WO2019018561A1

WO2019018561A1 - Solid-phase genomic library generation for high-throughput sequencing

Info

Publication number: WO2019018561A1
Application number: PCT/US2018/042746
Authority: WO
Inventors: Kristian Andersen; Glenn OLIVEIRA
Original assignee: The Scripps Research Institute
Priority date: 2017-07-19
Filing date: 2018-07-18
Publication date: 2019-01-24

Abstract

Provided herein are methods and apparatuses for generating a template library for a polynucleotide sequencing reaction, comprising: immobilizing a first primer sequence on a solid surface; appending a probe to the first primer sequence; hybridizing to the probe a polynucleotide that is complementary in sequence to the target polynucleotide to be sequenced; polymerizing the target polynucleotide; and attaching a second primer sequence to the target polynucleotide.

Description

SOLID-PHASE GENOMIC LIBRARY GENERATION FOR HIGH-THROUGHPUT

SEQUENCING

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/534,434 filed on July 19, 2017, the contents of which are hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT.

This invention was made with government support under grant U11TR001114 awarded by National Center for Advancing Translational Sciences (NCATS). The U.S. government has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure relates to solid phase genomic library generation for high- throughput sequencing.

BACKGROUND OF THE DISCLOSURE

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Clonal, high-throughput (HTP) sequencing technologies include a manual prerequisite step, commonly referred to as 'library preparation', before they are able to interpret a nucleic acid sequence. The traditional sequencing library preparation protocol consists of three primary steps: fragmentation, adaptor ligation, and amplification. DNA molecules are first mechanically or enzymatically fragmented into 200- 400 bp, and then sequencing adaptors are ligated to the fragments. As one example, for the Illumina sequencing technology, library preparation consists of appending universal adapter sequences, referred to as 'P5' and 'P7' adapters, to flank any nucleic acid fragments of interest. Finally, after several cycles of PCR, the DNA library is ready to go through several quality control steps and load into the NGS instrument. These steps typically take 8 to 10 hours of hands-on work and require expensive equipment (e.g. Covaris) and trained personnel. See Feng, K et al, "Next generation sequencing library construction on a surface," BMC Genomics, 2018, 19:416. Similarly Farias-Hesson, E. et al notes that DNA library preparation is time-consuming and requires highly trained and qualified personnel to perform nearly 60 sub-step operations to prepare usable samples, which can take about 12 hours to prepare just one sample. See Farias- Hesson, E. et al, "Semi- Automated Library Preparation for High-Throughput DNA Sequencing Platforms," Journal of Biomedicine and Biotechnology, Volume 2010.

Thus, there remains a need in the art for new methods for expediting the time consuming and laborious library preparation before loading a template on the flow-cell, and make library preparation faster and less laborious by reducing or eliminating some or all of the manual steps.

SUMMARY OF THE DISCLOSURE

Various embodiments disclosed herein comprise a method of generating a template library for a polynucleotide sequencing reaction, comprising: immobilizing a first primer sequence on a solid surface; appending a probe to the first primer sequence; hybridizing to the probe a polynucleotide that is complementary in sequence to the target polynucleotide to be sequenced; polymerizing the target polynucleotide; and attaching a second primer sequence to the target polynucleotide. In one embodiment, the first primer sequence is a P7 primer sequence. In one embodiment, the second primer sequence is a P5 primer sequence. In one embodiment, the solid surface comprises a flow cell surface. In one embodiment, the flow cell comprises a Hiseq flowcell. In one embodiment, the flow cell comprises a Miseq flowcell. In one embodiment, the polynucleotide complementary in sequence to the target polynucleotide is a RNA. In one embodiment, the target polynucleotide is a cDNA. In one embodiment, the polymerization step comprises reacting with a reaction mixture having nucleotides. In one embodiment, the nucleotides comprise a mixture of standard nucleotide triphosphates and modified dideoxynucleotides with an azido modification. In one embodiment, the polymerization reaction is terminated when a modified dideoxynucleotides having an azido modification is encountered, and the grafted polynucleotide comprises a terminal 3 '-azido group. In one embodiment, the attaching step comprises attachment by click chemistry ligation, and wherein the second primer sequence comprises a P5 primer sequence having a 5' alkyne group. In one embodiment, the polymerization step further comprises attaching poly-C residues at the 3' end of the polymerized DNA molecule. In one embodiment, the second primer sequence is attached to the target polynucleotide by using template switching, Tn5 transposition, ligation, DNA/RNA nanoballs/rolling circle amplification, bridge amplification, template walking, emulsion PCR on beads, and/or click reaction. In one embodiment, the probe is specific to the polynucleotide being sequenced. In one embodiment, the probe is a fragment of a genome sequence of an organism. In one embodiment, the organism is a human. In one embodiment, the organism is a bacteria or virus. In one embodiment, the probe comprises a polynucleotide comprising a random sequence that complements a variety of nucleotide sequences in a target polynucleotide. In one embodiment, the polymerization step further comprises polymerization by an enzyme. In one embodiment, the enzyme comprises a DNA polymerase. In one embodiment, the enzyme comprises a reverse transcriptase enzyme. In one embodiment, the reverse transcriptase enzyme is Moloney Murine Lukemia Virus (MMLV). In one embodiment, the method further comprises sequencing the population of target nucleotides by high throughput sequencing.

