Library quantification and identification

Abstract

Methods, compositions and kits for library quantification and identification are described herein. Some embodiments relate to a method for library quantification. For example, the method may include providing DNA fragments, amplifying the DNA fragments by polymerase chain reaction (PCR) in the presence of primers each labeled with a fluorophore. In these cases, only a predetermined number of fluorophores are attached to each DNA fragment. The method may further include detecting the fluorescent signal generated by the amplified DNA fragments, and calculating the number of amplified DNA fragments based on the detected fluorescent signal.

Description

[0001] Sequence Listing

[0002] The sequence table associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence table is Sequence_listing.txt. The text file is approximately 27KB and is submitted electronically via EFS-Web. Background Art

[0003] Next generation DNA sequencers usually use DNA fragments with known sequence ends. Two ends have known sequences so that DNA fragments can be amplified, immobilized, and provide the starting position (e.g., initiation site) for sequencing. The ends of known sequences are usually referred to as adapters; they adjust DNA fragments according to the needs of the sequencer. Not all DNA fragments in the solution have adapters for sequence determination at each end. The PCR amplification step using two different primers (each primer has specificity for one of the adapters) is usually used for enrichment of fragments with two different adapters at the end. This collection of DNA fragments with adapters at the end is usually referred to as a library. These libraries can be fixed on a solid support so that the spatial distance between library elements (e.g., adapters of different DNA fragments inserted therein) allows visualization (detection) and identification of individual elements to each other.

[0004] The distance between elements becomes more critical as individual elements must be amplified on the surface to increase their number and allow efficient detection of the fluorophore when the fragments are sequenced. This amplification is often referred to as bridge amplification and produces results often referred to as clusters. As the fragments are amplified, clusters of fragments with the same sequence are generated on the support.

[0005] For the sequence of DNA to be determined in the cluster, the cluster is homogeneous and does not include DNA from any other library element. If the clusters are too tight or overlap in extreme cases, the image analysis software may be difficult to distinguish the boundaries of the clusters, and it is difficult to combine them into a single feature for data extraction. Since the data of this cluster come from two different DNA fragments with two different sequences, the software may not be able to accurately measure the sequence. If these clusters are farther apart, each cluster can be analyzed separately and the sequence can be accurately measured. If these clusters are too far apart, sequencing becomes inefficient. The cost of processing samples is fixed, but the cost of each cluster will increase.

[0006] Since the spacing of clusters is determined by the concentration of individual library elements, the concentrations of these library elements need to be accurately determined. Summary of the invention

[0007] Methods, compositions and kits for library quantification and identification are described herein. Some embodiments relate to a method for library quantification. For example, the method may include providing DNA fragments, amplifying the DNA fragments by polymerase chain reaction (PCR) in the presence of primers each labeled with a fluorophore. In these cases, only a predetermined number of fluorophores are connected to each DNA fragment. The method may further include detecting the fluorescent signal generated by the amplified DNA fragments, and calculating the quantity of the amplified DNA fragments based on the detected fluorescent signal.

[0008] In some embodiments, before detecting the signal generated by the amplified DNA fragments, the method may further comprise removing primers not incorporated into the amplified DNA fragments or quenching the signal generated by the primers not incorporated into the amplified DNA fragments.

[0009] In some embodiments, only one fluorophore is attached to each DNA fragment.

[0010] In some embodiments, the method may further comprise the step of fluorescence-based sequencing of the amplified DNA fragments.

[0011] In some embodiments, the signal generated by the amplified DNA fragments can be detected by using a fluorometer to detect the fluorescent signal generated by the fluorophore incorporated into the amplified DNA fragments.

[0012] In some embodiments, the method may further comprise generating a standard curve indicating the relationship between the number of DNA fragments derived from the standard library and the fluorescent signals generated by the DNA fragments.

[0013] In some embodiments, the number of amplified DNA fragments can be calculated based on the detected signal by calculating the number of amplified DNA fragments based on the detected fluorescent signal and a standard curve.

[0014] In some embodiments, the method may further comprise diluting the amplified DNA fragments to a predetermined concentration suitable for fluorescence-based sequencing.

[0015] In some embodiments, the method may further comprise determining the identity of the amplified DNA fragments.

[0016] In some embodiments, the characteristics of the amplified DNA fragments can include the average size of the amplified DNA fragments.

