known in the art, for example, MagMAX™ DNA MultiSample Ultra Kit (Applied Biosystems, Thermo Fisher Scientific), the MagMAX™ Express-96 Magnetic Particle Processor and the KingFisher™ Flex Magnetic Particle Processor (Thermo Fisher Scientific), a RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE and PureLink™ FFPE RNA Isolation Kit (Ambion™, Thermo Fisher Scientific), the ABI Prism™ 6100 Nucleic Acid PrepStation and the ABI Prism™ 6700 Automated Nucleic Acid Workstation (Applied Biosystems, Thermo Fisher Scientific), and the like.

[0067] In some embodiments, the nucleic acid molecules within the obtained sample are selected from DNA molecules, genomic DNA molecules, cfDNA molecules, ctDNA molecules, cDNA molecules, RNA molecules, mRNA molecules, rRNA molecules, mtDNA, siRNA molecules, or any combination thereof. In some embodiments, the plurality of nucleic acid molecules comprises single stranded polynucleotides. In some embodiments, the nucleic acid molecules are cfDNA molecules. In some embodiments, the nucleic acid molecules are ctDNA molecules. In some embodiments, the nucleic acid molecules are cfDNA molecules; and the obtained sample is a liquid biopsy sample.

[0068] In some embodiments, the nucleic acid molecules within the obtained sample may be prepared for downstream processing (e.g., the ligation of one or more universal adapters) by fragmenting, cutting, or shearing the nucleic acids. In some embodiments, the fragmenting, cutting, and / or shearing may be accomplished using such procedures as mechanical force, sonication, restriction endonuclease cleavage, or any method known in the art. In other embodiments, nofragmentation is necessary (some genomic samples may already consist of appropriately sized fragments and will not require additional fragmentation).

[0069] In some embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 10 mer to about 1000 mer. In some embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 10 mer to about 550 mer. In other embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 15 mer to about 500 mer. In yet other embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 15 mer to about 450 mer. In further embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 15 mer to about 400 mer. In even further embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 15 mer to about 350 mer. In yet even further embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 15 mer to about 300 mer. In some embodiments, the nucleic acid molecules within the sample are fragmented to a platform-specific size range.

[0070] Following the fragmentation of the sample, in some embodiments, the fragmented nucleic acid molecules are end repaired and then a "tailing" reaction is performed. Tailing is an enzymatic method for adding a non-templated nucleotide to the 3' end of a blunt, double-stranded DNA molecule. In some embodiments, a Taq polymerase is utilized for A-tailing.

[0071] LIGATION OF UNIVERSAL ADAPTERS

[0072] After end-repair tailing, one or more universal adapters are ligated to the one or more nucleic acid molecules within the obtained sample to provide adapter ligated nucleic acid molecules (step 102). In some embodiments, the universal adapters of the present disclosure are adapters that are not specific to any sequencing instrument, i.e., the universal adapters of the present disclosure are not "platform-specific" adapters.

[0073] In some embodiments, the universal adapters of the present disclosure include (i) one or more unique molecular identifiers (UMI) and, and (ii) a universal primer binding site (UPBS). Both universal primers and platform-specific primers (each described in further detail herein) hybridize to at least a portion of the UPBS within the universal adapter.

[0074] In some embodiments, the universal adapters have the generic formula:5' - [UPBS] - [UMI] - 3'

[0075] In some embodiments, the universal adapter is substantially non-complementary to the 3' end or the 5' end of any nucleic acid molecule present in the obtained sample, facilitating its ligation to the nucleic acid molecules within the obtained sample.

[0076] In some embodiments, the universal adapters include a complementary region (forming a hybridized double-stranded region) and a non-complementary region (single-stranded). In some embodiments, the universal adapter is "Y" shaped including non-self-complementary singlestranded regions, in addition to single-stranded regions, where such adapters include a doublestranded region suitable for coupling (e.g., ligation) to double-stranded nucleic acid molecules. Such adapters are described in U.S. Pat. No. 6,346,399, U.S. Pat. No. 7,741,463, US Patent application US 2007 / 0172839, and International Publication No. W02007 / 111937; the disclosures of which are hereby incorporated by reference herein in their entireties.

[0077] Universal primer binding sites may be located in the single-stranded section, the double-stranded section, or a combination of both sections in embodiments of universal adapters including complementary and non-complementary regions. In some embodiments, the universal adapters include a blunt end for coupling (e.g., ligation). In other embodiments, the universal adapters include a sticky end for coupling (e.g., ligation).

[0078] In some embodiments, the universal adapters having a length ranging from about 10 to about 100 nucleotides, such as from about 10 to about 60 nucleotides, such as from about 10 to about 50 nucleotides, such as from about 12 to about 50 nucleotides, such as from about 15 to about 50 nucleotides.