Various embodiments disclosed herein further include an apparatus for sequencing a target polynucleotide, comprising: a solid surface comprising target polynucleotides immobilized on the surface as disclosed herein; an amplifier adapted to amplify the target polynucleotide sequences; and a polynucleotide sequencer adapted to sequentially and accurately sequence the amplified target polynucleotides. In one embodiment, the amplifier uses clonal amplification. In one embodiment, the polynucleotide sequencer uses single- molecule real time sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, sequencing by ligation, Nanopore sequencing, chain termination, shotgun sequencing and/or bridge PCR. In one embodiment, the solid surface comprises the surface of a flow cell. In one embodiment, the solid surface comprises the surface of a bead. In one embodiment, the target polynucleotide is a cDNA.

Embodiments of the present disclosure also include a method of preparing a target oligonucleotide for sequencing, comprising: fragmenting the target oligonucleotide to a desired length to form target fragments, preparing the target oligonucleotide for sequencing by attaching adapters to the ends of the target fragments according to the method disclosed herein. In one embodiment, the desired length of the target fragments is between 100 and 1000 nucleotides.

Other embodiments disclosed herein include a method of diagnosing a disease and/or diagnosing susceptibility to a disease in a subject, comprising: providing a nucleic acid sample of the subject; sequencing the nucleic acid sample according to methods disclosed herein; and diagnosing a disease and/or diagnosing susceptibility to a disease based on the presence or absence of biomarkers in the sequenced nucleic acid sample. In one embodiment, the method further comprises treating the subject who is diagnosed with a disease and/or susceptible to the disease. In one embodiment, the disease is a caused by a pathogen.

Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various embodiments of the invention.

DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

Figure 1 depicts, in accordance with embodiments herein, the template switching method disclosed herein by using the example of Zika. The steps as illustrated herein comprises (A) P5/P7 grafted oligos on the solid surface; (B) hybridize Zika Complement oligo; (C) Extend Zika complement; (D) denature complement oligo; (E) hybridize sika fragment to respective probe; (F) extend; (G) extension leads to poly C protrusion; (H) use poly C protrusion to add P5 sequence; (I) use poly C protrusion to add P5 sequence; and (J) fragment contains P5/P7 sequences and ready for bridge amplification.

Figure 2 depicts, in accordance with embodiments herein, (A) design of 35 DNA oligonucleotides specific to various regions of the Zika virus (ZIKV) genome; (B) library preparation using the steps (Bl) add Zika specific probe; (B2) hybridize synthetic sika- specific RNA and DNA oligos; (B3) polymerize strand via reverse transcriptase, poly C's are added by terminal transferase activity of RT enzyme; and (B4) template switching oligo used to complete library, oligo containing poly G sequence is annealed to the poly C stretch.

Figure 3 depicts, in accordance with embodiments herein, nearly full sequence coverage of the ZIKV genome using currently disclosed methods.

