CN110582577B

CN110582577B - Library quantification and identification

Info

Publication number: CN110582577B
Application number: CN201780090352.2A
Authority: CN
Inventors: D·A·阿莫雷塞; B·李; B·G·施罗德; R·A·费克特
Original assignee: Tecan Genomics Inc
Current assignee: Tecan Genomics Inc
Priority date: 2017-04-11
Filing date: 2017-04-11
Publication date: 2025-03-21
Anticipated expiration: 2037-04-11
Also published as: WO2018190814A1; EP3610031A1; EP3610031A4; JP2020516274A; JP6983906B2; CN110582577A

Abstract

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

Description

Library quantification and identification

Sequence listing

The sequence listing relevant to the present application is provided in text format to replace paper copies and is hereby incorporated by reference into this specification. The text file containing the Sequence listing is named sequence_listing. The text file is approximately 27KB and is submitted electronically via the EFS-Web.

Background

The next generation DNA sequencers typically use DNA fragments having known sequence ends. Having known sequences at both ends allows for amplification, immobilization of the DNA fragment, and provides a starting location (e.g., priming site) for sequencing. The ends of the known sequence are often referred to as adaptors, which adapt the DNA fragment as required by the sequencer. Not all DNA fragments in solution have adaptors at each end for sequencing. PCR amplification steps using two different primers, each primer specific for one of the adaptors, are typically used to enrich for fragments having two different adaptors at their ends. This collection of DNA fragments with adaptors at their ends is commonly referred to as a library. These libraries can be immobilized on a solid support such that the spatial distance between library elements (e.g., adaptors between which different DNA fragments are inserted) allows for the visualization (detection) and identification of individual elements relative to each other.

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

For the sequences of the DNA to be determined in the clusters, the clusters are homogenous and do not contain DNA from any other library elements. If the clusters are too tight or overlap occurs in extreme cases, the image analysis software may have difficulty distinguishing the boundaries of the clusters and combining them into a single feature for data extraction. Because the data for this cluster is from two different DNA fragments having two different sequences, the software may not be able to accurately determine the sequence. If the clusters are farther apart, each cluster can be analyzed separately and the sequence determined accurately. If the clusters are too far apart, sequencing becomes inefficient. The cost of handling the samples is fixed, but the cost per cluster increases.

Since the spacing of the clusters is determined by the concentration of individual library elements, it is necessary to accurately determine the concentration of these library elements.

Disclosure of Invention

Described herein are methods, compositions, and kits for library quantification and identification. Some embodiments relate to a method of library quantification. For example, the method may comprise providing DNA fragments that are amplified by Polymerase Chain Reaction (PCR) in the presence of respective fluorophore-labeled primers. In these cases, only a predetermined number of fluorophores are attached to each DNA fragment. The method may further comprise detecting a fluorescent signal generated by the amplified DNA fragments and calculating the number of amplified DNA fragments based on the detected fluorescent signal.

In some embodiments, the method may further comprise removing primers not incorporated into the amplified DNA fragments or quenching signals generated by primers not incorporated into the amplified DNA fragments prior to detecting signals generated by the amplified DNA fragments.

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

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

In some embodiments, the signal generated by the amplified DNA fragment may be detected by detecting a fluorescent signal generated by a fluorophore incorporated into the amplified DNA fragment using a fluorometer.

In some embodiments, the method may further comprise generating a standard curve indicative of a relationship between the number of DNA fragments derived from the standard library and the fluorescent signal generated by the DNA fragments.

In some embodiments, the number of amplified DNA fragments may 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.

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

In some embodiments, the method may further comprise determining a characteristic of the amplified DNA fragment.

In some embodiments, the characteristics of the amplified DNA fragments may comprise the average size of the amplified DNA fragments.

In some embodiments, the DNA fragment may comprise an adapter, and the primer is complementary to the adapter.

Some embodiments relate to a nucleic acid library comprising DNA fragments each having only a predetermined number of fluorophores attached such that the number of DNA fragments is calculated based on fluorescent signals generated by the attached DNA fragments.

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

Some embodiments relate to a method of sequencing a DNA sample. For example, the method may comprise generating a DNA fragment using a DNA sample, and amplifying the DNA fragment 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 comprise 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.

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

Some embodiments may further comprise a kit comprising an adapter capable of ligating to a DNA fragment and a primer complementary to the adapter. Each primer may be labeled with a fluorophore such that the DNA fragments are amplified using the primers to ligate each fragment to only a predetermined number of fluorophores.