[0079] A UMI is a barcode that identifies a nucleic acid molecule to which it is attached. As used herein, the term "barcode" refers to a nucleic acid sequence that can be detected and identified. In some embodiments, the barcodes include between about 5 and about 20 nucleotides, such that in a sample, the nucleic acids incorporating the barcodes can be distinguished or grouped according to the barcodes. In some embodiments, the barcodes include between about 5 and about 15 nucleotides. In some embodiments, the barcodes include between about 5 and about 10 nucleotides. In some embodiments, the barcodes include between about 10 and about 15 nucleotides. In some embodiments, the barcodes include about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 nucleotides. Non-limiting examples of barcodes and / or unique molecular identifiers (UMIs) are described in U.S. Publication No. 2020 / 0032244, and in U.S. Patent Nos. 7,393,665,8,168,385, 8,481,292, 8,685,678, and 8,722,368, and in PCT Publication No. WO / 2018 / 138237, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0080] In some embodiments, UMIs may be incorporated as part of an overall DNA amplification and sequencing workflow to perform error correction. In some embodiments, errors are introduced (1) by the polymerase during amplification, and (2) during sequencing (i.e., reading) of the amplified molecules. In some embodiments, UMIs ligated to nucleic acid molecules reduce the impact of one or both sources of error. For instance, UMIs incorporate a unique barcode onto each molecule within a given sample library. By incorporating individual barcodes on each original DNA fragment, variant alleles present in the original sample (true variants) can be distinguished from errors introduced during library preparation, target enrichment, or sequencing.

[0081] In some embodiments, the UMIs have the one of the general Formulas set forth below: (W)(N)(N)(N)(N)(N)(W)(N)(N)(N)(N)(N)(W), or (N)(W)(N)(N)(N)(W)(N)(N)(N),

[0082] where N includes (in the aggregate) about 25% adenosine; about 25% guanine; about 25% cytosine; and about 25% thymine; and W includes (in the aggregate) about 50% adenosine and about 50% thymine.

[0083] The coupling of a universal adapter to a nucleic acid molecule may be achieved through ligation or via tagmentation using transposase complexes (see, for example, International Publication No. WO / 2016 / 130704, the disclosure of which is hereby incorporated by reference herein in its entirety). Methods of ligating adapters to a nucleic acid molecule are described in U.S. Patent Publication Nos. 2017 / 0037459, 2018 / 0334709, 2018 / 0016630 and in PCT Publication No. WO2017021449, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0084] In some embodiments, universal adapters are ligated to the nucleic acid molecules with any suitable ligase, such as T4 ligase. In some embodiments, the ligase is a modified T4 ligase, a mutant of a T4 ligase, or a variant of a T4 ligase (collectively referred to herein as a "modified T4 ligase"), such as those disclosed in International Publication No. WO / 2024 / 123733, in U.S. Patent No. 10,837,009, or in U.S. Publication No. 2018 / 0320162, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0085] The coupling of the universal adapter complexes provides an adapter ligated nucleic acid molecule having the generic structure recited below:

[0086] 5' - ([UPBS] - [UMI]) - [Nucleic Acid Molecule] - ([UMI] - [UPBS]) - 3'

[0087] The skilled artisan will appreciate that the nucleic acid molecules are ligated to a pair of universal adapters including universal primer binding sites, where the universal primer binding sites are oriented so as to enable the amplification of the nucleic acid molecule located between the universal adapters.

[0088] FIRST ROUND OF AMPLIFICATION UTILIZING UNIVERSAL PRIMERS

[0089] Following the coupling, e.g., ligation, of the one or more universal adapters to the one or more nucleic acid molecules (step 102), the one or more universal adapter ligated nucleic acid molecules are amplified (step 103) with a set of universal primers specific for the universal primer binding sites within the universal adapters. In some embodiments, amplification of the one or more universal adapter ligated nucleic acid molecules is by polymerase chain reaction.

[0090] In some embodiments, a pool of universal primers is introduced to the sample including the adapter ligated nucleic acid molecules, where the introduced universal primers bind to and direct primer extension from the universal priming site. In some embodiments, the universal primer has exact complementarity with a portion of the universal primer binding site. In other embodiments, the universal primers have less than exact complementarity with the universal primer binding site of the universal adapter (e.g., less than 99% complementarity, less than 97% complementarity, less than 96% complementarity, less than 94% complementarity, less than 92% complementarity, less than 90% complementarity, less than 85% complementarity, less than 80% complementarity) provided that the universal primer can hybridize sufficiently with the universal primer binding site so as to be extendible by a DNA polymerase.

[0091] In some embodiments, amplification of the one or more adapter ligated nucleic acid molecules is based on template directed oligonucleotide primer extension using one or more polymerases. For instance, the sample including the one or more adapter ligated nucleic acid molecules is contacted with a polymerase and / or other amplification reagents to provide one or more amplified nucleic acid molecules (step 103).