DETAILED DESCRIPTION

All references, publications, and patents cited herein are incorporated by reference in their entirety as though they are fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Horny ak, et al, Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

As used herein, the term "nucleotide" refers to any ribonucleotide triphosphate and deoxyribonucleotide triphosphate with any natural or modified base in that structure that occurs in polymerized form as a component of a nucleic acid. The terms "rNTP" and "dNTP" refer to a ribonucleotide triphosphate and a deoxyribonucleotide triphosphate respectively with any natural or modified base. The terms "rNTPs" and "dNTPs" refer to a mixture of ribonucleotide triphosphates and deoxyribonucleotide triphosphates respectively consisting of at least two different ribonucleotide triphosphates or deoxyribonucleotide triphosphates. The term "NTP" is used to refer to a nucleoside triphosphate without reference to its specific sugar (e.g. a ribonucleoside triphosphate (rNTP), a deoxyribonucleoside triphosphate (dNTP), or a modified rNTP or dNTP).

The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably herein, and refer generally to linear polymers of natural or modified nucleotides, including deoxyribonucleotides, ribonucleotides, alpha-anomeric forms thereof, and the like, usually linked by phosphodiester bonds or analogs thereof ranging in size from a few monomeric units, e.g. 2-4, to several hundreds of monomelic units. When a polynucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5'- 3' order from left to right.

As used herein, the term "RNA" refers to a nucleic acid molecule comprising at least one ribose sugar, while the term "DNA" refers to a nucleic acid comprising at least one deoxyribose sugar. The oligonucleotides contemplated herein may also be DNA/RNA hybrids, comprising a mixture of dNTPs and rNTPs.

The term "immobilized" as used herein is intended to encompass direct or indirect, covalent or non-covalent attachment, unless indicated otherwise, either explicitly or by context. In certain embodiments of the invention, covalent attachment is preferred, but generally all that is required is that the molecules (e.g. nucleic acids) remain immobilized or attached to the support under conditions in which it is intended to use the support, for example in applications for amplification and/or hybridization.

The term "template switch" as used herein, refers to the ability of certain nucleic acid polymerases, such as a reverse transcriptase, to use a first nucleic acid strand as a template for polymerization, and then switching to a second template nucleic acid strand (which may be referred to as a "template switch nucleic acid" or an "acceptor template") while continuing the polymerization reaction. The result is the synthesis of a hybrid nucleic acid strand with a 5 ' region complementary to the first template nucleic acid strand and a 3 ' region complementary to the template switch nucleic acid. In certain aspects, the nucleotide sequence of all or a portion (e.g., a 5' region) of the template switch nucleic acid may be defined by a practitioner of the subj ect methods such that the newly-synthesized hybrid nucleic acid strand has a nucleotide sequence at its 3 ' end useful for a downstream application(s) of interest, e.g., PCR amplification, cloning, sequencing, and/or any other downstream application(s) of interest.

The terms "P5" and "P7" sequences as contemplated herein refers to oligomers that are appended to the ends of a nucleic acid fragment of interest before sequencing the nucleic acid fragment. The P5 sequence may also be referred to as a P5 aptamer or P5 primer. Similarly, the P7 sequence may also be referred to as P7 aptamer or P7 primer. The P5 and P7 sequences referred to herein may be a standard commercially available sequence, or it may be a custom designed sequence for a particular application.

The term "click chemistry" as used herein refers to the copper(I)-catalyzed [3+2]- Huisgen 1,3-dipolar cyclo-addition of terminal alkynes and azides leading to 1 ,2,3-triazoles. (see, e.g., Wang et al. J. Am. Chem. Soc. 125 : 11 164-11 165, 2003). It may also refer to a copper free variant of this reaction that might also be used. (See Baskin et al, Proc. Natl. Acad. Sci. U. S.A. 2007, 104, 16793.).

