WO2019100024A1 - Methods for reduction in required material for shotgun sequencing - Google Patents

Methods for reduction in required material for shotgun sequencing Download PDF

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Publication number
WO2019100024A1
WO2019100024A1 PCT/US2018/061870 US2018061870W WO2019100024A1 WO 2019100024 A1 WO2019100024 A1 WO 2019100024A1 US 2018061870 W US2018061870 W US 2018061870W WO 2019100024 A1 WO2019100024 A1 WO 2019100024A1
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Prior art keywords
nucleic acid
mixture
acid molecules
volume
pooled
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PCT/US2018/061870
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French (fr)
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Daniel DELUBAC
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Freenome Holdings, Inc.
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Publication of WO2019100024A1 publication Critical patent/WO2019100024A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the methods further comprise providing a second nucleic acid sample; using said second nucleic acid sample to generate a second mixture of nucleic acid molecules, wherein said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules have a combined maximal volume of about 5 m ⁇ ; pooling said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules, thereby producing a pooled mixture of nucleic acid molecules; and transferring said pooled mixture of nucleic acid molecules of nucleic acid molecules onto said sequencer.
  • each mixture of nucleic acid molecules comprises a mixture barcode, wherein each mixture barcode is identical among nucleic acids within the mixture of nucleic acid molecules and unique between mixtures of nucleic acid molecules.
  • the first mixture of nucleic acid molecules may require about 2 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods.
  • the first mixture of nucleic acid molecules may require about 3 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods.
  • the first mixture of nucleic acid molecules may require about 4 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods.
  • the first mixture of nucleic acid molecules may require about 5 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods.
  • the first mixture of nucleic acid molecules may require about 6 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods.
  • the dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1.2 ml at most.
  • the dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1.1 ml at most.
  • the dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1 ml at most.
  • the dilution can be a dilution to a maximal volume of about 0.5 ml.
  • the dilution can be a dilution to a maximal volume of about 0.6 ml.
  • the dilution can be a dilution to a maximal volume of about 0.7 ml.
  • the pooled volume of the plurality of nucleic acid libraries may be at least about 1 m ⁇ .
  • the pooled volume of the plurality of nucleic acid libraries may be at least about 4.5 m ⁇ .
  • the pooled volume of the plurality of nucleic acid libraries may be at least about 5 m ⁇ .
  • the pooled volume of the plurality of nucleic acid libraries may be at least about 10 m ⁇ .
  • the pooled volume of the plurality of nucleic acid libraries may be at least about 15 m ⁇ .
  • the pooled volume of the plurality of nucleic acid libraries may be at least about 20 m ⁇ .
  • the pooled volume of the plurality of nucleic acid libraries may be at least about 100 m ⁇ .
  • the pooled volume of the plurality of nucleic acid libraries may be at least about 200 m ⁇ .
  • the pooled volume of the plurality of nucleic acid libraries may be at least about 300 m ⁇ .
  • the method may comprise loading at least about 10% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer.
  • the method may comprise loading at least about 20% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer.
  • the method may comprise loading at least about 30% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer.
  • the method may comprise loading at least about 40% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer.
  • the pooled volume of a plurality of nucleic acid libraries can be about 0.3% of the diluted pooled volume.
  • the pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 1% at most of the diluted pooled volume.
  • the pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 0.9% at most of the diluted pooled volume.
  • the pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 0.8% at most of the diluted pooled volume.
  • the pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 0.7% at most of the diluted pooled volume.
  • the method may comprise transferring the first mixture of nucleic acid molecules or diluted first mixture of nucleic acid molecules onto the sequencer.
  • the method may comprise loading more than 10% of the first mixture of nucleic acid molecules onto the sequencer.
  • the method may comprise providing a second nucleic acid sample and using the second nucleic acid sample to generate a second mixture of nucleic acid molecules.
  • the method may comprise pooling the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules, thereby producing a pooled mixture of nucleic acid molecules.
  • the method may comprise transferring the pooled mixture of nucleic acid molecules or diluted pooled mixture of nucleic acid molecules onto the sequencer.
  • the method may comprise loading more than 10% of the pooled mixture of nucleic acid molecules onto the sequencer.
  • the method may comprise a first mixture of nucleic acid molecules with a maximal volume of about 5 m ⁇ .
  • FIG. 4 shows a computer system 401 that is programmed or otherwise configured to direct the extraction and transferring of one or more mixtures of nucleic acid molecules into a container.
  • the computer system 401 can regulate various aspects of sample preparation of the present disclosure, such as, for example, generating a pooled mixture of nucleic acid molecules and a nucleic acid library template that can be ready for loading onto a sequencer.
  • the computer system 401 can generate a mixture of nucleic acid molecule samples.
  • the computer system 401 can direct a liquid handler to dilute a mixture of nucleic acid molecule samples.

Abstract

The present disclosure provides methods for nucleic acid sequencing that require less material than present sequencing methods. The methods can use, for example, a low volume liquid handler to extract and transfer volumes of mixtures of nucleic acid samples, and pooling and diluting samples so as to allow a user to sequence a greater amount of the sample than might otherwise be possible. In so doing, the user can achieve greater nucleic acid sequencing efficiency.

Description

METHODS FOR REDUCTION IN REQUIRED MATERIAL FOR
SHOTGUN SEQUENCING
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No. 62/588,892, filed November 20, 2017, which is entirely incorporated herein by reference.
BACKGROUND
[0002] Nucleic acid sequence data can provide important information for medical and scientific research endeavors. For example, sequence information can facilitate detection and/or risk analysis of active disease and genetic disease states, assist in rational design of drugs (e.g., targeting specific diseases, avoiding unwanted side effects, improving potency, and the like), and serve as a basis for genomic and evolutionary studies and genetic engineering applications.
[0003] Sequencing technologies and systems, such as, for example, those provided by Applied Biosystems/Life Technologies (SOLiD Sequencing System), Illumina, and 454 Life Sciences can provide high throughput DNA/RNA sequencing capabilities. Applications that can benefit from these sequencing technologies include, but are not limited to, targeted resequencing, miRNA analysis, DNA methylation analysis, whole-transcriptome analysis, and cancer genomics research.
[0004] Sequencing platforms can vary from one another in their mode of operation (e.g., sequencing by synthesis, sequencing by ligation, pyrosequencing, etc.) and the type/form of raw sequencing data that they generate. However, attributes that are typically common to all these platforms is that the sequencing runs performed on the platforms tend to require an amount of nucleic acid material to be generated in excess compared to the amount of nucleic acid material that is actually sequenced. Thus, the methods accompanying these platforms result in higher costs and time requirements, and can lack sensitivity or accuracy.
SUMMARY
[0005] Many current tabletop, next-generation sequencers (e.g., the Illumina NextSeq® and MiSeq® sequencing systems) rely on a plurality of dilution steps prior to loading a nucleic acid (e.g. a library template) sample onto a sequencer. For example, a sample nucleic acid library once generated, undergoes nucleic acid denaturation and one or more serial dilutions. These dilution steps lead to about an 88% sample loss. This high sample loss implies that at least about eight times more nucleic acid material needs to be generated than is actually sequenced. In amplification-free processes, this implies the requirement is to start the sequencing process with at least about eight times more material.
[0006] The nucleic acid sequencing methods described herein provide for low input volume requirements while maintaining a high sensitivity. This technology allows for simple and efficient sequencing that may prevent about an 88% sample loss. The nucleic acid sequencing methods described herein may not require any modifications to current library preparation methods. Moreover, the nucleic acid sequencing methods described herein provides for a low minimum amount of nucleic acid material that has to be generated for sequencing. Thus, the nucleic acid sequencing methods described herein may enable the sequencing of amplification- free processes that are currently not viable due to the high nucleic acid input material required. Additionally, the nucleic acid sequencing methods described herein may enable fewer cycles of nucleic acid amplification that consequently, may reduce amplification-related biases during sequencing.