[0092] Non-limiting examples of polymerases include prokaryotic DNA polymerases (e.g., Pol I, Pol II, Pol III, Pol IV, and Pol V), eukaryotic DNA polymerase, archaeal DNA polymerase, etc. In some embodiments, suitable polymerases may be derived from: archaea (e.g., Thermococcus litoralis (Vent, GenBank: AAA72101), Pyrococcus furiosus (Pfu, GenBank: D12983, BAA02362), Pyrococcus woesii, Pyrococcus GB-D (Deep Vent, GenBank: AAA67131), Thermococcus kodakaraensis KODI (KOD, GenBank: BD175553, BAA06142; Thermococcus sp. strain KOD (Pfx, GenBank: AAE68738)), Thermococcus gorgonarius (Tgo, Pdb: 4699806), Sulfolobus solataricus(GenBank: NC002754, P26811), Aeropyrum pernix (GenBank: BAA81109), Archaeglobus fulgidus (GenBank: 029753), Pyrobaculum aerophilum (GenBank: AAL63952), Pyrodictium occultum (GenBank: BAA07579, BAA07580), Thermococcus 9 degree Nm (GenBank: AAA88769, Q56366), Thermococcus fumicolans (GenBank: CAA93738, P74918), Thermococcus hydrothermalis (GenBank: CAC18555), Thermococcus sp. GE8 (GenBank: CAC12850), Thermococcus sp. JDF-3 (GenBank: AX135456; WOOl 32887), Thermococcus sp. TY (GenBank: CAA73475), Pyrococcus abyssi (GenBank: P77916), Pyrococcus glycovorans (GenBank: CAC12849), Pyrococcus horikoshii (GenBank: NP 143776), Pyrococcus sp. GE23 (GenBank: CAA90887), Pyrococcus sp. ST700 (GenBank: CAC 12847), Thermococcus pacificus (GenBank: AX411312.1), Thermococcus zilligii (GenBank: DQ3366890), Thermococcus aggregans, Thermococcus barossii, Thermococcus celer (GenBank: DD259850.1), Thermococcus profundus (GenBank: E14137), Thermococcus siculi (GenBank: DD259857.1), Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfolobus acidocaldarium, Sulfolobus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum (GenBank: AAF27815), Methanococcus jannaschii (GenBank: Q58295), Desulforococcus species TOK, Desulforococcus, Pyrolobus, Pyrodictium, Staphylothermus, Vulcanisaetta, Methanococcus (GenBank: P52025) and other archaeal B polymerases, such as GenBank AAC62712, P956901, BAAA07579)), thermophilic bacteria Thermus species (e.g., flavus, ruber, thermophilus, lacteus, rubens, aquaticus), Bacillus stearothermophilus, Thermotoga maritima, Methanothermus fervidus, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, phi29, Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp. GT, P. sp. GB-D, KOD, Pfu, T. gorgonarius, T. zilligii, T. litoralis and Thermococcus sp. 9N-7 polymerases.

[0093] To effectuate amplification, the one or more adapter ligated nucleic acid molecules are heat denatured. Melting temperatures for heat denaturation are dependent upon several variables including the GC content of the nucleic acid molecule and / or the size of the nucleic acid molecule, but in general may be about 95°C or higher, such as for about 15 seconds to about 2 minutes. Following heat denaturation, the universal primers are annealed to the universal primer binding sites of the one or more adapter ligated nucleic acid molecules at a lower temperature, (typically between about 40°C and about 60°C, such as for about 30 to about 60 seconds). The annealing temperature, like the heat denaturation temperature, is dependent upon the GC content and / or length of the primers. Subsequently, a corresponding nucleic acid strand to the template is synthesized from the universal primer through use of the polymerase and deoxynucleotide triphosphates (dNTPs) (also referred to as "primer extension"). In some embodiments, the temperature is raised for the polymerase, which inthe case of commonly used thermostable polymerases is about 74° C, primer extension then lasts approximately 1 to 2 minutes. Reactions take place in a PCR master mixture which includes the adapter ligated nucleic acid molecule, a polymerase, universal primers, deoxynucleotide triphosphates (dNTPs), reaction buffer, magnesium and / or optional additives.

[0094] TARGET ENRICHMENT

[0095] Following the preparation of the amplified nucleic acid molecules (step 103), the sample including the amplified nucleic acid molecules is enriched for one or more target nucleic acid molecules (step 104). During the step of enrichment, non-target nucleic acid molecules are removed from the sample to provide for an enriched sample.