[0017] In some embodiments, the DNA fragments may comprise adaptors, and the primers are complementary to the adaptors.

[0018] Some embodiments relate to a nucleic acid library comprising DNA fragments each of which is linked only to a predetermined number of fluorophores, such that the number of DNA fragments is calculated based on fluorescent signals generated by the linked DNA fragments.

[0019] In some embodiments, the DNA fragments are PCR amplicons generated using primers, each primer being labeled with a fluorophore such that only a predetermined number of fluorophores are attached to each PCR amplicon fragment.

[0020] In some embodiments, the DNA fragments may comprise adaptors, and the primers are complementary to the adaptors.

[0021] Some embodiments relate to a method for sequencing a DNA sample. For example, the method may include generating DNA fragments using the DNA sample, and amplifying the DNA fragments by polymerase chain reaction (PCR) in the presence of primers each labeled with a fluorophore. In these cases, only a predetermined number of fluorophores are attached to each DNA fragment. The method may further include detecting a fluorescent signal generated by the amplified DNA fragments, calculating the number of amplified DNA fragments based on the detected fluorescent signal, diluting the amplified DNA fragments to a predetermined concentration suitable for fluorescence-based sequencing, and sequencing at least a portion of the amplified DNA fragments using a fluorescence-based sequencing technique.

[0022] In some embodiments, before detecting the signal generated by the amplified DNA fragments, the method may further comprise removing primers not incorporated into the amplified DNA fragments or quenching the signal generated by the primers not incorporated into the amplified DNA fragments.

[0023] In some embodiments, only one fluorophore is attached to each DNA fragment.

[0024] In some embodiments, the signal generated by the amplified DNA fragments can be detected by using a fluorometer to detect the fluorescent signal generated by the fluorophore incorporated into the amplified DNA fragments.

[0025] In some embodiments, the method may further comprise generating a standard curve indicating the relationship between the number of DNA fragments derived from the standard library and the fluorescent signals generated by the DNA fragments.

[0026] In some embodiments, the number of amplified DNA fragments can be calculated based on the detected signal by calculating the number of amplified DNA fragments based on the detected fluorescent signal and a standard curve.

[0027] In some embodiments, the method may further comprise determining the identity of the amplified DNA fragments.

[0028] In some embodiments, the characteristics of the amplified DNA fragments can include the average size of the amplified DNA fragments.

[0029] In some embodiments, the DNA fragments may comprise adaptors, and the primers are complementary to the adaptors.

[0030] Some embodiments may further comprise a kit comprising an adaptor capable of being ligated to a DNA fragment and a primer complementary to the adaptor. Each primer may be labeled with a fluorophore so that the DNA fragments are amplified using the primers so that each fragment is ligated only to a predetermined number of fluorophores.

[0031] In some embodiments, the kit may comprise one or more polymerases.

[0032] In some embodiments, the kit may include reagents for amplification.

[0033] In some embodiments, the kit may include reagents for sequencing.

[0034] In some embodiments, the kit may include written instructions for using the kit.

[0035] In some embodiments, the kit may comprise dATP, dCTP, dGTP, dTTP, or any mixture thereof. DETAILED DESCRIPTION

[0036] Methods, compositions and kits for library quantification and identification are described herein. Embodiments of the present disclosure relate to a surprising discovery that attaching fluorescent dyes to DNA fragments for library quantification and identification does not interfere with subsequent sequence determination. In certain embodiments, primers attached with fluorescent dyes are used for library quantification and identification. When primers attached with fluorescent dyes are retained in the library, the library can be subsequently sequenced using fluorescence-based sequencing techniques.

[0037] Various methods for quantifying NGS libraries have been reported. Some methods rely on electrophoretic separation of library elements and quantification of fragments of different lengths (area under the curve evaluation). Bioanalyzers (Agilent Technologies) are commonly used for this method. In some cases, scientists may estimate the mass of a fragment of a given size and apply a correction factor based on their experience to determine the extent of dilution of the library so that it is within the appropriate range of the sequencer. Some people use this information in conjunction with the total nucleic acid mass determined by UV spectrophotometry, and also derive correction factors based on practical experience. Still others use qPCR to more accurately determine the mass of the actual library (rather than total nucleic acid), and use this mass in conjunction with the fragment size measured by the bioanalyzer to more accurately determine the number of clustered units and how to properly dilute the sample to obtain the desired concentration for application to the DNA sequencer.