[0007] Disclosed herein, in some aspects, are methods for sequencing a nucleic acid sample, comprising: using said nucleic acid sample to generate a first mixture of nucleic acid molecules, wherein said first mixture of nucleic acid molecules has a maximal volume of about 5 mΐ;
transferring said first mixture of nucleic acid molecules or diluted first mixture of nucleic acid molecules onto said sequencer; and using said sequencer to subject said first mixture of nucleic acid molecules to sequencing to generate one or more sequences of said nucleic acid sample at a efficiency greater than 90% . In some aspects, the methods further comprise providing a second nucleic acid sample; using said second nucleic acid sample to generate a second mixture of nucleic acid molecules, wherein said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules have a combined maximal volume of about 5 mΐ; pooling said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules, thereby producing a pooled mixture of nucleic acid molecules; and transferring said pooled mixture of nucleic acid molecules of nucleic acid molecules onto said sequencer. In some aspects, each mixture of nucleic acid molecules comprises a mixture barcode, wherein each mixture barcode is identical among nucleic acids within the mixture of nucleic acid molecules and unique between mixtures of nucleic acid molecules. In some aspects, step (a) further comprises diluting said first mixture of nucleic acid molecules to a maximal volume of about 5 mΐ, thereby producing a diluted first mixture of nucleic acid molecules. In some aspects, said first mixture of nucleic acid molecules is not amplified prior to said transferring. In some aspects, said first mixture of nucleic acid molecules comprises tagged nucleic acid molecules. In some aspects, said tagged nucleic acid molecules are non-uniquely tagged. In some aspects, the methods further comprise denaturing said first mixture of nucleic acid molecules. In some aspects, the methods further comprise diluting said first mixture of nucleic acid molecules. In some aspects, said dilution is a dilution to a maximal volume of about 1.3 ml. In some aspects, more than 10% of said first mixture of nucleic acid molecules is loaded onto said sequencer.
[0008] Disclosed herein, in some aspects, are methods for sequencing a plurality of nucleic acid samples, comprising: using said plurality of nucleic acid samples to generate a first mixture of nucleic acid molecules and a second mixture of nucleic acid molecules; using a low volume liquid handler to extract and transfer a first volume of said first mixture of nucleic acid molecules and a second volume of said second mixture of nucleic acid molecules into a container to yield a pooled volume, wherein said pooled volume comprises a plurality of nucleic acid libraries comprising equimolar quantities of said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules, wherein said pooled volume is less than about 5 mΐ; diluting said pooled volume of a plurality of nucleic acid libraries to generate a diluted pooled volume of a plurality of nucleic acid libraries; and transferring said diluted pooled volume of nucleic acid libraries onto said sequencer. In some aspects, more than 10% of said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules is loaded onto said sequencer. In some aspects, said pooled volume of a plurality of nucleic acid libraries is about 0.3% of said diluted pooled volume. In some aspects, said low volume liquid handler is an acoustic liquid handler, a piezoelectric pipette, an ink-type dispenser, a
microsyringe, a pressure-sensing system, or a pin tool.
[0009] Disclosed herein, in some aspects, are methods for sequencing a nucleic acid sample, comprising: using said nucleic acid sample to generate a first mixture of nucleic acid molecules; transferring said first mixture of nucleic acid molecules or diluted first mixture of nucleic acid molecules onto said sequencer, wherein more than 10% of said first mixture of nucleic acid molecules is loaded onto said sequencer. In some aspects, the methods further provide a second nucleic acid sample; using said nucleic acid sample to generate a second mixture of nucleic acid molecules; pooling said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules, thereby producing a pooled mixture of nucleic acid molecules; and transferring said pooled mixture of nucleic acid molecules or diluted pooled mixture of nucleic acid molecules onto said sequencer; wherein more than 10% of said pooled mixture of nucleic acid molecules is loaded onto said sequencer. In some aspects, each mixture of nucleic acid molecules comprises a mixture barcode, wherein each mixture barcode is identical among nucleic acids within the mixture of nucleic acid molecules and unique between mixtures of nucleic acid molecules. In some aspects, step (a) further comprises diluting said first mixture of nucleic acid molecules, thereby producing a diluted first mixture of nucleic acid molecules. In some aspects, said first mixture of nucleic acid molecules or diluted first mixture of nucleic acid molecules is not amplified prior to said transferring. In some aspects, said first mixture of nucleic acid molecules comprises tagged nucleic acid molecules. In some aspects, said tagged nucleic acid molecules are non-uniquely tagged. In some aspects, the method further comprises denaturing said first mixture of nucleic acid molecules or diluted first mixture of nucleic acid molecules. In some aspects, said first mixture of nucleic acid molecules has a maximal volume of about 5 mΐ.
[0010] Disclosed herein, in some aspects, are methods for sequencing a plurality of nucleic acid samples, comprising: using said plurality of nucleic acid samples to generate a first mixture of nucleic acid molecules and a second mixture of nucleic acid molecules; using a low volume liquid handler to extract and transfer a first volume of said first mixture of nucleic acid molecules and a second volume of said second mixture of nucleic acid molecules into a container to yield a pooled volume, wherein said pooled volume comprises a plurality of nucleic acid libraries comprising equimolar quantities of said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules; diluting said pooled volume of said plurality of nucleic acid libraries to generate a diluted pooled volume of said plurality of nucleic acid libraries, wherein said pooled volume of said plurality of nucleic acid libraries is from about 0.01% to 20% of said diluted pooled volume; and transferring said diluted pooled volume of nucleic acid libraries onto said sequencer. In some aspects, said pooled volume of said plurality of nucleic acid libraries is diluted once. In some aspects, the total volume of said pool of nucleic acid libraries is less than about 5 mΐ. In some aspects, said low volume liquid handler is an acoustic liquid handler, a piezoelectric pipette, an ink-type dispenser, a microsyringe, a pressure- sensing system, or a pin tool.
[0011] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE
[0012] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative
embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also“Figure” and“FIG.” herein), of which:
[0014] FIGS. 1A and IB illustrate different methods of nucleic acid library preparation immediately prior to sequencing. FIG. 1A illustrates a conventional method of preparing a nucleic acid library immediately prior to sequencing. FIG. IB illustrates a method described herein of preparing a nucleic acid library immediately prior to sequencing.
[0015] FIGS. 2A and 2B show insert size histograms of nucleic acid samples. FIG. 2A shows an insert size histogram of a nucleic acid sample prepared using a conventional method (i.e., using multiple dilutions). FIG. 2B shows an insert size histogram of a nucleic acid sample prepared using a method described herein.
[0016] FIGS. 3A and 3B show GC bias and normalized coverage. Read coverage with respect to GC content, calculated in 100 base pair (bp) windows (shown as vertical bars) and in each window fraction of normalized coverage (shown as circles) is plotted against the left y- axis. Mean base quality at GC % (shown as a line graph) is calculated and plotted against the right y-axis. FIG. 3A shows read coverage of a nucleic acid sample prepared using the conventional nucleic acid library preparation methods. FIG. 3B shows read coverage of a nucleic acid sample prepared using the conventional nucleic acid library preparation methods.
[0017] FIG. 4 shows a computer control system that is programmed or otherwise configured to implement methods provided herein.
[0018] FIGS. 5A and 5B show GC bias and the normalized coverage. Read coverage with respect to GC content, calculated in 100 base pair (bp) windows (shown as vertical bars) and in each window fraction of normalized coverage (shown as circles) is plotted against the left y- axis. Mean base quality at GC % (shown as a line graph) is calculated and plotted against the right y-axis. FIG. 5A shows read coverage of a nucleic acid sample prepared using the conventional nucleic acid library preparation methods. FIG. 5B shows read coverage of a nucleic acid sample prepared using the conventional nucleic acid library preparation methods.