[0096] Any method may be utilized to enrich the prepared sample for the presence of one or more target nucleic acid molecules. In some embodiments, a hybridization-based target enrichment workflow may be utilized to enrich the prepared sample. In hybridization-based target enrichment workflows, a target area of a target nucleic acid molecule is captured by one or more hybridization probes that can selectively bind to a capture surface. This capture allows the removal of non-target nucleic acids and subsequent release and collection of captured target molecules. Hybridization of target regions may occur either on a solid surface (microarray) or in solution. Hybridization-based target enrichment workflows are described in United States Patent No. 8,383,338, the disclosure of which is hereby incorporated by reference herein in its entirety. Commercial hybridization-based target enrichment workflows are available from Roche Sequencing Solutions, Inc. (e.g., KAPA HyperCap Workflow). Other commercial hybridization-based target enrichment workflows include SECAP EZ Target Enrichment System (ROCHE) and SURESELECT Target Enrichment System (AGILENT).

[0097] By way of example, hybridization-based target enrichment may be performed by capturing the target nucleic acid molecules in a sample with one or more introduced target-specific probes. In some embodiments, the one or more target nucleic molecules in an obtained sample may be denatured and contacted with single-stranded target-specific probes. In some embodiments, the single-stranded target-specific probes may comprise a ligand for an affinity capture moiety such that following the formation of hybridization complexes, the hybridization complexes are captured by contacting the sample with the affinity capture moiety. In some embodiments, the affinity capture moiety is avidin or streptavidin and the ligand is biotin. In some embodiments, the moiety is bound to solid support. In some embodiments, the solid support may comprise superparamagnetic spherical polymer particles such as DYNABEADS™ magnetic beads or magnetic glass particles.

[0098] In other embodiments, a primer extension target enrichment (PETE) workflow may be utilized to enrich the prepared sample. PETE workflows are described in United Patent Application Publication Nos. 2021 / 0207211 and 2020 / 0392483; in United States Patent Nos. 10,907,204 and 11,499,180; and in International Publication Nos. WO / 2018 / 013710 and WO / 2022 / 008578, the disclosures of which are each incorporated by reference herein in their entireties. Commercial PETE workflows are available from Roche (e.g., HAPA HyperPETE Workflow). By way of example only, a PETE workflow may be utilized to enrich a sample with one or more target nucleic acid molecules by: a) providing a reaction mixture comprising the sample and a first target-specific primer, wherein the sample comprises a single-stranded target nucleic acid molecule having a 3' and a 5' end and nontarget nucleic acid molecules; b) hybridizing a first target-specific primer to the single-stranded target nucleic acid molecules in the reaction mixture, wherein the first target-specific primer hybridizes at least 6 nucleotides from the 3' end of the single-stranded target nucleic acid molecule and comprises an affinity ligand; c) extending the hybridized first target-specific primer with a DNA polymerase to form a first double-stranded product comprising the target nucleic acid molecule hybridized to the extended first target-specific primer, wherein the hybridized target nucleic acid molecule comprises a single-stranded overhang region of at least 6 consecutive nucleotides at the 3' end; d) removing singlestranded target and non-target nucleic acid molecules from the reaction mixture by capturing the affinity ligand of the first double-stranded product; e) hybridizing a second target-specific primer to the single-stranded overhang region at the 3' end of the hybridized target polynucleotide of the captured first double stranded product, wherein the second target-specific primer comprises a 3' hybridizing region and a barcode region; and f) extending the hybridized second target-specific primer with a DNA polymerase, wherein the DNA polymerase comprises strand displacement activity, 5 '-3' double stranded DNA exonuclease activity, or a combination thereof, thereby displacing or degrading the extended first target-specific primer and forming a second double-stranded product comprising a barcode, wherein the second double-stranded product comprises the target nucleic acid molecule hybridized to an extended second target-specific primer, wherein the extended second target- specific primer comprises: i) a complement of at least a portion of the target nucleic acid molecule; and, ii) a single-stranded 5' overhang region comprising the barcode.

[0099] One exemplary method of enriching a sample for one or more target nucleic acid molecules includes the following steps. An obtained sample is contacted with one or more oligonucleotide probes. In some embodiments, the one or more oligonucleotide probes comprise reference nucleic acid sequences capable of hybridizing to complementary target nucleic acidsequences within one or more target nucleic acid molecules, and wherein the oligonucleotide probes comprise a first member of a pair of specific binding entities. In some embodiments, the oligonucleotide probes are designed to target desired genes, exons, and / or other regions of interest within the one or more target nucleic acid molecules. In some embodiments, the oligonucleotide probes are selected such that the oligonucleotide probes relate to, by way of non-limiting examples, a set of genes of interest, all the exons of a genome, particular genetic regions of interest, disease, or physiological states and the like.

[0100] In some embodiments, the oligonucleotide probes are DNA capture probes. In some embodiments, the DNA capture probes include a pool of Roche SeqCap EZ Probes (available from Roche Sequencing and Life Sciences, Indianapolis, IND). In some embodiments, a pool of Roche SeqCap EZ Probes include a mixture of different biotinylated single-stranded DNA oligonucleotides in solution, each with a specific sequence, where the length of individual oligonucleotides can range from about 50 nucleotides to about 100 nucleotides with a typical size of about 75 nucleotides. In some embodiments, a Roche SeqCap EZ Probe Pool can be used in sequence capture experiments to hybridize to targeted complementary fragments of a DNA sequencing library and thus to capture and enrich them relative to untargeted fragments of the same DNA sequencing library prior to sequencing. The DNA sequencing library may be constructed from genomic DNA for genome analysis, or from cDNA prepared from RNA or mRNA for transcriptome analysis, and it may be constructed from the DNA or cDNA of any species of organism from which these nucleic acids can be extracted.