[0038] While these methods can be effective, they rely on learning / judgment, estimation, and / or costly and time-consuming qPCR and bioanalyzer processing. Libraries contain fragments of varying lengths, and length information is critical to determining the number of fragments of a given assay quality. Therefore, the accuracy of these methods can vary between individuals and libraries. For example, if the size of a clustering element is half of the estimated value, the concentration may be twice the expected value. Furthermore, since not all fragments present in solution have two adapters or two different adapters, these fragments can mask the true clustering elements and lead to inaccurate average size estimates.

[0039] The present disclosure provides techniques for determining the number of elements capable of generating clusters.Some embodiments of the present disclosure relate to a method for library quantification and characterization that is independent of techniques such as UV spectrophotometry, qPCR, and average fragment size estimation.

[0040] In certain embodiments, fluorescently labeled PCR primers can be used in the library enrichment step. Because the enrichment step produces an amplicon with a single fluorophore or a predetermined number of fluorophores for each fragment, the number of molecules can be determined. Each amplified product (e.g., an amplicon) can have a predetermined number of fluorophores that are independent of its length. For example, only those elements that have been amplified have fluorophore binding. After PCR enrichment, unincorporated primers are removed and the amount of fluorescent primers / amplicons is determined by fluorescence. A standard curve can be generated to determine the number of fluorescent fragments in the solution.

[0041] Unless otherwise indicated, the terminology and notation of biochemistry, nucleic acid chemistry, molecular biology, and molecular genetics follow those of standard treatises and texts in the field.

[0042] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymerase" may refer to one reagent or a mixture of such reagents, and reference to "the method" includes reference to the same steps and / or methods known to those skilled in the art, and so forth.

[0043] The term "adaptor" as used herein may refer to an oligonucleotide with a known sequence, the ligation of which to a specific nucleic acid sequence or a target polynucleotide strand of interest can produce an amplification-ready product of the specific nucleic acid or the target polynucleotide strand of interest. Prior to adding at least one adaptor, the specific nucleic acid sample may be fragmented or unfragmented.

[0044] A variety of adapter designs are contemplated that are suitable for generating amplification-ready products of a particular sequence region / strand of interest. For example, when a double-stranded adapter is used, the two strands of the adapter can be self-complementary, non-complementary, or partially complementary. The adapter can contain at least a portion of a forward sequence priming site and a random sequence.

[0045] As used herein, the terms "amplifying," "amplification," and "amplify" a particular nucleic acid as used herein may refer to the process of producing multiple copies of a nucleic acid sample of interest, for example in the form of DNA copies. Many methods and protocols for amplifying nucleic acids are known in the art, such as PCR and qPCR.

[0046] As used herein, the term "cDNA" used herein may refer to complementary DNA.DNA can be synthesized from a messenger RNA (mRNA) template in a reaction catalyzed by reverse transcriptase and DNA polymerase.

[0047] As used herein, the term "complementary" used herein can refer to all or only a part of a sequence that is complementary. The quantity of nucleotides in the hybridizable sequence of specific oligonucleotide primers or probes can be the quantity that the stringent condition for hybridizing oligonucleotide primers or probes can prevent excessive random non-specific hybridization. The number of nucleotides in the hybridization portion of oligonucleotide primers or probes can be at least as many as the determined sequence of the target polynucleotide of oligonucleotide primers or probe hybridizations, generally about 20 to about 50 nucleotides. The target polynucleotide / oligonucleotide can be greater than oligonucleotide primers, primers or probes.

[0048] As used herein, the term "denaturation" used herein may refer to the separation of double-stranded nucleic acids into single strands. Denaturation may be achieved using any method known in the art, including but not limited to physical denaturation, thermal denaturation, and / or chemical denaturation.

[0049] As used herein, the phrase "genomic DNA" used herein may refer to chromosomal DNA, abbreviated as gDNA to represent genomic deoxyribonucleic acid. gDNA contains the genetic material of an organism.

[0050] As used herein, the term "genome" used herein may refer to DNA, RNA or cDNA sequences originating from a patient, tissue, organ, single cell, tumor, organic fluid sample taken from a patient, free-circulating nucleic acid, fungi, prokaryotes and viruses.