DETAILED DESCRIPTION
[0019] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
[0020] Because many current tabletop next-generation sequencers rely on multiple dilution steps prior to loading nucleic acid samples onto a sequencer, valuable sequencing information can be lost because an entire sample is unable to be run on the sequencer. These dilution steps can lead to, for example, an approximately 88% loss of total starting nucleic acid sample material prior to sequencing. Thus, there is a need for alternative nucleic acid sample preparation methods that reduce nucleic acid sample material loss and improve efficiency of the nucleic acid sequencing methods.
[0021] The methods and systems provided herein improve the efficiency of nucleic acid sequencing methods by reducing the initial nucleic acid sample material required because less (or no) nucleic acid sample material is lost prior to sequencing, as compared to current systems. The methods and systems provided herein can further decrease sample preparation time and/or reduce (including eliminate) amplification processes, thereby reducing amplification-driven biases and the overall cost of nucleic acid library preparation and sequencing. Efficiency is also a measurable metric calculated as the division of the number of unique molecules for which sequences will be available after sequencing over the number of unique molecules originally present in the primary sample.
[0022] In one aspect, the present disclosure provides a method for sequencing a nucleic acid sample, comprising: using the nucleic acid sample to generate a first mixture of nucleic acid molecules, wherein the first mixture of nucleic acid molecules has a maximal volume of about 5 mΐ; transferring the first mixture of nucleic acid molecules or diluted first mixture of nucleic acid molecules onto the sequencer; and using the sequencer to subject the first mixture of nucleic acid molecules to sequencing to generate one or more sequences of the nucleic acid sample at an efficiency, accuracy, sensitivity, precision, or specificity greater than 70%, greater than 71%, greater than 72%, greater than 73%, greater than 74%, greater than 75%, greater than 76%, greater than 77%, greater than 78%, greater than 79%, greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.
[0023] In one aspect, the present disclosure provides a method for sequencing a plurality of nucleic acid samples, comprising: using the plurality of nucleic acid samples to generate a first mixture of nucleic acid molecules and a second mixture of nucleic acid molecules; using a low volume liquid handler to extract and transfer a first volume of the first mixture of nucleic acid molecules and a second volume of the second mixture of nucleic acid molecules into a container to yield a pooled volume, wherein the pooled volume comprises a plurality of nucleic acid libraries comprising equimolar (or approximately equimolar) quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules, wherein the pooled volume is less than about 5 mΐ; diluting the pooled volume of a plurality of nucleic acid libraries to generate a diluted pooled volume of a plurality of nucleic acid libraries; and transferring the diluted pooled volume of nucleic acid libraries onto the sequencer.
[0024] In one aspect, the present disclosure provides a method for sequencing a nucleic acid sample, comprising: using the nucleic acid sample to generate a first mixture of nucleic acid molecules; transferring the first mixture of nucleic acid molecules or diluted first mixture of nucleic acid molecules onto the sequencer, wherein more than 10% of the first mixture of nucleic acid molecules is loaded onto the sequencer.
[0025] In one aspect, the present disclosure provides a method for sequencing a plurality of nucleic acid samples, comprising: using the plurality of nucleic acid samples to generate a first mixture of nucleic acid molecules and a second mixture of nucleic acid molecules; using a low volume liquid handler to extract and transfer a first volume of the first mixture of nucleic acid molecules and a second volume of the second mixture of nucleic acid molecules into a container to yield a pooled volume, wherein the pooled volume comprises a plurality of nucleic acid libraries comprising equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules; diluting the pooled volume of the plurality of nucleic acid libraries to generate a diluted pooled volume of the plurality of nucleic acid libraries, wherein the pooled volume of the plurality of nucleic acid libraries is from about 0.01% to about 20% of the diluted pooled volume; and transferring the diluted pooled volume of nucleic acid libraries onto the sequencer.
Definitions
[0026] As used herein, the term“biological sample,” refers to any suitable biological or contrived sample (for example, cells in centrifugation media) that comprises a nucleic acid, a protein, or any other biological analyte. The biological sample may be obtained from a subject.
A biological sample may be solid matter (e.g., biological tissue) or may be a fluid (e.g., a biological fluid). In general, a biological fluid can include any fluid associated with living organisms. Non-limiting examples of a biological sample include blood (or components of blood -e.g., white blood cells, red blood cells, platelets) obtained from any anatomical location (e.g., tissue, circulatory system, bone marrow) of a subject, cells obtained from any anatomical location of a subject, skin, heart, lung, kidney, breath, bone marrow, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, breast, pancreas, cerebral spinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, cavity fluids, sputum, pus, microbiota, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluid, tears, ocular fluids, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, fingernails, skin cells, plasma, nasal swab or nasopharyngeal wash, spinal fluid, cord blood, emphatic fluids, and/or other excretions or body tissues.
[0027] A biological sample may be obtained from a subject by any means known in the art. Non-limiting examples of means to obtain a biological sample directly from a subject include accessing the circulatory system (e.g., intravenously or intra-arterially via a syringe or other needle), collecting a secreted biological sample (e.g., feces, urine, sputum, saliva, etc.), surgically (e.g., biopsy), swabbing (e.g., buccal swab, oropharyngeal swab), pipetting, and breathing. Moreover, a biological sample may be obtained from any anatomical part of a subject where a desired biological sample is located. Alternatively, a sample can be constructed by mixing biological and non-biological substances.
[0028] As used herein, the term“subject,” generally refers to an entity or a medium that has testable or detectable biological information. A biological sample can be obtained from a subject. A subject can be a person or individual. A subject can be an invertebrate or a vertebrate, such as, for example, a mammal. Non-limiting examples of mammals include murines, simians, humans, farm animals, sport animals, and pets. [0029] As used herein, the term a "nucleic acid sample" refers to a collection of nucleic acid molecules. In some instances, the nucleic acid sample may be from a single biological source, e.g. one individual or one tissue sample, and in other instances, the nucleic acid sample may be a pooled sample, e.g., containing nucleic acids from more than one organism, individual or tissue. In some instances, the nucleic acid sample may be a recombinant nucleic acid. Non-limiting examples of synthetic nucleic acids include plasmids, viral vectors, and shRNAs. In some instances, the nucleic acid sample may be a synthetic nucleic acid. Non-limiting examples of synthetic nucleic acids include synthetic RNA such as RNA spike-ins, synthetic DNA such as sequins, primers, modified analogs of nucleotides such as morpholinos, and siRNA.
[0030] The term nucleic acid sample encompasses "nucleic acid library" or“library” which, as used herein, includes a nucleic acid library that has been prepared by any method known in the art. In some instances, providing the nucleic acid library may include the steps required for preparing the library, for example, including the process of incorporating one or more nucleic acid samples into a vector-based collection, such as by ligation into a vector and transformation of a host. In some instances, providing a nucleic acid library may include the process of incorporating a nucleic acid sample into a non-vector-based collection, such as by ligation to adaptors. The adaptors may anneal to PCR primers to facilitate amplification by PCR or may be universal primer regions such as, for example, sequencing tail adaptors. The adaptors may be universal sequencing adaptors. As used herein, the term“efficiency,” may refer to a measurable metric calculated as the division of the number of unique molecules for which sequences will be available after sequencing over the number of unique molecules originally present in the primary sample. Additionally, the term“efficiency” may also refer to reducing initial nucleic acid sample material required, decreasing sample preparation time, decreasing amplification processes, and/or reducing overall cost of nucleic acid library preparation. .
[0031] As used herein, the terms“accuracy,”“specificity”,“sensitivity,” and“precision” generally refers to sequencing or base calling accuracy, specificity, sensitivity, or precision, respectively. Accuracy, specificity, sensitivity, and precision are functions of the number of true positive base calls (TP), true negative base calls (TN), false positive base calls (FP), and false negative base calls (FN). A true positive is a base call for a particular base that correctly identifies the base. A true negative is a base call ruling out a particular base that correctly rules out the base. A false positive is a base call for a particular base that incorrectly identifies the base. A false negative is a base call ruling out a particular base that incorrectly rules out the base. Accuracy is measured as (TP + TN)/(TP + TN + FP +FN). Specificity is measured as (TN)/(TN + FP). Sensitivity is measured as (TP)/(TP + FN). Precision is measured as (TP)/(TP + FP).