[0101] In some embodiments, the oligonucleotide probes hybridize to complementary nucleic acid sequences within the one or more target nucleic acid molecules in the sample which include the desired genes, exons, and / or other genomic regions of interest to form target-probe complexes having a first member of a pair of specific binding entities. In some embodiments, one or more non-target nucleic acid molecules in the obtained sample that do not include the desired genes, exons, and / or other genomic regions of interest do not form target-probe complexes. As such, following the introduction of the oligonucleotide probes, the sample (or any solution derived from the sample) may include formed target-probe complexes, non-target nucleic acid molecules, and / or free probes (assuming that an excess amount of oligonucleotide probes are provided to any solution including adapter-ligated DNA fragments).

[0102] In some embodiments, the sample is further contacted with one or more universal oligonucleotide blockers. Oligonucleotide blockers are short pieces of DNA that are designed to hybridize to any nucleic acid molecule. They are typically used in target enrichment experiments toprevent non-target nucleic acids from binding to the oligonucleotide probes. In some embodiments, the sample is first hybridized with oligonucleotide probes that are designed to hybridize to the desired genes, exons, and / or other genomic regions of interest (e.g., Poly(dT) blockers that are specifically designed to bind to the poly(A) tails). In some embodiments, the non-target nucleic acid molecules in the sample will not form target-probe complexes. However, they could still bind to the oligonucleotide probes, which would interfere with the sequencing process. To prevent this, the sample is further contacted with one or more universal blockers. The universal blockers will bind to any non-target nucleic acid molecules in the sample, preventing them from binding to the oligonucleotide probes.

[0103] In some embodiments, the formed target-probe complexes are then bound to a solid support. For instance, in some embodiments the sample is contacted with a solution including one or more functionalized beads, where the functionalized beads include a second member of the pair of specific binding entities. In some embodiments, the first members of the pair of specific binding entities of the target-probe complexes react with the second members of the pair of specific binding entities of the functionalized beads such that the target-probe complexes within the solution for enrichment become bound to the functionalized beads. Likewise, in some embodiments the first members of the pair of specific binding entities of any free probes in the solution for enrichment become bound to the functionalized beads. In this manner, the target-probe complexes and / or free probes become bound to the beads within the chamber. As such, the beads within the sample include target-probe complexes and free probes. Also included within the sample, in some embodiments, are unbound, non-target nucleic acid molecules.

[0104] Following the binding of the target-probe complexes to the functionalized beads and / or the binding of free-probes to the beads, unbound non-target nucleic acid molecules, reagents, and / or impurities in the sample are then removed (step 304). In some embodiments, the non-target nucleic acid molecules, reagents, and / or impurities are removed by washing the sample one or more times (such as two or more times, such as three or more times, etc.), such as with a buffer or other was reagent. Non-liming examples of buffers include citric acid, potassium dihydrogen phosphate, boric acid, diethyl barbituric acid, piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsinic acid, 2-(N- morpholino)ethanesulfonic acid, tris(hydroxymethyl)methylamine (TRIS), 2-(N- morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine(Bicine), N- tris(hydroxymethyl)methylglycine (Tri cine), 4-2-hy dr oxy ethyl- 1 -piperazineethanesulfonic acid (HEPES), 2- {[tris(hydroxymethyl)methyl]amino} ethanesulfonic acid (TES), and combinationsthereof. In other embodiments, the buffer may be comprised of tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine(Bicine), N - tris(hydroxymethyl)methylglycine (Tri cine), 4-2-hy dr oxy ethyl- 1 -piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), or a combination thereof. In some embodiments, removal of the non-target nucleic acid molecules that were not complementary to any the oligonucleotide probes introduced to the sample enriches the sample for the presence of immobilized target nucleic acid molecules.

[0105] Following the removal of substantially all non-target nucleic acid molecules, reagents, and / or impurities from the sample, the target molecules are, released from the solid support (e.g., beads) to provide an enriched sample. In some embodiments, the target nucleic acid molecule is released by contacting the bead bound target-probe complexes with a fluid or reagent suitable for releasing the target nucleic acid molecule from the bead. In some embodiments, the target molecules are removed by introducing a heated buffer to the sample. In other embodiments, a reagent (e.g., an enzyme) is introduced to effectuate release. Examples of suitable enzymes include trypsin (which cleaves the peptide bonds at the carboxyl end of lysine and arginine residues) and clostripain (which cleaves at the carboxyl side of arginine residues).