As described herein, in accordance with the various embodiments herein, the inventors have developed a novel method for reducing or eliminating the need for a laborious and time consuming manual library preparation before loading a template on the flow cell. For example, in one non-limiting embodiment, the inventors have used a system, referred to as 'template switching', to build libraries (append P5 and P7 sequences to flank nucleic acid fragments of interest) directly on a sequencing flow cell, eliminating the need for a manual library preparation before loading a template on the flow cell. In one embodiment, a Tn5 transposes can be used to add the P5 sequence, eliminating the need for a manual library preparation before loading a template on the flow cell. In one embodiment, the present disclosure provides a method of preparing a population of target polynucleotides for sequencing comprising: immobilizing a first primer sequence on a flow cell surface; appending a polynucleotide probe fragment to the first primer sequence; hybridizing to the polynucleotide probe fragment a polynucleotide that is complementary in sequence to a target polynucleotide; polymerizing the target polynucleotide; and adding a second primer sequence to the target polynucleotide. In one embodiment, the first primer sequence is a P7 primer sequence. In one embodiment, the second primer sequence is a P5 primer sequence. In one embodiment, the polynucleotide complementary in sequence to the target polynucleotide is a RNA. In one embodiment, the target polynucleotide is a cDNA. In one embodiment, the polymerization step further comprises adding poly-C residues at the 3' end of the polymerized DNA molecule. In one embodiment, the P5 primer sequence is added to the second polynucleotide by using template switching, Tn5 transposition, ligation, DNA/RNA nanoballs/rolling circle amplification, bridge amplification, template walking, and/or emulsion PCR on beads. In one embodiment, the method is illustrated in Figure 1. In one embodiment, the polymerization step comprises reacting with a reaction mixture having nucleotides. In one embodiment, the nucleotides comprise a mixture of standard nucleotide triphosphates and modified dideoxynucleotides with an azido modification. In one embodiment, the polymerization reaction is terminated when a modified dideoxynucleotides having an azido modification is encountered, and the grafted polynucleotide comprises a terminal 3 '-azido group. In one embodiment, the attaching step comprises attachment by click chemistry ligation, and wherein the second primer sequence comprises a P5 primer sequence having a 5' alkyne group. In one embodiment, the appended nucleotide probe fragment is a fragment of a genome sequence of an organism. In one embodiment, the organism is a human. In another embodiment, the organism is a bacteria or virus. In one embodiment, the appended nucleotide probe fragment is a fragment of a specific region of a genome. In one embodiment, the polymerization step comprises a reverse transcription reaction step. In one embodiment, the polymerization step further comprises polymerization by an enzyme. In one embodiment, the polymerization step further comprises polymerization by a reverse transcriptase enzyme. In one embodiment, the reverse transcriptase enzyme is Moloney Murine Lukemia Virus (MMLV). In one embodiment, the method further comprises sequencing the population of target nucleotides by high throughput sequencing.

Further, as will be readily appreciated by those skilled in the art, the library preparation methods as described herein are not restricted to template switching. It is contemplated that other methods of grafting libraries to sequencing flow cell, besides template switching, could be used to avoid manual library preparation. Such methods include, but are not limited to, Tn5 transposition, ligation, DNA/RNA Nanoballs/rolling circle amplification, bridge amplification, template walking, rolling circle amplification, and/or emulsion PCR on beads. In one embodiment, the ligation method involves ligating DNA or RNA templates directly to oligonucleotides that are grafted to sequencing flow cells. This could include single or double stranded ligations. This could be followed by a subsequent ligation of a third second molecule to complete the required library molecule - an insert flanked by two adapters. In one embodiment, DNA/RNA Nanoballs/Rolling circle amplification comprises DNA molecules to be sheared and circularized, for example via ligation, and then hybridized to primers that are grafted to the flowcell. Rolling circle amplification is used to generate clusters. The fragmentation and circularization can be considered library preparation, but these steps may be minimal, compared to the presently available methods. In another embodiment, randomers may be used as an alternative to template switching. In this embodiment, instead of using the terminal transferase activity that is used by M-MuLV reverse transcriptase for template switching, randomers are used to bind to the end of their transcribed templates to add the P5 Illumina sequence.

In one preferred embodiment, the distal P5 oligonucleotide can be added via a chemical ligation, such as, for example, Click Chemistry. In this embodiment, dideoxynucleotides with azido modifications are mixed with standard nucleotide triphosphates. When a modified dideoxynucleotide is incorporated, polymerization is terminated resulting in a grafted cDNA/DNA molecule with a 3' azido group. In the presence of a copper-based catalyst, a P5 oligonucleotide with a 5 ' alkyne group is covalently bound to the grafted nucleic acid molecule. Click chemistry does not require a prior fragmentation step, has far less off target effects when compared to enzymatic ligation, and is compatible with most polymerases, including reverse transcriptase.