[0032] As used herein, the term“barcode” may be a known sequence used to associate a polynucleotide fragment with the input polynucleotide or target polynucleotide from which it is produced. It can be a sequence of synthetic nucleotides or natural nucleotides. A barcode sequence may be contained within adapter sequences such that the barcode sequence is contained in the sequencing reads. Each barcode sequence may include at least 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, or more nucleotides in length. In some cases, barcode sequences may be of sufficient length and may be sufficiently different from one another to allow the identification of samples based on barcode sequences with which they are associated. In some cases, barcode sequences are used to tag and subsequently identify an“original” nucleic acid molecule (a nucleic acid molecule present in a sample from a subject). In some cases, a barcode sequence, or a combination of barcode sequences, is used in conjunction with endogenous sequence information to identify an original nucleic acid molecule. For example, a barcode sequence (or combination of barcode sequences) can be used with endogenous sequences adjacent to the barcodes (e.g., the beginning and end of the endogenous sequences) and/or with the length of the endogenous sequence.
[0033] As used herein, the term "next-generation sequencer" refers to a sequencer which is capable of next-generation sequencing. A next-generation sequencer can include a number of different sequencers, such as Illumina sequencers.
[0034] In some embodiments, nucleic acid molecules used herein can be subjected to a “tagmentation” or“ligation” reaction. “Tagmentation” combines the fragmentation and ligation reactions into a single step of the library preparation process. The tagged polynucleotide fragment is "tagged" with transposon end sequences during tagmentation and may further include additional sequences added during extension during a few cycles of amplification. Alternatively, the biological fragment can directly be“tagged,” for example, with ligation adapters, with or without a preceding“end preparation” reaction.
Methods for Reduction in Required Material for Shotgun Sequencing
[0035] In one aspect, the present disclosure provides a method for sequencing a nucleic acid sample. The nucleic acid sample may be a collection of nucleic acid molecules. The nucleic acid molecules may be DNA, plasmid DNA, RNA, messenger RNA (mRNA), transfer RNA (tRNA), microRNA (miRNA), or ribosomal RNA (rRNA). The nucleic acid sample may be isolated from a biological sample. The biological sample may be isolated from a subject. Non- limiting examples of the biological sample include blood, saliva, serum, plasma, circulating cells. The methods for nucleic acid sequencing, described herein, may be based on the reduction of nucleic acid sample volume required for sequencing.
[0036] The method for sequencing a nucleic acid sample, described herein, can use a nucleic acid sample to generate a first mixture of nucleic acid molecules. The first mixture of nucleic acid molecules can have a maximal volume of about 5 mΐ or more. The first mixture of nucleic acid molecules can have a maximal volume of about 10 mΐ or more. The first mixture of nucleic acid molecules can have a maximal volume of about 20 mΐ or more. The first mixture of nucleic acid molecules can have a maximal volume of about 30 mΐ or more. The first mixture of nucleic acid molecules can have a maximal volume of about 40 mΐ or more. The first mixture of nucleic acid molecules may have a volume of at least about 0.1 mΐ to about 1 mΐ at most. The first mixture of nucleic acid molecules can have a volume of at least about 1 mΐ to about 2 mΐ at most. The first mixture of nucleic acid molecules can have a volume of at least about 2 mΐ to about 3 mΐ at most. The first mixture of nucleic acid molecules can have a volume of at least about 3 mΐ to about 4 mΐ at most. The first mixture of nucleic acid molecules can have a volume of at least about 4 mΐ to about 5 mΐ at most. The first mixture of nucleic acid molecules can have a volume of at least about 1 mΐ to about 3 mΐ at most. The first mixture of nucleic acid molecules can have a volume of at least about 1 mΐ to about 4 mΐ at most. The first mixture of nucleic acid molecules can have a volume of at least about 1 mΐ to about 5 mΐ at most. The first mixture of nucleic acid molecules can have a volume of at least about 2 mΐ to about 3 mΐ at most. The first mixture of nucleic acid molecules can have a volume of at least about 2 mΐ to about 4 mΐ at most. The first mixture of nucleic acid molecules can have a volume of at least about 2 mΐ to about 5 mΐ at most. The first mixture of nucleic acid molecules can have a volume of at least about 3 mΐ to about 4 mΐ at most. The first mixture of nucleic acid molecules can have a volume of at least about 3 mΐ to about 5 mΐ or more. The first mixture of nucleic acid molecules can have a volume of about 0.1 mΐ. The first mixture of nucleic acid molecules can have a volume of about 0.2 mΐ. The first mixture of nucleic acid molecules can have a volume of about 0.3 mΐ. The first mixture of nucleic acid molecules can have a volume of about 0.4 mΐ. The first mixture of nucleic acid molecules can have a volume of about 0.5 mΐ. The first mixture of nucleic acid molecules can have a volume of about 0.6 mΐ. The first mixture of nucleic acid molecules can have a volume of about 0.7 mΐ. The first mixture of nucleic acid molecules can have a volume of about 0.8 mΐ. The first mixture of nucleic acid molecules can have a volume of about 0.9 mΐ. The first mixture of nucleic acid molecules can have a volume of about 1 mΐ. The first mixture of nucleic acid molecules can have a volume of about 2 mΐ. The first mixture of nucleic acid molecules can have a volume of about 3 mΐ. The first mixture of nucleic acid molecules can have a volume of about 4 mΐ. The first mixture of nucleic acid molecules can have a volume of about 4.5 mΐ. The first mixture of nucleic acid molecules can have a volume of about 5 mΐ.
[0037] The method for sequencing a nucleic acid sample, described herein, may comprise transferring the first mixture of nucleic acid molecules or a diluted first mixture of nucleic acid molecules onto a sequencer. The diluted first mixture of nucleic acid molecules may be prepared by diluting the first mixture with a first agent that denatures the nucleic acid molecules. The first agent that denatures the nucleic acid molecules may be sodium hydroxide.
Additionally, the first diluted mixture of nucleic acid molecules may be prepared by diluting the first mixture with a second agent that normalizes the pH of the first mixture. The agent that normalizes the pH of the first mixture may be hydrochloric acid. The first diluted mixture of nucleic acid molecules may be prepared by diluting the first mixture with a third agent to increase the total volume of the first diluted mixture. The third agent that increases the total volume of the first diluted mixture may be HT1 hybridization buffer.
[0038] The method for sequencing a nucleic acid sample, described herein, may comprise using the sequencer to subject the first mixture of nucleic acid molecules to sequencing to generate one or more sequences of the nucleic acid sample at an efficiency, accuracy, sensitivity, precision, or specificity greater than about 90%.
[0039] The efficiency may be greater than about 20% and less than 30%. The efficiency may be greater than about 30% and less than 40%. The efficiency may be greater than about 40% and less than 50%. The efficiency may be greater than about 50% and less than 60%. The efficiency may be greater than about 60% and less than 70%. The efficiency may be greater than about 70%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about 71%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about 72%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about 73%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about 74%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about 75%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about 76%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about 77%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about 78%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about 79%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about 80%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
81%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
82%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
83%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
84%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
85%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
86%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
87%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
88%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
89%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
91%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
92%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
93%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
94%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
95%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
96%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
97%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
98%. The efficiency, accuracy, sensitivity, precision, or specificity may be greater than about
99%.
[0040] The method may comprise providing a second nucleic acid sample. The method may comprise using the second nucleic acid sample to generate a second mixture of nucleic acid molecules.