[0106] Following release of the target nucleic acid molecules from the beads, the target nucleic molecules are single stranded. In some embodiments, a second strand synthesis is conducted to form double stranded target nucleic acid molecules which may be used for downstream processes. In some embodiments, the second strand is synthesized via a primer extension method, such as described in United States Patent Application No. 2020 / 0032244. For instance, a primer may be hybridized to a portion of the single stranded target nucleic acid molecule and the hybridized primer may be extended via an enzyme (e.g., a polymerase) to provide a double stranded target nucleic acid molecule. The term "extend," or any grammatical equivalents thereof refers to the addition of dNTPs to a primer, polynucleotide, or other nucleic acid molecule by an extension enzyme, such as a polymerase. The step of second-strand synthesis does not increase a copy number of any of the single stranded and / or double stranded target nucleic acid molecules, i.e., the step of second-strand synthesis does not result in amplification of any of the target nucleic acid molecules following enrichment.

[0107] SUBSEQUENT ROUND OF AMPLIFICATION UTILIZING INDEXED, PLATFORM-SPECIFIC PRIMERS

[0108] Following the preparation of an enriched sample comprising one or more target nucleic acid molecules, a subsequent round of amplification is performed utilizing platform-specific primers to provide a nucleic acid library suitable for sequencing, such as with a next-generation sequencing platform (e.g., a sequencing-by-synthesis platform) (step 105). In some embodiments, the subsequent round of amplification utilizing indexed, platform- specific primers is a second round of amplification. In other embodiments, the subsequent round of amplification utilizing indexed, platform-specific primers is a third round of amplification. In those embodiments where the subsequent round of amplification is a third round of amplification, a second round of amplification may utilize universal primers (while the third round of amplification utilizes indexed, platform-specific primers).

[0109] In some embodiments, the platform-specific primers comprise a sample identifier or index sequence which, when incorporated into a nucleic acid molecule, serves as characteristic marker of a sample from which the nucleic acid molecule was derived. In some embodiments, the sample identifier or index sequence allows nucleic acids from different sources or samples to be mixed and sequenced simultaneously. The index sequence may have any length, e.g., 4 mer, 6 mer, 8 mer, 12 mer, 16 mer, 20 mer, 24 mer, etc. Examples sample identifiers or index sequences are described in U.S. Publication No. 2020 / 0032244, and in U.S. Patent Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678, and 8,722,368, and in PCT Publication No. WO / 2018 / 138237, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0110] In some embodiments, the platform-specific primers further include a platformspecific anchoring site. For instance, the platform- specific primers include one or more sequences which, when incorporated into a nucleic acid molecule, serve to either facilitate the attachment of the nucleic acid molecule to a flow cell or to ensure platform- specific compatibility. In other embodiments, the platform-specific primers include a platform-specific anchoring site and a sequencing primer binding site.

[0111] When the one or more target nucleic acid molecules are amplified using the platformspecific primer, the indexing sequencing, the platform- specific anchoring site, and / or the sequencing primer binding site are incorporated into the one or more target nucleic acid molecules (see, e.g., FIG. 1).

[0112] In some embodiments, a pool of platform-specific primers is introduced to the target enriched sample where the introduced platform-specific primers bind to and direct primer extension from the universal priming site within the universal adapter. In some embodiments, the platformspecific primers have exact complementarity with a portion of the primer site within the universaladapter. In other embodiments, the platform-specific primers have less than exact complementarity with the primer binding site within the universal adapter (e.g., less than 99% complementarity, less than 97% complementarity, less than 96% complementarity, less than 94% complementarity, less than 92% complementarity, less than 90% complementarity, less than 85% complementarity, less than 80% complementarity) provided that the platform-specific primer can hybridize sufficiently with the primer binding site so as to be extendible by a DNA polymerase.

[0113] To effectuate amplification, the amplified target nucleic acid molecules in the enriched sample are heat denatured. Following heat denaturation, the platform-specific primers are annealed to the primer binding sites of the amplified target nucleic acid molecules at a lower temperature, (typically between about 40°C and about 60°C, such as for about 30 to about 60 seconds). Subsequently, a corresponding nucleic acid strand to the template is synthesized from the platform- speciifc primer through use of the polymerase and deoxynucleotide triphosphates (dNTPs) (also referred to as "primer extension"). In some embodiments, the temperature is raised for the polymerase, which in the case of commonly used thermostable polymerases is about 74° C, primer extension then lasts approximately 1 to 2 minutes. Reactions take place in a PCR master mixture which includes the amplified target nucleic acid molecule, a polymerase, platform, -specific primers, deoxynucleotide triphosphates (dNTPs), reaction buffer, magnesium and / or optional additives.