[0051] As used herein, the term "transcriptome" used herein may be all RNA sequences that can reflect part or all of the expressed genome of an organism.

[0052] As used herein, the term "test kit" used herein may refer to any system for delivering materials. In the context of a reaction test, such a delivery system may include elements that allow reaction components (such as oligonucleotides, buffer components, additives, reaction enhancers, enzymes, etc.) to be stored, transported, or delivered to another location in a suitable container, usually with written instructions for measuring. The test kit may include one or more housings or boxes containing related reaction reagents and support materials. The test kit may include two or more separate containers, each of which includes a portion of the entire test kit components. The containers may be delivered to the intended recipient together or separately.

[0053] As used herein, the phrase "nucleic acid (NA) modifying enzyme" used herein may refer to a DNA specific modifying enzyme. For the specificity of double-stranded DNA, a NA modifying enzyme may be selected. The enzyme may be a double-stranded specific nuclease, a blunt-end frequent cutting restriction enzyme, or another restriction enzyme.

[0054] As used herein, the phrases "nucleic acid fragment" and "specific nucleic acid" are used interchangeably and as used herein can refer to a portion of a nucleic acid sample. The nucleic acid in the input sample can be divided into a population of fragmented nucleic acid molecules or one or more polynucleotides of a specific size range.

[0055] As used herein, the phrase "specific nucleic acid sequence" or "specific sequence" used herein can be a polynucleotide sequence of interest, which needs to be digitally measured and / or quantified, including but not limited to nucleic acid fragments. With respect to its actual sequence, the specific sequence can be known or unknown. "Template" used herein can be a polynucleotide containing a specific nucleic acid sequence. The terms "specific sequence", "specific nucleic acid sequence", "specific nucleotide sequence", "region of interest" or "sequence of interest" and variants thereof are used interchangeably.

[0056] As used herein, the phrases "qualified nucleic acid" and "identified target nucleic acid fragment" used herein can refer to a fragment of a gDNA or RNA sequence, that is: i.) an acceptable template for DNA polymerase, that is, the template can be free of crosslinks or inhibitors of DNA polymerase, or ii.) the template has a modification, including but not limited to at least one of a polynucleotide sequence attached to the 5' and / or 3' end, that is, a barcode, an adapter, a sequence complementary to a primer, etc., so that the fragment can be modified for quantification, amplification, detection or other methods known to technicians in the field of gDNA and cDNA sequence analysis.

[0057] As used herein, the term "oligonucleotide" can refer to a polynucleotide chain, typically less than 200 residues in length, such as between 15 and 100 nucleotides in length, but may also include longer polynucleotide chains. Oligonucleotides can be single-stranded or double-stranded. As used in the present disclosure, the term "oligonucleotide" can be used interchangeably with the terms "primer," "probe," and "adapter."

[0058] As used herein, "PCR" is an abbreviation of the term "polymerase chain reaction", which is a commonly used nucleic acid amplification technique. In certain embodiments, PCR uses two oligonucleotide primers for each designed chain, such as an extension of one primer to provide a template for another primer in the next PCR cycle. In order to distinguish oligonucleotide primers in the discussion, any one of a pair of oligonucleotide primers can be named as "forward" or "reverse" primers in this article. PCR can be composed of: repeating (or cycling) (i) a denaturation step of separating the chains of double-stranded nucleic acids, followed by (ii) an annealing step allowing primers to anneal to the side positions of the sequence of interest; and then (iii) an extension step, which extends the primers in the 5' to 3' direction, thereby forming a nucleic acid fragment complementary to the target sequence. Each of the above steps can be performed at different temperatures using an automatic thermal cycler. The PCR cycle can be repeated at a desired frequency, resulting in an exponential accumulation of target DNA fragments, the ends of which are usually defined by the 5' ends of the primers used.

[0059] As used herein, the phrase "quantitative PCR" or "qPCR" may refer to a PCR designed to measure the abundance of one or more specific target sequences in a sample. Quantitative measurements may be made using one or more reference nucleic acid sequences, which may be determined alone or together with the target nucleic acid.

[0060] The term "portion" as used herein may refer to less than the total length of a nucleic acid sequence, a nucleic acid sequence fragment, a specific nucleic acid sequence, a specific nucleic acid fragment, a probe, a primer, and the like.