The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined maximal volume of about 5 mΐ or more. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined maximal volume of about 10 mΐ or more. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined maximal volume of about 20 mΐ or more. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined maximal volume of about 30 mΐ or more. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined maximal volume of about 40 mΐ or more. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined maximal volume of 40 mΐ to 200mI.. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about 0.1 mΐ to about 1 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about 1 mΐ to about 2 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about 2 mΐ to about 3 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about 3 mΐ to about 4 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about 4 mΐ to about 5 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about 1 mΐ to about 3 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about
1 mΐ to about 4 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about 1 mΐ to about 5 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about 2 mΐ to about 3 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about 2 mΐ to about 4 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about
2 mΐ to about 5 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about 3 mΐ to about 4 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of at least about 3 mΐ to about 5 mΐ at most. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of about 0.1 mΐ. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of about 0.2 mΐ. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of about 0.3 mΐ. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of about 0.4 mΐ. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of about 0.5 mΐ. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of about 0.6 mΐ. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of about 0.7 mΐ. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of about 0.8 mΐ. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of about 0.9 mΐ. The first mixture of nucleic acid molecules can have a combined volume of about 1 mΐ. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of about 2 mΐ. The first mixture of nucleic acid molecules can have a combined volume of about 3 mΐ. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a combined volume of about 4 mΐ. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a volume of about 4.5 mΐ. The first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules can have a volume of about 5 mΐ.
[0041] The method can comprise pooling the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules, thereby producing a pooled mixture of nucleic acid molecules. The method can comprise transferring the pooled mixture of nucleic acid molecules of nucleic acid molecules onto the sequencer.
[0042] In some instances, the mixture of nucleic acid molecules can comprise a mixture barcode. The mixture barcode can be identical among nucleic acids within the mixture of nucleic acid molecules. The mixture barcode can be unique between mixtures of nucleic acid molecules. Adapters comprising barcode sequences may be attached to the mixture of nucleic acid molecules using a variety of standard techniques known and available in the art. For example, adapters can be attached to polynucleotide fragments by a ligase or a polymerase. The ligase may be any enzyme capable of ligating an adapter sequence or any oligonucleotide to polynucleotides. Suitable ligases include T4 DNA ligase, which is commercially available. Methods for using ligases are also well known in the art.
[0043] The method can comprise diluting the first mixture of nucleic acid molecules to a maximal volume of about 5 mΐ, thereby producing a diluted first mixture of nucleic acid molecules. In some cases, the first mixture of nucleic acid molecules is not amplified prior to transferring onto a sequencer. In some cases, amplification of the first mixture of nucleic acid molecules is not required prior to producing a pooled mixture of nucleic acid libraries.
Amplification, for example by polymerase chain reaction (PCR), is usually performed in order to generate sufficient starting material to complete the sequencing process. However, the methods provided herein allow for sequencing without the need for amplification (or with reduced amplification) to produce enough starting material for sequencing because of decreased sample loss during pooling of one or more nucleic acid libraries prior to sequencing. The methods provided herein can require fewer amplification cycles because a decreased amount of starting nucleic acid material is required. The methods provided herein can require less amplification and thus, yield better library complexity and/or reduce PCR amplification-driven biases. In some cases, the first mixture of nucleic acid molecules may be amplified prior to transferring onto a sequencer. In some cases, the first mixture of nucleic acid molecules may be amplified prior to producing a pooled mixture of nucleic acid libraries. In some cases, the first mixture of nucleic acid molecules may not require additional PCR cycles prior to producing a pooled mixture of nucleic acid libraries or prior to producing a pooled mixture of nucleic acid libraries.
[0044] The first mixture of nucleic acid molecules may require about 1 to about 10 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods. The first mixture of nucleic acid molecules may require about 1 to about 5 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods. The first mixture of nucleic acid molecules may require about 1 to about 3 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods. The first mixture of nucleic acid molecules may require about 1 less additional PCR cycle prior to sequencing a nucleic acid sample, compared to current sequencing methods. The first mixture of nucleic acid molecules may require about 2 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods. The first mixture of nucleic acid molecules may require about 3 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods. The first mixture of nucleic acid molecules may require about 4 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods. The first mixture of nucleic acid molecules may require about 5 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods. The first mixture of nucleic acid molecules may require about 6 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods. The first mixture of nucleic acid molecules may require about 7 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods. The first mixture of nucleic acid molecules may require about 8 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods. The first mixture of nucleic acid molecules may require about 9 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods. The first mixture of nucleic acid molecules may require about 10 less additional PCR cycles prior to sequencing a nucleic acid sample, compared to current sequencing methods.
[0045] The first mixture of nucleic acid molecules can comprise tagged nucleic acid molecules. The tagged nucleic acid molecules can be non-uniquely tagged.“Tagmentation” is a method to prepare genomic nucleic acid libraries by using a transposase enzyme that simultaneously fragments and tags nucleic acid molecules in a single reaction. Tagmentation is possible due to the transposome complex which comprises an engineered transposase pre-loaded with two double-stranded sequencing adapters. The transposome simultaneously fragments the DNA and inserts the adapters. The full adapter sequences are completed during subsequent PCR cycling, after which the library template is ready for quantitation and loading onto the sequencer.
[0046] The method can comprise denaturing the first mixture of nucleic acid molecules. For example, the first mixture of nucleic acid molecules may be denatured by contacting the first mixture of nucleic acid molecules with sodium hydroxide. In some instances, the sodium hydroxide is at a concentration of about 0.2 N. The total denaturation time may be about 5 minutes. After denaturation, the first mixture of nucleic acid molecules may be neutralized by contacting the first mixture of nucleic acid molecules with hydrochloric acid or Tris-HCl. The Tris-HCl may be at a concentration of about 200 mM and a pH of about 7.
[0047] The method may comprise diluting the first mixture of nucleic acid molecules. The dilution can be a dilution to a maximal volume of about 1.3 ml. The dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 2 ml at most. The dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1.9 ml at most. The dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1.8 ml at most. The dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1.7 ml at most. The dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1.6 ml at most. The dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1.5 ml at most. The dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1.4 ml at most. The dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1.3 ml at most. The dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1.2 ml at most. The dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1.1 ml at most. The dilution can be a dilution to a maximal volume of at least about 0.5 ml to about 1 ml at most. The dilution can be a dilution to a maximal volume of about 0.5 ml. The dilution can be a dilution to a maximal volume of about 0.6 ml. The dilution can be a dilution to a maximal volume of about 0.7 ml. The dilution can be a dilution to a maximal volume of about 0.8 ml. The dilution can be a dilution to a maximal volume of about 0.9 ml. The dilution can be a dilution to a maximal volume of about 1 ml. The dilution can be a dilution to a maximal volume of about 1.1 ml. The dilution can be a dilution to a maximal volume of about 1.2 ml. The dilution can be a dilution to a maximal volume of about 1.3 ml. The dilution can be a dilution to a maximal volume of about 1.4 ml. The dilution can be a dilution to a maximal volume of about 1.5 ml. The dilution can be a dilution to a maximal volume of about 1.6 ml. The dilution can be a dilution to a maximal volume of about 1.7 ml. The dilution can be a dilution to a maximal volume of about 1.8 ml. The dilution can be a dilution to a maximal volume of about 1.9 ml. The dilution can be a dilution to a maximal volume of about 2 ml. The dilution can be a dilution to a maximal volume of about 2.1 ml. The dilution can be a dilution to a maximal volume of about 2.2 ml. The dilution can be a dilution to a maximal volume of about
2.3 ml. The dilution can be a dilution to a maximal volume of about 2.4 ml. The dilution can be a dilution to a maximal volume of about 2.5 ml. The dilution can be a dilution to a maximal volume of about 2.6 ml. The dilution can be a dilution to a maximal volume of about 2.7 ml. The dilution can be a dilution to a maximal volume of about 2.8 ml. The dilution can be a dilution to a maximal volume of about 2.9 ml. The dilution can be a dilution to a maximal volume of about 3 ml. The dilution can be a dilution to a maximal volume of about 3.1 ml. The dilution can be a dilution to a maximal volume of about 3.2 ml. The dilution can be a dilution to a maximal volume of about 3.3 ml. The dilution can be a dilution to a maximal volume of about
3.4 ml. The dilution can be a dilution to a maximal volume of about 3.5 ml. The dilution can be a dilution to a maximal volume of about 3.6 ml. The dilution can be a dilution to a maximal volume of about 3.7 ml. The dilution can be a dilution to a maximal volume of about 3.8 ml. The dilution can be a dilution to a maximal volume of about 3.9 ml. The dilution can be a dilution to a maximal volume of about 4 ml. The dilution can be a dilution to a maximal volume of about 5 ml. The dilution can be a dilution to a maximal volume of about 5 ml to about 10 ml. The dilution can be a dilution to a maximal volume of about 5 ml to about 10 ml. The dilution can be a dilution to a maximal volume of about 10 ml. The dilution can be a dilution to a maximal volume of about 15 ml.