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

In some embodiments, the kit may comprise reagents for amplification.

In some embodiments, the kit may comprise reagents for sequencing.

In some embodiments, the kit may comprise written instructions for using the kit.

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

Detailed Description

Described herein are methods, compositions, and kits for library quantification and identification. The examples of the present disclosure relate to the surprising discovery that attaching fluorescent dyes to DNA fragments for library quantification and identification does not interfere with subsequent sequencing. In some embodiments, the primer with the fluorescent dye attached is used for library quantification and identification. When the primer with the fluorescent dye attached remains in the library, the library can be subsequently sequenced using fluorescence-based sequencing techniques.

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 assessment). Bioanalyzers (Agilent technologies (Agilent Technologies)) are commonly used for this method. In some cases, the scientist may estimate the mass of a fragment of a given size and apply correction factors based on his experience to determine the extent to which the library is diluted to be within the proper range of the sequencer. Some use this information in combination with the total nucleic acid mass determined by ultraviolet spectrophotometry, again based on practical experience to derive correction factors. Still others use qPCR to more accurately determine the mass of the actual library (rather than the total nucleic acid) and use this mass in combination 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.

While these methods may be effective, they rely on learning/judgment, estimation and/or cost and time consuming qPCR and bioanalyzer processing. The library contains fragments of different lengths and the length information is crucial for determining the number of fragments of a given assay quality. Thus, the accuracy of these methods may vary from individual to individual and from library to library. For example, if the size of the clustered elements is half the estimated value, the concentration may be twice the expected value. Furthermore, since not all fragments present in solution have two adaptors or two different adaptors, these fragments can mask the true clustered elements and result in inaccurate average size estimates.

The present disclosure provides techniques for determining a number of elements capable of generating a cluster. Some embodiments of the present disclosure relate to a method for library quantification and identification that is not dependent on techniques such as ultraviolet spectrophotometry, qPCR, and average fragment size estimation.

In some embodiments, fluorescently labeled PCR primers can be used in the library enrichment step. Because the enrichment step produces amplicons with a single fluorophore or a predetermined number of fluorophores per fragment, the number of molecules can be determined. Each amplification product (e.g., amplicon) may have a predetermined number of fluorophores 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 amount of fluorescent fragments in solution.

Unless otherwise indicated, biochemical, nucleic acid chemistry, molecular biology and molecular genetics terms and symbols follow those of standard papers and texts in the art.

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 an agent or a mixture of such agents, and reference to "the method" includes reference to the same steps and/or methods known to those of skill in the art, and so forth.

The term "adapter" as used herein may refer to an oligonucleotide of known sequence, the ligation of which to a specific nucleic acid sequence or target polynucleotide strand of interest is capable of producing an amplification ready product of said specific nucleic acid or said target polynucleotide strand of interest. The specific nucleic acid sample may or may not be fragmented prior to the addition of the at least one adapter.

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

As used herein, the terms "amplify (amplifying)", "amplification" and "amplification" specific nucleic acids 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. Numerous methods and protocols for amplifying nucleic acids are known in the art, such as PCR and qPCR.

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

As used herein, the term "complementary" as used herein may refer to being complementary to all or only a portion of a sequence. The number of nucleotides in the hybridizable sequence of the specific oligonucleotide primer or probe may be such that stringent conditions used to hybridize the oligonucleotide primer or probe can prevent excessive random non-specific hybridization. The number of nucleotides in the hybridizing portion of the oligonucleotide primer or probe may be at least as large as the defined sequence of the target polynucleotide to which the oligonucleotide primer or probe hybridizes, typically about 20 to about 50 nucleotides. The target polynucleotide/oligonucleotide may be larger than the oligonucleotide primer, primer or probe.

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

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

As used herein, the term "genome" as used herein may refer to DNA, RNA or cDNA sequences derived from a patient, tissue, organ, single cell, tumor, organic liquid sample taken from a patient, free circulating nucleic acids, fungi, prokaryotes and viruses.

As used herein, the term "transcriptome" as used herein may be any RNA sequence capable of reflecting the partial or complete expression of a genome of an organism.