[0061] The term "primer" as used herein may refer to an oligonucleotide generally having a free 3' hydroxyl group that can hybridize or anneal with a template (such as a specific polynucleotide, a target DNA, a target RNA, a primer extension product, or a probe extension product), and can also promote polymerization of a polynucleotide complementary to the template. A primer may contain a non-hybridizing sequence constituting the tail of the primer. Even if the primer sequence is not completely complementary to the target, the primer can still hybridize with the target.

[0062] The primer used herein may be an oligonucleotide used in an extension reaction performed by a polymerase along a polynucleotide template, such as PCR, qPCR, extension reaction, etc. An oligonucleotide primer may be a synthetic polynucleotide, possibly single-stranded, containing a sequence at its 3' end that is capable of hybridizing to a sequence of a target polynucleotide.

[0063] The 3' region of a primer that hybridizes to a specific nucleic acid may comprise at least 80%, preferably 90%, more preferably 95%, and most preferably 100% complementarity to the sequence or primer binding site.

[0064] The term "sample" as used herein may refer to any substance that contains or is presumed to contain the nucleic acid of interest, and thus includes nucleic acids, cells, organisms, tissues, body fluids (e.g., cerebrospinal fluid or lymph fluid), samples of organic fluids taken from patients, and samples including but not limited to: blood, plasma, serum, urine, tears, feces, respiratory and urogenital tracts, saliva, fragments of different organs, tissues, blood cells, circulating tumor cells (CTC) or disseminated tumor cells (CTD), bones, in vitro cell culture samples, or specimens suspected of containing nucleic acid molecules.

[0065] As used herein, the term "PCR duplicate" may refer to any sequencing read that is derived from the same original nucleic acid molecule and therefore from the same primer / probe extension product sequence as another sequencing read, and therefore does not represent a unique nucleic acid molecule.

[0066] Additional information related to definitions, procedures, method structures, and other embodiments is given in U.S. Patent Publication No. US20160203259 assigned to Nugen Corp., which is incorporated by reference in its entirety.

[0067] Embodiments of the present disclosure relate to methods, compositions, and kits for library quantification and characterization.

[0068] Some embodiments relate to a method for library quantification. In some embodiments, the method may include providing DNA fragments, and amplifying the DNA fragments by polymerase chain reaction (PCR) in the presence of primers each labeled with a fluorophore. In these cases, only a predetermined number of fluorophores are attached to each DNA fragment. The method may further include detecting a fluorescent signal generated by the amplified DNA fragments, and calculating the number of amplified DNA fragments based on the detected fluorescent signal.

[0069] Some embodiments relate to a method for library quantification using two or more types of primers. Each primer type can have a related single fluorophore, multiple fluorophores, or no fluorophore at all. For those embodiments with two types of primers, the first type will have a related single fluorophore, and the second type of primer will not have a fluorophore. DNA fragments can be amplified by polymerase chain reaction (PCR) in the presence of at least one primer, wherein at least one primer is labeled with a fluorophore, resulting in a predetermined number of fluorophores being connected to each DNA fragment. Detect the fluorescent signal generated from the amplified DNA fragments, and calculate the number of amplified DNA fragments based on the detected fluorescent signal. In certain embodiments, the DNA fragments for fluorescent sequencing can be further prepared by diluting the amplified DNA fragments to a predetermined concentration.

[0070] In some embodiments, before detecting the signal generated by the amplified DNA fragments, the method may further comprise removing primers not incorporated into the amplified DNA fragments or quenching the signal generated by the primers not incorporated into the amplified DNA fragments.

[0071] In some embodiments, unincorporated fluorescent PCR primers can be removed before quantitative measurement. For example, the fluorescence of the primer can be quenched. After the PCR reaction, the quenching of unincorporated dyes can be achieved by annealing short oligonucleotides, which are complementary to the fluorescent oligonucleotides and attached with compounds that can quench the fluorophore. When it is planned to bring multiple samples together before sequencing, some embodiments of the present disclosure can enable the crude sample to be accurately quantified, mixed in a suitable ratio, and then purified as a whole rather than separately.