[0048] The method can comprise loading more than 10% of the first mixture of nucleic acid molecules onto the sequencer. The sequencer may be a next-generation sequencer, such as an Illumina sequencer. Non-limiting examples of sequencers include the MiniSeq Sequencing System, MiSeq Series, NextSeq Series, HiSeq Series, HiSeq X Series, and NovaSeq 6000 System. [0049] In another aspect, the present disclosure provides another method for sequencing a plurality of nucleic acid samples. The method can comprise using the plurality of nucleic acid samples to generate a first mixture of nucleic acid molecules and a second mixture of nucleic acid molecules.
[0050] The method can comprise using a low volume liquid handler to extract and transfer a first volume of the first mixture of nucleic acid molecules and a second volume of the second mixture of nucleic acid molecules into a container to yield a pooled volume. The low volume liquid handler may be an acoustic liquid handler, a piezoelectric pipette, an ink-type dispenser, a microsyringe, a pressure-sensing system, or a pin tool. Then, the method can comprise extracting and transferring the first volume of the first mixture and the second volume of the second mixture via any low volume liquid handling device known in the art. An acoustic liquid handler transfers and handles liquid volumes by using pulses of acoustic energy (i.e. acoustic volume transfer). The acoustic energy pulses move nanoliter to microliter range of fluids, without any physical contact (i.e. without the use of pipettes). Acoustic volume transfer can precisely transfer volumes of at least about 2.5 nl to about 25 nl. Utilizing a low volume liquid handling technology to extract and transfer the first volume of the first mixture and the second volume of the second mixture of nucleic acid molecules into a container provides for a method to pool a large number of mixtures of nucleic acid molecules (i.e. nucleic acid libraries) without the need for multiple or serial dilutions.
[0051] The pooled volume can comprise a plurality of nucleic acid libraries comprising equimolar (or approximately equimolar) quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules. In some cases, the plurality of nucleic acid libraries can comprise non-equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules. The plurality of nucleic acid libraries can comprise approximately equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules. Approximately equimolar quantities are equimolar within a margin of error (e.g., within a 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% margin of error).
[0052] The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 20% (i.e. about ± 20%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin about 19% (i.e. about ± 19%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 18% (i.e. about ± 18%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 17% (i.e. about ± 17%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 16% (i.e. about ± 16%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 15% (i.e. about ± 15%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 14% (i.e. about ± 14%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 13% (i.e. about ± 13%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 12% (i.e. about ± 12%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an % error margin of about 11 (i.e. about ±
11%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 10% (i.e. about ± 10%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 9% (i.e. about ± 9%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 8% (i.e. about ± 8%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 7% (i.e. about ± 7%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 6% (i.e. about ± 6%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 5% (i.e. about ± 5%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 4% (i.e. about ± 4%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 3% (i.e. about ± 3%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 2% (i.e. about ± 2%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 1% (i.e. about ± 1%). The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 20% to about 15%. The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 15% to about 10%. The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 10% to about 5%. The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 5% to about 1%. The plurality of nucleic acid libraries may comprise equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules within an error margin of about 1% to about 0.1%.
[0053] The pooled volume of the plurality of nucleic acid libraries may be at least about 0.1 nl to about 300 mΐ or more. The pooled volume of the plurality of nucleic acid libraries may be at least about 1 nl to about 5 mΐ or more. The pooled volume of the plurality of nucleic acid libraries may be at least about 5 mΐ to about 10 mΐ or more. The pooled volume of the plurality of nucleic acid libraries may be at least about 10 mΐ to about 50 mΐ or more. The pooled volume of the plurality of nucleic acid libraries may be at least about 50 mΐ to about 100 mΐ or more. The pooled volume of the plurality of nucleic acid libraries may be at least about 100 mΐ to about 200 mΐ or more. The pooled volume of the plurality of nucleic acid libraries may be at least about 200 mΐ to about 300 mΐ or more.
[0054] The pooled volume of the plurality of nucleic acid libraries may be at least about 1 mΐ. The pooled volume of the plurality of nucleic acid libraries may be at least about 4.5 mΐ. The pooled volume of the plurality of nucleic acid libraries may be at least about 5 mΐ. The pooled volume of the plurality of nucleic acid libraries may be at least about 10 mΐ. The pooled volume of the plurality of nucleic acid libraries may be at least about 15 mΐ. The pooled volume of the plurality of nucleic acid libraries may be at least about 20 mΐ. The pooled volume of the plurality of nucleic acid libraries may be at least about 100 mΐ. The pooled volume of the plurality of nucleic acid libraries may be at least about 200 mΐ. The pooled volume of the plurality of nucleic acid libraries may be at least about 300 mΐ.
[0055] The pooled volume can be less than about 5 mΐ. The pooled volume can have a volume of at least about 0.1 mΐ to about 1 mΐ at most. The pooled volume can have a volume of at least about 1 mΐ to about 2 mΐ at most. The pooled volume can have a volume of at least about 2 mΐ to about 3 mΐ at most. The pooled volume can have a volume of at least about 3 mΐ to about 4 mΐ at most. The pooled volume can have a volume of at least about 4 mΐ to about 5 mΐ at most. The pooled volume can have a volume of at least about 1 mΐ to about 3 mΐ at most. The pooled volume can have a volume of at least about 1 mΐ to about 4 mΐ at most. The pooled volume can have a volume of at least about 1 mΐ to about 5 mΐ at most. The pooled volume can have a volume of at least about 2 mΐ to about 3 mΐ at most. The pooled volume can have a volume of at least about 2 mΐ to about 4 mΐ at most. The pooled volume can have a volume of at least about 2 mΐ to about 5 mΐ at most. The pooled volume can have a volume of at least about 3 mΐ to about 4 mΐ at most. The pooled volume can have a volume of at least about 3 mΐ to about 5 mΐ at most.
The pooled volume can have a volume of about 0.1 mΐ. The pooled volume can have a volume of about 0.2 mΐ. The pooled volume can have a volume of about 0.3 mΐ. The pooled volume can have a volume of about 0.4 mΐ. The pooled volume can have a volume of about 0.5 mΐ. The pooled volume can have a volume of about 0.6 mΐ. The pooled volume can have a volume of about 0.7 mΐ. The pooled volume can have a volume of about 0.8 mΐ. The pooled volume can have a volume of about 0.9 mΐ. The pooled volume can have a volume of about 1 mΐ. The pooled volume can have a volume of about 2 mΐ. The pooled volume can have a volume of about 3 mΐ. The pooled volume can have a volume of about 4 mΐ. The pooled volume can have a volume of about 4.5 mΐ. The pooled volume can have a volume of about 5 mΐ.
[0056] The method can comprise diluting the pooled volume of a plurality of nucleic acid libraries to generate a diluted pooled volume of a plurality of nucleic acid libraries. The method can comprise transferring the diluted pooled volume of nucleic acid libraries onto the sequencer. The method can comprise loading more than 10% of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method can comprise loading more than about 20% of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method can comprise loading more than about 30% of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method can comprise loading more than about 30% of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method can comprise loading more than about 40% of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method can comprise loading more than about 50% of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method can comprise loading more than about 60% of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method can comprise loading more than about 70% of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method can comprise loading more than about 80% of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method can comprise loading more than about 90% of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method can comprise loading about 100% of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer.