As used herein, the term "kit" as used herein may refer to any system for delivering materials. In the context of a reaction assay, such a delivery system may comprise elements that allow for storage, transport or delivery of reaction components (e.g., oligonucleotides, buffer components, additives, reaction enhancers, enzymes, etc.) from one location to another in a suitable container, typically with written instructions for performing the assay. The kit may comprise one or more housings or cassettes containing the relevant reagents and support materials. The kit may comprise two or more separate containers, wherein each container comprises a portion of the entire kit component. The containers may be delivered to the intended recipient together or separately.

As used herein, the phrase "Nucleic Acid (NA) modifying enzyme" as used herein may refer to a DNA-specific modifying enzyme. For the specificity of double-stranded DNA, NA-modifying enzymes may be selected. The enzyme may be a double-strand specific endonuclease, a blunt-ended frequent cutting restriction enzyme, or another restriction enzyme.

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. Nucleic acids entered into a sample may be partitioned into a population of fragmented nucleic acid molecules or one or more polynucleotides of a particular size range.

As used herein, the phrase "specific nucleic acid sequence" or "specific sequence" as used herein may be a polynucleotide sequence of interest, which requires digital measurement and/or quantification, including but not limited to nucleic acid fragments. In terms of its actual sequence, the particular sequence may be known or unknown. As used herein, a "template" may be a polynucleotide comprising a particular 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.

As used herein, the phrases "qualified nucleic acid" and "identifying a target nucleic acid fragment" as used herein may refer to fragments of a gDNA or RNA sequence, i.e., i.) acceptable templates for DNA polymerase, i.e., templates may be free of cross-linking or inhibitors of DNA polymerase, or ii.) templates having modifications, including but not limited to at least one of the attachment of polynucleotide sequences at the 5 'and/or 3' ends, i.e., barcodes, adaptors, sequences complementary to primers, etc., such that fragments may be modified for quantification, amplification, detection, or other methods known to those of skill in the art of gDNA and cDNA sequence analysis.

As used herein, the term "oligonucleotide" may refer to a polynucleotide strand, typically less than 200 residues in length, e.g., between 15 and 100 nucleotides in length, but may also include longer polynucleotide strands. The oligonucleotides may be single-stranded or double-stranded. As used in this disclosure, the term "oligonucleotide" may be used interchangeably with the terms "primer," probe, "and" adapter.

As used herein, "PCR" is an abbreviation for the term "polymerase chain reaction" and is a commonly used technique for nucleic acid amplification. In some embodiments, PCR uses two oligonucleotide primers for each designed strand, such as extension of one primer provides a template for the other primer in the next PCR cycle. To distinguish between oligonucleotide primers in the discussion, either one of a pair of oligonucleotide primers may be referred to herein as a "forward" or "reverse" primer. PCR may consist of repeating (or cycling) (i) a denaturation step that separates strands of double-stranded nucleic acid, followed by (ii) an annealing step that allows the primer to anneal to flanking locations of the sequence of interest, and then (iii) an extension step that extends the primer in the 5 'to 3' direction, thereby forming a nucleic acid fragment that is complementary to the target sequence. Each of the above steps may be performed at a different temperature using an automatic thermal cycler. The PCR cycle can be repeated as often as desired, resulting in exponential accumulation of target DNA fragments, the ends of which are typically defined by the 5' ends of the primers used.

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

The term "portion" as used herein may refer to a portion that is less than the total length of a nucleic acid sequence, nucleic acid sequence fragment, specific nucleic acid sequence, specific nucleic acid fragment, probe, primer, etc.

The term "primer" as used herein may refer to an oligonucleotide, typically having a free 3' hydroxyl group, that is capable of hybridizing or annealing to a template (e.g., a particular polynucleotide, target DNA, target RNA, primer extension product, or probe extension product) and also is capable of promoting polymerization of a polynucleotide complementary to the template. The primer may contain non-hybridizing sequences that constitute the primer tail. Even if the primer sequence is not fully complementary to the target, the primer can still hybridize to the target.

The primer used herein may be an oligonucleotide used in an extension reaction by a polymerase along a polynucleotide template, such as PCR, qPCR, extension reaction, and the like. The oligonucleotide primer may be a synthetic polynucleotide, possibly single stranded, containing at its 3' end a sequence capable of hybridizing to the sequence of the target polynucleotide.

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

The term "sample" as used herein may refer to any substance that contains or is supposed to contain a nucleic acid of interest, and thus comprises nucleic acids, cells, organisms, tissues, bodily fluids (e.g., spinal fluid or lymph fluid), samples taken from an organic fluid of a patient, as well as samples including, but not limited to, blood, plasma, serum, urine, tears, stool, respiratory and genitourinary tracts, saliva, fragments of different organs, tissues, blood cells, circulating Tumor Cells (CTCs) or disseminated tumor Cells (CTDs), bones, in vitro cell culture samples, or samples suspected of containing nucleic acid molecules.