[0072] In certain embodiments, the functional elements in the crude mixture can be measured using a single oligonucleotide with a quencher, and the oligonucleotides with a hairpin structure and a fluorophore and a quencher are used for enrichment. When the oligonucleotide is in the hairpin structure, the fluorophore and the quencher are very close to interfere with the fluorescence detection. When the oligonucleotide structure is relaxed, and the oligonucleotide is annealed to the PCR template, the spacing between the fluorophore and the quencher increases, so that the fluorophore can be detected. After PCR, when the solution is cooled, the hairpin structure is reformed in the unincorporated oligonucleotide. When measuring, the oligonucleotides incorporated into the amplicon are detected, but the unincorporated oligonucleotides are dark.

[0073] In some embodiments, only one fluorophore is attached to each DNA fragment.

[0074] In some embodiments, the method may further comprise the step of fluorescence-based sequencing of the amplified DNA fragments.

[0075] In some embodiments, the signal generated by the amplified DNA fragments can be detected by using a fluorometer (alternatively spelled "fluorimeter") to detect fluorescent signals generated by fluorophores incorporated into the amplified DNA fragments.

[0076] In some embodiments, the method may further comprise generating a standard curve indicating the relationship between DNA fragments derived from a standard library and fluorescent signals generated by the DNA fragments.

[0077] In some embodiments, the number of amplified DNA fragments can be calculated based on the detected signal by calculating the number of amplified DNA fragments based on the detected fluorescent signal and a standard curve.

[0078] In some embodiments, the method may further comprise diluting the amplified DNA fragments to a predetermined concentration suitable for fluorescence-based sequencing.

[0079] In certain embodiments, the method may further include determining the characteristics of the amplified DNA fragments. For example, after measuring the fluorescent primers, a fluorescent intercalating dye may be added to the sample. The fluorescent intercalating dye may be combined in proportion to the total mass of the double-stranded DNA. The absolute mass may then be determined by comparing this fluorescent reading with a standard curve. By accurately determining the number of elements and the total mass, the average size of the library fragments may be determined. The measurement may provide data associated with the quality of the library and whether the library is correctly formed.

[0080] In some embodiments, the characteristics of the amplified DNA fragments can include the average size of the amplified DNA fragments.

[0081] In some embodiments, the DNA fragments may comprise adaptors, and the primers are complementary to the adaptors.

[0082] Some embodiments relate to a nucleic acid library comprising DNA fragments each of which is linked only to a predetermined number of fluorophores, such that the number of DNA fragments is calculated based on fluorescent signals generated by the linked DNA fragments.

[0083] In some embodiments, the DNA fragments are PCR amplicons generated using primers, each primer being labeled with a fluorophore such that only a predetermined number of fluorophores are attached to each PCR amplicon fragment.

[0084] In some embodiments, the DNA fragments may comprise adaptors, and the primers are complementary to the adaptors.

[0085] Some embodiments relate to a method for sequencing a DNA sample. For example, the method may include generating DNA fragments using the DNA sample, and amplifying the DNA fragments by polymerase chain reaction (PCR) in the presence of primers each labeled with a fluorophore. In these cases, only a predetermined number of fluorophores are attached to each DNA fragment. The method may further include detecting a fluorescent signal generated by the amplified DNA fragments, calculating the number of amplified DNA fragments based on the detected fluorescent signal, diluting the amplified DNA fragments to a predetermined concentration suitable for fluorescence-based sequencing, and sequencing at least a portion of the amplified DNA fragments using a fluorescence-based sequencing technique.

[0086] In some embodiments, before detecting the signal generated by the amplified DNA fragments, the method may further comprise removing primers not incorporated into the amplified DNA fragments or quenching the signal generated by the primers not incorporated into the amplified DNA fragments.

[0087] In some embodiments, only one fluorophore is attached to each DNA fragment.

[0088] In some embodiments, the signal generated by the amplified DNA fragments can be detected by using a fluorometer to detect the fluorescent signal generated by the fluorophore incorporated into the amplified DNA fragments.

[0089] In some embodiments, the method may further comprise generating a standard curve indicating the relationship between DNA fragments derived from a standard library and fluorescent signals generated by the DNA fragments.

[0090] In some embodiments, the number of amplified DNA fragments can be calculated based on the detected signal by calculating the number of amplified DNA fragments based on the detected fluorescent signal and a standard curve.