[0057] The method may comprise loading at least about 10% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 20% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 30% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 40% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 50% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 60% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 70% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 80% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 90% to about 100% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer.
[0058] The method may comprise loading at least about 10% to about 90% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 20% to about 90% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 30% to about 90% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 40% to about 90% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 50% to about 90% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 60% to about 90% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 70% to about 90% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 80% to about 90% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer.
[0059] The method may comprise loading at least about 10% to about 80% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 20% to about 80% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 30% to about 80% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 40% to about 80% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 50% to about 80% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 60% to about 80% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 70% to about 80% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer.
[0060] The method may comprise loading at least about 10% to about 70% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 20% to about 70% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 30% to about 70% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 40% to about 70% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 50% to about 70% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 60% to about 70% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer.
[0061] The method may comprise loading at least about 10% to about 60% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 20% to about 60% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 30% to about 60% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 40% to about 60% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 50% to about 60% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer.
[0062] The method may comprise loading at least about 10% to about 50% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 20% to about 50% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 30% to about 50% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 40% to about 50% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer.
[0063] The method may comprise loading at least about 10% to about 40% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 20% to about 40% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 30% to about 40% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 10% to about 30% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 20% to about 30% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer. The method may comprise loading at least about 10% to about 20% at most of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules onto the sequencer.
[0064] The pooled volume of a plurality of nucleic acid libraries can be about 0.3% of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 1% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 0.9% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 0.8% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 0.7% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 0.6% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 0.5% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 0.4% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 0.3% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be at least about 0.1% to about 0.2% at most of the diluted pooled volume.
[0065] The pooled volume of a plurality of nucleic acid libraries may be about 0.1% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be about 0.2% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be about 0.3% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be about 0.4% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be about 0.6% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be about 0.7% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be about 0.8% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be about 0.9% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be about 1% at most of the diluted pooled volume. The pooled volume of a plurality of nucleic acid libraries may be about 1% to about 10% of the diluted pool volume.
[0066] In another aspect, the present disclosure provides yet another method for sequencing a nucleic acid sample. The method may comprise using the nucleic acid sample to generate a first mixture of nucleic acid molecules.
[0067] The method may comprise transferring the first mixture of nucleic acid molecules or diluted first mixture of nucleic acid molecules onto the sequencer. The method may comprise loading more than 10% of the first mixture of nucleic acid molecules onto the sequencer. The method may comprise providing a second nucleic acid sample and using the second nucleic acid sample to generate a second mixture of nucleic acid molecules. The method may comprise pooling the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules, thereby producing a pooled mixture of nucleic acid molecules. The method may comprise transferring the pooled mixture of nucleic acid molecules or diluted pooled mixture of nucleic acid molecules onto the sequencer. The method may comprise loading more than 10% of the pooled mixture of nucleic acid molecules onto the sequencer. The method may comprise a first mixture of nucleic acid molecules with a maximal volume of about 5 mΐ.
[0068] In another aspect, the present disclosure provides yet another method for sequencing a plurality of nucleic acid samples. The method may comprise using the plurality of nucleic acid samples to generate a first mixture of nucleic acid molecules and a second mixture of nucleic acid molecules.
[0069] Next, the method may comprise using a low volume liquid handler to extract and transfer a first volume of the first mixture of nucleic acid molecules and a second volume of the second mixture of nucleic acid molecules into a container to yield a pooled volume, wherein the pooled volume comprises a plurality of nucleic acid libraries comprising equimolar quantities of the first mixture of nucleic acid molecules and the second mixture of nucleic acid molecules.
[0070] The method may comprise diluting the pooled volume of the plurality of nucleic acid libraries to generate a diluted pooled volume of the plurality of nucleic acid libraries. The pooled volume of the plurality of nucleic acid libraries may be from about 0.1% to 20% of the diluted pooled volume.
[0071] Next, the method may comprise transferring the diluted pooled volume of nucleic acid libraries onto the sequencer. The pooled volume of the plurality of nucleic acid libraries may be diluted once. The total volume of the pool of nucleic acid libraries may be less than about 40 mΐ.
Computer control systems
[0072] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 4 shows a computer system 401 that is programmed or otherwise configured to direct the extraction and transferring of one or more mixtures of nucleic acid molecules into a container. The computer system 401 can regulate various aspects of sample preparation of the present disclosure, such as, for example, generating a pooled mixture of nucleic acid molecules and a nucleic acid library template that can be ready for loading onto a sequencer. The computer system 401 can generate a mixture of nucleic acid molecule samples. The computer system 401 can direct a liquid handler to dilute a mixture of nucleic acid molecule samples. The computer system 401 can direct a liquid handler to extract and transfer first and second mixtures of nucleic acid molecule samples to generate a pooled volume of nucleic acid libraries. The computer system 401 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.
[0073] The computer system 401 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 405, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 401 also includes memory or memory location 410 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 415 (e.g., hard disk), communication interface 420 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 425, such as cache, other memory, data storage and/or electronic display adapters. The memory 410, storage unit 415, interface 420 and peripheral devices 425 are in communication with the CPU 405 through a communication bus (solid lines), such as a motherboard. The storage unit 415 can be a data storage unit (or data repository) for storing data. The computer system 401 can be operatively coupled to a computer network (“network”) 430 with the aid of the communication interface 420. The network 430 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 430 in some cases is a telecommunication and/or data network. The network 430 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 430, in some cases with the aid of the computer system 401, can implement a peer-to-peer network, which may enable devices coupled to the computer system 401 to behave as a client or a server.
[0074] The CPU 405 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 410. The instructions can be directed to the CPU 405, which can subsequently program or otherwise configure the CPU 405 to implement methods of the present disclosure. Examples of operations performed by the CPU 405 can include fetch, decode, execute, and writeback.
[0075] The CPU 405 can be part of a circuit, such as an integrated circuit. One or more other components of the system 401 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).
[0076] The storage unit 415 can store files, such as drivers, libraries and saved programs. The storage unit 415 can store user data, e.g., user preferences and user programs. The computer system 401 in some cases can include one or more additional data storage units that are external to the computer system 401, such as located on a remote server that is in communication with the computer system 401 through an intranet or the Internet.
[0077] The computer system 401 can communicate with one or more remote computer systems through the network 430. For instance, the computer system 401 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 401 via the network 430.
[0078] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 401, such as, for example, on the memory 410 or electronic storage unit 415. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 405. In some cases, the code can be retrieved from the storage unit 415 and stored on the memory 410 for ready access by the processor 405. In some situations, the electronic storage unit 415 can be precluded, and machine-executable instructions are stored on memory 410.
[0079] The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
[0080] Aspects of the systems and methods provided herein, such as the computer system 401, can be embodied in programming. Various aspects of the technology may be thought of as “products” or“articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.
“Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine“readable medium” refer to any medium that participates in providing instructions to a processor for execution.
[0081] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
[0082] The computer system 401 can include or be in communication with an electronic display 435 that comprises a user interface (Ed) 440 for providing, for example, sample tracking, which indicates an amount of sample diluted, extracted, or transferred into a container. The UI 440 may also provide a protocol or a plurality of protocols that direct a liquid handler. Examples of UFs include, without limitation, a graphical user interface (GUI) and web-based user interface.
[0083] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 405. The algorithm can, for example, direct the dilution, extraction, and generation of a pooled volume of nucleic acid libraries.