The term "PCR repetition" as used herein may refer to any sequencing read that is derived from the same original nucleic acid molecule and thus from the same primer/probe extension product sequence as another sequencing read and thus does not represent a unique nucleic acid molecule.

Additional information regarding definitions, processes, method structures, and other embodiments is given in U.S. patent publication No. US20160203259 assigned to the core age company (Nugen corp.), which is incorporated by reference in its entirety.

Embodiments of the present disclosure relate to methods, components, and kits for library quantification and identification.

Some embodiments relate to a method of library quantification. In some embodiments, the method may comprise providing a DNA fragment, and amplifying the DNA fragment by Polymerase Chain Reaction (PCR) in the presence of each fluorophore-labeled primer. In these cases, only a predetermined number of fluorophores are attached to each DNA fragment. The method may further comprise detecting a fluorescent signal generated by the amplified DNA fragments and calculating the number of amplified DNA fragments based on the detected fluorescent signal.

Some embodiments relate to methods of library quantification using two or more types of primers. Each primer type may have an associated single fluorophore, multiple fluorophores, or no fluorophores at all. For those embodiments with two types of primers, the first type will have a single fluorophore associated therewith, and the second type of primer will have no fluorophore. The DNA fragments may 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 attached to each DNA fragment. Fluorescent signals generated from the amplified DNA fragments are detected, and the number of amplified DNA fragments is calculated based on the detected fluorescent signals. In some embodiments, the DNA fragments for fluorescent sequencing may be further prepared by diluting the amplified DNA fragments to a predetermined concentration.

In some embodiments, unincorporated fluorescent PCR primers may be removed prior to performing the quantitative measurement. For example, the fluorescence of the primer may be quenched. After the PCR reaction, quenching without dye incorporation can be achieved by annealing a short oligonucleotide that is complementary to the fluorescent oligonucleotide and to which a compound capable of quenching a fluorophore is attached. When planning to pool multiple samples together prior to sequencing, some embodiments of the present disclosure may enable crude samples to be accurately quantified, mixed in appropriate proportions, and then purified as a whole rather than individually.

In some embodiments, a single oligonucleotide with a quencher may be used to measure the functional elements in the crude mixture, and an oligonucleotide with a hairpin structure and with both a fluorophore and a quencher will be used for enrichment. When the oligonucleotide is in a hairpin structure, the fluorophore and quencher are in close proximity to interfere with fluorescent detection. When the oligonucleotide structure is relaxed and the oligonucleotide anneals to the PCR template, the spacing between the fluorophore and the quencher increases such that the fluorophore can be detected. After PCR, hairpin structures reform in unincorporated oligonucleotides when the solution cools. When the measurement is made, the oligonucleotides that incorporate the amplicon are detected, but the unincorporated oligonucleotides are dark.

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

In some embodiments, the signal generated by the amplified DNA fragment may be detected by detecting a fluorescent signal generated by a fluorophore incorporated into the amplified DNA fragment using a fluorometer (alternatively spelled "fluorometer").

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

In some embodiments, the method may further comprise determining a characteristic of the amplified DNA fragment. For example, a fluorescent intercalating dye may be added to the sample after measuring the fluorescent primer. Fluorescent intercalating dyes can bind in proportion to the total mass of double stranded DNA. The absolute mass can then be determined by comparing this fluorescence reading to a standard curve. The average size of the library fragments can be determined by the exact number of elements and the total mass. The measurements may provide data associated with the quality of the library and whether the library is properly formed.

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

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

In some embodiments, the kit may comprise reagents for amplification.

In some embodiments, the kit may comprise reagents for sequencing.

The 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 specified. Accordingly, the present disclosure should in no way be construed as limited to the following examples, but rather should be construed to cover any and all variations that become apparent from the teachings provided herein.

Example 1

Two lllumina TruSeq DNA libraries (BC 11 and BC 13) were PCR amplified using fluorescent labeled PCR primers (/ 56-FAM/CAA GCAGAA GAC GGC ATA CG (SEQ ID: 1)). The purified library was analyzed on an Agilent bioanalyzer to determine the average size of the library. The library was also quantified by a NanoDrop uv-vis spectrophotometer and KAPA library quantification kit. The molar concentration was calculated using the amounts determined by KAPA library quantification kit and the average library size determined by bioanalyzer. 2 to 8. Mu.l of library was mixed with 200. Mu.l of TE buffer and the fluorescence was read on a qubit 2.0 fluorometer.