[0091] In some embodiments, the method may further comprise determining the identity of the amplified DNA fragments.

[0092] In some embodiments, the characteristics of the amplified DNA fragments can include the average size of the amplified DNA fragments.

[0093] In some embodiments, the DNA fragments may comprise adaptors, and the primers are complementary to the adaptors.

[0094] Some embodiments may further comprise a kit comprising an adaptor capable of being ligated to a DNA fragment and a primer complementary to the adaptor. Each primer may be labeled with a fluorophore so that the DNA fragments are amplified using the primers so that each fragment is ligated only to a predetermined number of fluorophores.

[0095] In some embodiments, the kit may comprise one or more polymerases.

[0096] In some embodiments, the kit may include reagents for amplification.

[0097] In some embodiments, the kit may include reagents for sequencing.

[0098] In some embodiments, the kit may include written instructions for using the kit.

[0099] In some embodiments, the kit may comprise dATP, dCTP, dGTP, dTTP, or any mixture thereof.

[0100] The present disclosure is further described with reference to the following examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the present disclosure should never be construed as being limited to the following examples, but rather should be construed as covering any and all variations that become apparent as a result of the teachings provided herein.

[0101] Example 1

[0102] Two lllumina TruSeq DNA libraries (BC11 and BC13) were PCR amplified using fluorescently labeled PCR primers ( / 56-FAM / CAA GCAGAA GAC GGC ATA CG (SEQ ID: 1)). The purified libraries were analyzed on an Agilent Bioanalyzer to determine the average size of the libraries. The libraries were also quantified by a NanoDrop UV-Vis spectrophotometer and a KAPA library quantification kit. The molar concentration was calculated using the amount determined by the KAPA library quantification kit and the average size of the libraries determined by the bioanalyzer. 2 to 8 microliters of the library were mixed with 200 μL of TE buffer and the fluorescence was read on a Qubit 2.0 fluorometer.

[0103] Two lllumina TruSeq DNA libraries (BC12 and BC14) were PCR amplified using fluorescently labeled PCR primers ( / 56-FAM / CAA GCA GAA GAC GGC ATA CG (SEQ ID: 1)). 5 microliters of the library were mixed with 200 μL of TE buffer and the fluorescence was read on a Qubit 2.0 fluorometer. Library BC13 was used as a standard to calculate the molar concentration of libraries BC12 and BC14. Equimolar libraries of the four libraries were sequenced on an lllumina MiSeq sequencer.

[0104] Example 2

[0105] Six DNA libraries were formed by using fluorescently labeled PCR primers ( / 56-FAM / CAA GCA GAAGAC GGCATACG (SEQ ID: 1)) in the final PCR amplification. The average size of the library fragments can be determined by the accurate number of elements and the total mass. Accordingly, the purified libraries were analyzed on an Agilent bioanalyzer to determine the average size of the libraries. 2 microliters of the library were mixed with 198 μL of the Qubit dsDNA HS reagent or 198 μL of TE buffer and the fluorescence was read on a Qubit 2.0 fluorometer. The results are shown in Table 1.

[0106] Table 1

[0107]

[0108]

[0109] Although the subject matter has been described in language specific to structural features and / or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts are disclosed as example forms of the claims. Sequence Listing <110> Tecan Genomics <120> Library quantification and identification <130> NUGEN 00101 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 1 caagcagaag acggcatacg 20

Claims

1. A method for library quantification, the method comprising: providing a DNA fragment, wherein the DNA fragment comprises at least two adaptors; amplifying the DNA fragments by polymerase chain reaction (PCR) in the presence of at least two primers to obtain a plurality of amplified DNA fragments, wherein at least one primer is labeled with a fluorophore, wherein each amplified DNA fragment has a predetermined number of fluorophores attached, and wherein the primers are complementary to the adaptors; removing primers that are not incorporated into the amplified DNA fragments to prepare for fluorescent sequencing; detecting a fluorescent signal generated from the amplified DNA fragment; generating a standard curve indicating the relationship between the quantity of DNA fragments derived from a reference sample and the fluorescent signals generated by the DNA fragments; Calculating the number of the amplified DNA fragments based on the detected fluorescent signal; and The amplified DNA fragments are prepared for solid phase ligation by diluting them to a predetermined concentration. 2 . The method of claim 1 , wherein the at least one primer comprises a first primer type and a second primer type, and the first primer type has a single attached fluorophore. The method of claim 1 , wherein a single fluorophore is attached to each DNA fragment. 4 . The method of claim 1 , wherein the detecting the signal generated by the amplified DNA fragment comprises detecting the fluorescent signal generated by a fluorophore incorporated into the amplified DNA fragment using a fluorometer. 5 . The method according to claim 1 , wherein the calculating the number of the amplified DNA fragments based on the detected signal comprises calculating the number of the amplified DNA fragments based on the detected fluorescent signal and the standard curve.