[0114] SEQUENCING

[0115] Following the preparation of the nucleic acid library, the nucleic acid molecules with the library are prepared for sequencing and then sequenced. In some embodiments, sequencing methods include Sanger sequencing and dye-terminator sequencing, as well as next-generation sequencing technologies such as pyrosequencing, nanopore sequencing, micropore-based sequencing, nanoball sequencing, MPSS, SOLiD, Illumina, Ion Torrent, Starlite, SMRT, tSMS, sequencing by synthesis, sequencing by ligation, mass spectrometry sequencing, polymerase sequencing, RNA polymerase (RNAP) sequencing, microscopy-based sequencing, microfluidic Sanger sequencing, microscopy-based sequencing, RNAP sequencing, etc. Instruments and methods of sequencing are disclosed, for example, in PCT Publication Nos. WO2014144478, W02015058093, W02014106076 and WO2013068528, the disclosures of which are hereby incorporated by reference in their entireties.

[0100] Sequencing by synthesis is defined as any sequencing method which monitors the generation of side products upon incorporation of a specific deoxynucleoside-triphosphate during the sequencing reaction (Hyman, 1988, Anal. Biochem. 174:423-436; Rhonaghi et al., 1998, Science281:363-365). An example of a sequencing by synthesis reaction is the pyrophosphate sequencing method. In this method, the generation of a pyrophosphate during nucleotide incorporation is monitored by an enzymatic cascade which results in the generation of a chemo-luminescent signal. The 454 Genome Sequencer System (Roche Applied Science cat. No. 04 760 085 001), an example of sequence by synthesis, is based on the pyrophosphate sequencing technology. For sequencing on a 454 GS20 or 454 FLX instrument, the average genomic DNA fragment size is in the range of 200 or 600 bp, respectively, as described in the product literature.

[0101] In some embodiments, a sequencing by synthesis reaction can alternatively be based on a terminator dye type of sequencing reaction. In this case, the incorporated dye deoxynucleotriphosphates (ddNTPs) building blocks comprise a detectable label, which is preferably a fluorescent label that prevents further extension of the nascent DNA strand. The label is then removed and detected upon incorporation of the ddNTP building block into the template / primer extension hybrid for example by using a DNA polymerase comprising a 3 '-5' exonuclease or proofreading activity.

[0116] In some embodiments, and in the case of the Genome Sequencer workflow (Roche Applied Science Catalog No. 04 896 548 001), in a first step, (clonal) amplification is performed by emulsion PCR. Thus, it is also within the scope of the present disclosure, that the step of amplification is performed by emulsion PCR methods. The beads carrying the clonally amplified target nucleic acid molecules may then become arbitrarily transferred into a picotiter plate according to the manufacturer's protocol and subjected to a pyrophosphate sequencing reaction for sequence determination.

[0117] In some embodiments, sequencing is performed using a next-generation sequencing method such as that provided by Illumina, Inc. (the "Illumina Sequencing Method"). Without wishing to be bound by any particular theory, the Illumina next-generation sequencing technology uses clonal amplification and sequencing by synthesis (SBS) chemistry to enable rapid, accurate sequencing. The process simultaneously identifies DNA bases while incorporating them into a nucleic acid chain. Each base emits a unique fluorescent signal as it is added to the growing strand, which is used to determine the order of the DNA sequence.

[0118] Examples of sequencing-by-synthesis platforms include the Pacific BioSciences platform utilizing SMRT sequencing (Pacific Biosciences, Menlo Park, CA) or a platform utilizing nanopore technology such as those manufactured by Oxford Nanopore Technologies (Oxford, UK) or Roche Sequencing Solutions (Genia) (Santa Clara, Calif.).

[0119] EXAMPLE

[0120] Materials

[0121] cfDNA extracted from normal healthy donors

[0122] AVENIO Expanded Panel

[0123] KAPA HyperPREP Library Preparation Kit

[0124] KAPA HyperCapture Reagent and Bead Kits

[0125] Specialized Universal UMI Adapters

[0126] KAPA UDI Primer Mixes

[0127] Non-platform Specific Universal Primers

[0128] Method

[0129] Library preparation and target enrichment followed the steps detailed in the KAPA HyperCap cfDNA Workflow vl .1 (available from Roche Molecular Systems, Inc.) with the deviations listed below. In some embodiments, there are two Polymerase Chain Reaction (PCR) amplification events in the workflow. The first is after DNA ligation with the adapters (PCR1); and the second is after target enrichment (PCR2). Additionally, a third amplification event (PCR3) can be added if the desire is to generate libraries that have not been indexed with platform specific primers at PCR2. PCR3 is not part of the HyperCap cfDNA workflow.

[0130] Three sets of 24 libraries were made from twelve cfDNA samples (lOng of input, 2 replicates each) using the KAPA HyperPREP Library Preparation Kit. Specialized Universal UMI adapters were ligated onto the samples. All three sets follow the same workflow prior to PCR1. Workflow deviations at the PCR1 step are listed below:

[0131] Set 1 : At PCR1 , the samples were indexed with platform-specific primers (KAPA UDI Primer Mixes.)