Two lllumina TruSeq DNA libraries (BC 12 and BC 14) were PCR amplified using fluorescent labeled PCR primers (/ 56-FAM/CAA GCA GAA GAC GGC ATA CG (SEQ ID: 1)). 5 microliters of library was 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 for calculating the molar concentration of libraries BC12 and BC 14. Equimolar libraries of the four libraries were sequenced on a lllumina MiSeq sequencer.

Example 2

Six DNA libraries were formed by using fluorescent labeled PCR primers (/ 56-FAM/CAA GCA GAAGAC GGC ATACG (SEQ ID: 1)) in the final PCR amplification. The average size of the library fragments can be determined by the exact number of elements and the total mass. Accordingly, the purified library was analyzed on an Agilent bioanalyzer to determine the average size of the library. Mu.l of the library was mixed with 198. Mu.l of qubit dsDNA HS reagent or 198. Mu.l of TE buffer and the fluorescence was read on a qubit 2.0 fluorometer. The results are shown in table 1.

TABLE 1

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 claims.

Sequence listing

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Claims

1. A method of 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, wherein the primer is complementary to the adapter;

removing primers not incorporated into the amplified DNA fragments in preparation for fluorescence sequencing;

Detecting a fluorescent signal generated from the amplified DNA fragments;

generating a standard curve indicative of a relationship between the number of DNA fragments derived from a reference sample and a fluorescent signal generated by said DNA fragments;

calculating the number of the amplified DNA fragments based on the detected fluorescent signal, and

Solid phase ligation is prepared by diluting the amplified DNA fragments 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.

3. 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 fragments comprises detecting the fluorescent signal generated by fluorophores incorporated into the amplified DNA fragments using a fluorometer.

5. The method of claim 1, wherein said calculating the number of amplified DNA fragments based on the detected signal comprises calculating the number of amplified DNA fragments based on the detected fluorescent signal and the standard curve.

6. The method as recited in claim 1, further comprising:

a second measurement is performed to determine a characteristic of the amplified DNA fragment.

7. The method of claim 1, further comprising determining the total mass of DNA in the sample.

8. The method of claim 7, wherein the characteristics of the amplified DNA fragments comprise an average size of the amplified DNA fragments, the average size resulting from a ratio between the number of fragments and the mass of DNA.

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

10. The nucleic acid library of claim 9, wherein the DNA fragments are PCR amplicons generated using two different primers, and wherein one primer is a fluorescent-labeled primer.

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

12. A method of sequencing a DNA sample, the method comprising:

Generating a DNA fragment using a DNA sample, wherein the DNA fragment comprises at least two adaptors;

Amplifying the DNA fragments by polymerase chain reaction, PCR, in the presence of two different primers, wherein one primer is labeled with a fluorophore to provide a plurality of amplified DNA fragments, wherein each amplified DNA fragment has a predetermined number of fluorophores attached, wherein the primer is complementary to the adapter;

Detecting a fluorescent signal generated by the amplified DNA fragments;

generating a standard curve indicative of a relationship between the number of DNA fragments derived from a standard library and a fluorescent signal generated by said DNA fragments;

calculating the number of amplified DNA fragments based on the detected fluorescent signal,

Diluting the amplified DNA fragment to a predetermined concentration suitable for fluorescence-based sequencing, and

Sequencing at least a portion of the amplified DNA fragments using fluorescence-based sequencing techniques.

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 fluorophores incorporated into the amplified DNA fragments using a fluorometer.

15. The method of claim 12, wherein the calculating the number of amplified DNA fragments based on the detected signal comprises calculating the number of amplified DNA fragments based on the detected fluorescent signal and the standard curve.

16. The method as recited in claim 12, further comprising:

determining the characteristics of the amplified DNA fragments.

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

18. Use of a kit for performing the method of any one of claims 1-8 or the method of any one of claims 12-17, the kit comprising:

two different primers complementary to library adaptors, wherein at least one primer is labeled with a fluorophore such that a DNA fragment amplified using the primers has a predetermined number of attached fluorophores.

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

20. The use of claim 18, wherein the kit further comprises reagents for amplification.

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

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

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

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