6. The method according to claim 1, further comprising: A second measurement is performed to determine the identity of the amplified DNA fragment.

7. The method according to claim 1, further comprising: Determine the total mass of DNA in the sample.

8. The method of claim 7, wherein the properties of the amplified DNA fragments include an average size of the amplified DNA fragments, the average size being derived from a ratio between the number of fragments and the mass of the DNA.

9. A nucleic acid library prepared by the method of any one of claims 1 to 8, the nucleic acid library comprising a large number of DNA fragments for nucleic acid sequencing at a certain concentration in a solution, each DNA fragment being an amplicon of a PCR reaction and attached with a predetermined number of fluorophores from at least one fluorescently labeled primer used in the PCR reaction, so that the number of the DNA fragments is calculated based on the fluorescent signals generated by the fluorophores attached to the DNA fragments, and the solution is diluted based on the calculated number to reach the concentration.

10. The nucleic acid library of claim 9, wherein the DNA fragment is a PCR amplicon generated using two different primers, and one of the primers is a fluorescently labeled primer.

11. The nucleic acid library of claim 9, wherein the DNA fragments include at least two adapters, and the primers are complementary to the adapters.

12. A method for sequencing a DNA sample, the method comprising: using a DNA sample to generate DNA fragments, wherein the DNA fragments include at least two adaptors; amplifying the DNA fragments by polymerase chain reaction (PCR) in the presence of two different primers, one of which is labeled with a fluorophore, to provide a plurality of amplified DNA fragments, each of which has a predetermined number of fluorophores attached, wherein the primers are complementary to the adaptors; removing primers that are not incorporated into the amplified DNA fragments to prepare for fluorescent sequencing; detecting a fluorescent signal generated by the amplified DNA fragment; generating a standard curve indicating the relationship between the number of DNA fragments derived from the standard library and the fluorescent signals generated by the DNA fragments; The number of the amplified DNA fragments is calculated based on the detected fluorescent signal, diluting the amplified DNA fragments to a predetermined concentration suitable for fluorescence-based sequencing; and At least a portion of the amplified DNA fragments are sequenced using a fluorescence-based sequencing technique.

13. The method of claim 12, wherein only a single fluorophore is attached to each DNA fragment.

14. The method of claim 12, wherein the detecting the signal generated by the amplified DNA fragments comprises detecting the fluorescent signal generated by a fluorophore incorporated into the amplified DNA fragments using a fluorometer. 15 . The method according to claim 12 , wherein the calculating the number of the amplified DNA fragments based on the detected signal comprises calculating the number of the amplified DNA fragments based on the detected fluorescent signal and the standard curve.

16. The method of claim 12, further comprising: The identity of the amplified DNA fragment is determined.

17. The method of claim 16, wherein the characteristics of the amplified DNA fragments include an average size of the amplified DNA fragments.

18. Use of a kit for performing the method of any one of claims 1 to 8 or any one of claims 12 to 17, the kit comprising: Two different primers, the two different primers are complementary to the library adaptors, wherein at least one of the primers is labeled with a fluorophore such that a DNA fragment amplified using the primers has a predetermined number of attached fluorophores.

19. The use according to claim 18, wherein the kit further comprises one or more polymerases.

20. The use according to claim 18, wherein the kit further comprises a reagent for amplification.

21. The use according to claim 18, wherein the kit further comprises reagents for sequencing.

22. The use according to claim 18, wherein the kit further comprises written instructions for using the kit.

23. The use according to claim 18, wherein the kit further comprises dATP, dCTP, dGTP, dTTP or any mixture thereof.

24. The use according to claim 18, wherein the kit further comprises an adaptor capable of ligating to the DNA fragment.

Images