[0132] Set 2: At PCR1, the samples were amplified with non-platform specific universal primers. The platform specific KAPA UDI Primer mixes were used to index samples in PCR2.

[0133] Set 3: At PCR1, the samples were amplified with non-platform specific primers. The non-specific platform primers were also used at PCR2. After PCR2, the libraries underwent another 3 rounds of index amplification with the KAPA UDI Primer Mixes. Set 3 allowed for direct comparisons of different sequencing platforms from the same target enrichment libraries.

[0134] The target enrichment steps were the same for all three sets, and all were captured with the probes included in the AVENIO Expanded Panel (available from Roche Molecular Systems, Inc.).

[0135] All of the libraries from the three sets were pooled and sequenced on an Illumina Novaseq using the S4 flowcell. The samples were subsampled to 100M paired end reads and analyzed using a bioinformatics pipeline.

[0136] FIGS. 4 and 5 demonstrate that using a Universal PCR approach at PCR1, and adding indexed, platform-specific primers in PCR2 or 3, results in a boost in duplex recovery and GER. In particular, FIG. 4 illustrates that the Average % Duplex Recovery for libraries where indexed, platform-specific primers were added in different PCR reactions (PCR1 - 3): PCR1 36.2%, PCR2: 43.5%, PCR3: 43.7%. FIG. 5 depicts the Average GE recovery for libraries where indexed, platformspecific primers were added in different PCR reactions (PCR1 - 3): PCR1 : 66%, PCR275.8%, PCR3 : 76.5%

[0137] Although the present disclosure has been described with reference to several illustrative embodiments, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More particularly, reasonable variations and modifications are possible in the component parts and / or arrangements of the subject combination arrangement within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the disclosure. In addition to variations and modifications in the component parts and / or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

- 32 -CLAIMS1. A method of preparing a library comprising one or more target nucleic acid molecules, comprising:(a) obtaining a sample comprising one or more nucleic acid molecules;(b) ligating one or more universal adapters to each of the one or more nucleic acid molecules within the obtained sample to provide one or more universal adapter ligated nucleic acid molecules;(c) performing a first amplification, wherein the first amplification comprises amplifying the one or more universal adapter ligated nucleic acid molecules within the sample with one or more universal primers to provide one or more amplified nucleic acid molecules;(d) enriching the one or more amplified nucleic acid molecules in the sample for one or more target nucleic acid molecules to provide a target enriched sample;(e) performing a second amplification, wherein the second amplification comprises amplifying the one or more target nucleic acid molecules within the target enriched sample with one or more indexed, platform-specific primers to provide the library comprising the one or more target nucleic acid molecules.

2. The method of claim 1 , further comprising sequencing the library comprising the one or more target nucleic acid molecules.

3. The method of claims 1 - 2, wherein the one or more universal primers comprise one or more barcodes.

4. The method of claims 1 - 3, wherein the one or more universal primers comprise a universal primer binding site.

5. A method of preparing a library comprising one or more target nucleic acid molecules, comprising:(a) obtaining a sample comprising one or more nucleic acid molecules;(b) ligating one or more universal adapters to each of the one or more nucleic acid molecules within the obtained sample to provide one or more universal adapter ligated nucleic acid molecules;(c) performing a first amplification, wherein the first amplification comprises amplifying the one or more universal adapter ligated nucleic acid molecules within the- 33 - sample with one or more universal primers to provide one or more amplified nucleic acid molecules;(d) enriching the one or more amplified nucleic acid molecules in the sample for one or more target nucleic acid molecules to provide a target enriched sample;(e) performing a second amplification on the target enriched sample with the one or more universal primers to provide an amplified target enriched sample; and(f) performing a third amplification, wherein the third amplification comprises amplifying the one or more target nucleic acid molecules within the amplified target enriched sample with one or more teed, platform-specific primers to provide the library comprising the one or more target nucleic acid molecules.

6. The method of claim 5, further comprising sequencing the library comprising the one or more target nucleic acid molecules.

7. The method of any one of claims 5 - 6, wherein the one or more universal primers comprise one or more barcodes.

8. The method of any one of claims 5 - 7, wherein the one or more universal primers comprise a universal primer binding site.

9. A kit comprising: (a) one or more universal primers; and (b) one or more indexed, platformspecific primers.

10. The kit of claim 9, wherein the one or more universal primers comprise one or more barcodes and a universal primer binding site.

11. The kit of any one of claims 9- 10, wherein the one or more indexed, platform-specific primers include a sample identifier or index sequence.

12. The kit of any one of claims 9 - 11, wherein one or more indexed, platform-specific primers include a platform-specific anchoring site.

13. The kit of any one of claims 9 - 12, further comprising a polymerase.

14. The kit of claim 13, wherein the polymerase is an archaeal DNA polymerase.

15. The kit of any one of claims 10 - 14, further comprising a ligase.

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