EXAMPLES
Example 1 - Loading of a Plurality of Sample Libraries onto a Sequencer Using a
Conventional Method
[0084] FIG. 1A shows a conventional method for preparing a sample nucleic acid library prior to loading onto a sequencer. As shown in FIG. 1A, a plurality of sample nucleic acid libraries are pooled while maintaining an equimolar concentration and yield a library pool. The library pool is then denatured by adding sodium hydroxide, neutralized by adding hydrochloric acid, and diluted to a final volume of 1000 mΐ with HT1 hybridization buffer. A serial dilution is performed by acquiring an aliquot of 117 mΐ of the denatured and diluted library pool and further diluting with HT1 hybridization buffer to a final volume of 1300 mΐ, thereby generating a library template that is ready for loading onto a sequencer. The method illustrated by FIG. 1A leads to a loss of about 88% of a sample nucleic acid library. Example 2 - Loading of a Plurality of Sample Libraries onto a Sequencer Using a Method Described Herein
[0085] FIG. IB shows a method disclosed herein for preparing a sample nucleic acid library prior to loading onto a sequencer. As shown in FIG. IB, a plurality of sample nucleic acid libraries are transferred into a container, while maintaining an approximately equimolar concentration, and in order to generate a library pool. The sample library pool volume is then increased to a total of 4.5 mΐ, as needed. While omitting any additional transfer steps to additional containers, the library pool is then denatured by adding 4.5 mΐ sodium hydroxide, neutralized by adding 4.5 mΐ hydrochloric acid, and diluted to a final diluted volume of 1300 mΐ with HT1 hybridization buffer, thereby generating a library template that is ready for loading onto a sequencer. A serial dilution is not performed nor required, as shown in FIG IB. In addition, the nucleic acid library pool volume comprises about 0.3% of the final diluted volume. The method illustrated by FIG. IB, also described herein, prevents a loss of about 88% of a sample nucleic acid library.
[0086] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the
embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A method for sequencing a nucleic acid sample, comprising:
(a) using said nucleic acid sample to generate a first mixture of nucleic acid molecules, wherein said first mixture of nucleic acid molecules has a maximal volume of about 5 mΐ;
(b) transferring said first mixture of nucleic acid molecules or diluted first mixture of nucleic acid molecules onto said sequencer; and
(c) using said sequencer to subject said first mixture of nucleic acid
molecules to sequencing to generate one or more sequences of said nucleic acid sample at a efficiency greater than 90% .
2. The method of claim 1, further comprising providing a second nucleic acid
sample; using said second nucleic acid sample to generate a second mixture of nucleic acid molecules, wherein said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules have a combined maximal volume of about 5 mΐ; pooling said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules, thereby producing a pooled mixture of nucleic acid molecules; and transferring said pooled mixture of nucleic acid molecules of nucleic acid molecules onto said sequencer.
3. The method of claim 2, wherein each mixture of nucleic acid molecules comprises a mixture barcode, wherein each mixture barcode is identical among nucleic acids within the mixture of nucleic acid molecules and unique between mixtures of nucleic acid molecules.
4. The method of claim 1, wherein (a) further comprises diluting said first mixture of nucleic acid molecules to a maximal volume of about 5 mΐ, thereby producing a diluted first mixture of nucleic acid molecules.
5. The method of claim 1, wherein said first mixture of nucleic acid molecules is not amplified prior to said transferring.
6. The method of claim 1, wherein said first mixture of nucleic acid molecules
comprises tagged nucleic acid molecules.
7. The method of clam 6, wherein said tagged nucleic acid molecules are non- uniquely tagged.
8. The method of claim 1, further comprising denaturing said first mixture of nucleic acid molecules.
9. The method of claim 1, further comprising diluting said first mixture of nucleic acid molecules.
10. The method of claim 9, wherein said dilution is a dilution to a maximal volume of about 1.3 ml.
11. The method of claim 1, wherein more than 10% of said first mixture of nucleic acid molecules is loaded onto said sequencer.
12. A method for sequencing a plurality of nucleic acid samples, comprising:
(a) using said plurality of nucleic acid samples to generate a first mixture of nucleic acid molecules and a second mixture of nucleic acid molecules;
(b) using a low volume liquid handler to extract and transfer a first volume of said first mixture of nucleic acid molecules and a second volume of said second mixture of nucleic acid molecules into a container to yield a pooled volume, wherein said pooled volume comprises a plurality of nucleic acid libraries comprising equimolar quantities of said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules, wherein said pooled volume is less than about 5 mΐ;
(c) diluting said pooled volume of a plurality of nucleic acid libraries to
generate a diluted pooled volume of a plurality of nucleic acid libraries; and
(d) transferring said diluted pooled volume of nucleic acid libraries onto said sequencer.
13. The method of claim 12, wherein more than 10% of said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules is loaded onto said sequencer.
14. The method of claim 12, wherein said pooled volume of a plurality of nucleic acid libraries is about 0.3% of said diluted pooled volume.
15. The method of claim 12, wherein said low volume liquid handler is an acoustic liquid handler, a piezoelectric pipette, an ink-type dispenser, a microsyringe, a pressure-sensing system, or a pin tool.
16. A method for sequencing a nucleic acid sample, comprising: (a) using said nucleic acid sample to generate a first mixture of nucleic acid molecules;
(b) transferring said first mixture of nucleic acid molecules or diluted first mixture of nucleic acid molecules onto said sequencer,
wherein more than 10% of said first mixture of nucleic acid molecules is loaded onto said sequencer.
17. The method of claim 16, further comprising providing a second nucleic acid sample; using said nucleic acid sample to generate a second mixture of nucleic acid molecules; pooling said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules, thereby producing a pooled mixture of nucleic acid molecules; and transferring said pooled mixture of nucleic acid molecules or diluted pooled mixture of nucleic acid molecules onto said sequencer; wherein more than 10% of said pooled mixture of nucleic acid molecules is loaded onto said sequencer.
18. The method of claim 17, wherein each mixture of nucleic acid molecules
comprises a mixture barcode, wherein each mixture barcode is identical among nucleic acids within the mixture of nucleic acid molecules and unique between mixtures of nucleic acid molecules.
19. The method of claim 16, wherein (a) further comprises diluting said first mixture of nucleic acid molecules, thereby producing a diluted first mixture of nucleic acid molecules.
20. The method of claim 16, wherein said first mixture of nucleic acid molecules or diluted first mixture of nucleic acid molecules is not amplified prior to said transferring.
21. The method of claim 16, wherein said first mixture of nucleic acid molecules comprises tagged nucleic acid molecules.
22. The method of clam 21, wherein said tagged nucleic acid molecules are non- uniquely tagged.
23. The method of claim 16, further comprising denaturing said first mixture of nucleic acid molecules or diluted first mixture of nucleic acid molecules.
24. The method of claim 16, wherein said first mixture of nucleic acid molecules has a maximal volume of about 5 mΐ.
25. A method for sequencing a plurality of nucleic acid samples, comprising: (a) using said plurality of nucleic acid samples to generate a first mixture of nucleic acid molecules and a second mixture of nucleic acid molecules;
(b) using a low volume liquid handler to extract and transfer a first volume of said first mixture of nucleic acid molecules and a second volume of said second mixture of nucleic acid molecules into a container to yield a pooled volume, wherein said pooled volume comprises a plurality of nucleic acid libraries comprising equimolar quantities of said first mixture of nucleic acid molecules and said second mixture of nucleic acid molecules;
(c) diluting said pooled volume of said plurality of nucleic acid libraries to generate a diluted pooled volume of said plurality of nucleic acid libraries, wherein said pooled volume of said plurality of nucleic acid libraries is from about 0.01% to 20% of said diluted pooled volume; and
(d) transferring said diluted pooled volume of nucleic acid libraries onto said sequencer.
26. The method of claim 25, wherein said pooled volume of said plurality of nucleic acid libraries is diluted once.
27. The method of claim 25, wherein the total volume of said pool of nucleic acid libraries is less than about 5 mΐ.
28. The method of claim 25, wherein said low volume liquid handler is an acoustic liquid handler, a piezoelectric pipette, an ink-type dispenser, a microsyringe, a pressure-sensing system, or a pin tool.
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