The instantly disclosed methods and apparatuses have several applications. In one embodiment, the instant methods may be used in field-based pathogen detection, where an automated, easy-to-use sequence device is deployed outside of high tech laboratories and further into the field where outbreaks are more likely to originate. In another embodiment, the instant methods may find use in clinical sequencing. For example, with the absence of manual library preparation, HTP sequencing may be conducted by hospital staff. Upon a biological sample being loaded, the rest of the processing may be automated by a sequencing instrument. In one embodiment, disclosed herein is an apparatus for sequencing a target polynucleotide, comprising: a solid surface comprising target polynucleotides immobilized on the surface; an amplifier adapted to amplify the target polynucleotide sequences; and a polynucleotide sequencer adapted to sequentially and accurately sequence the amplified target polynucleotides. In one embodiment, the amplifier uses clonal amplification. In one embodiment the polynucleotide sequencer uses single-molecule real time sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, sequencing by ligation, Nanopore sequencing, chain termination, shotgun sequencing and/or bridge PCR. In one embodiment, the surface comprises the surface of a flow cell. In one embodiment, the surface comprises the surface of a bead. In one embodiment the target polynucleotide is a cDNA.

In one aspect, disclosed herein are methods of preparing a target oligonucleotide for sequencing, comprising: fragmenting the target oligonucleotide to a desired length to form target fragments, preparing the target oligonucleotide for sequencing by attaching adapters to the ends of the target fragments according to the method disclosed herein. In one embodiment, the desired length of the target fragments is between 10-50 nucleotides, or 50- 100 nucleotides, or 100-500 nucleotides or 100-1000 nucleotides or any combination thereof.

In one embodiment, disclosed herein is a method of diagnosing a disease and/or diagnosing susceptibility to a disease in a subject, comprising: providing a nucleic acid sample of the subject; sequencing the nucleic acid sample according to methods disclosed herein; and diagnosing a disease and/or diagnosing susceptibility to a disease based on the presence or absence of biomarkers in the sequenced nucleic acid sample. In one embodiment, the method further comprises treating the subject who is diagnosed with a disease and/or susceptible to the disease. In one embodiment, the disease is a caused by a pathogen.

In one embodiment, disclosed herein is a kit comprising: a first primer sequence; a polynucleotide probe fragment; a second primer sequence; a reverse transcriptase enzyme; and dNTPs. In one embodiment, the kit is useful for practicing the inventive method of preparing a population of target polynucleotides for high throughput sequencing. The kit is an assemblage of materials or components, including at least one of the inventive compositions. Thus, in some embodiments the kit contains a composition including NTPs, primers, a polynucleotide probe fragment, and/or a reverse transcriptase enzyme, as described above.

The exact nature of the components configured in the inventive kit depends on its intended purpose. For example, some embodiments are configured for the purpose of diagnosing, prognosing, treating, or monitoring treatment of a disease. In one embodiment, the kit is configured particularly for the purpose of treating mammalian subjects. In another embodiment, the kit is configured particularly for the purpose of treating human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.

Instructions for use may be included in the kit. "Instructions for use" typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to diagnosing, prognosing, treating, or monitoring treatment of a disease. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.

The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. The packaging materials employed in the kit are those customarily utilized in the diagnostic, forensic, and/or medical field. As used herein, the term "package" refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.

Embodiments of the present disclosure are further described in the following examples. The examples are merely illustrative and do not in any way limit the scope of the invention as claimed.

EXAMPLES

Example 1

Solid-phase Library Preparation

In one embodiment, the inventors appended highly-multiplexed, application-specific nucleotide sequences to existing P7 oligonucleotides that were grafted to an Illumina sequencing flow cell (Figures 1 A-D). These appended nucleotide sequences may complement an entire genome sequence of any organism or multiple organisms (bacterial, viral, mammalian, etc.) or they may complement specific regions of any genome (human BRCA gene, human HLA region, eukaryotic poly A region, etc.) They may also consist of random sequence in order to complement any nucleic acid present in a sample. Upon introduction to an Illumina flow cell, the nucleotide fragments of interest, either DNA or RNA, are hybridized to complementary, grafted nucleotide sequences (Figure IE). A reverse transcriptase enzyme, Moloney Murine Lukemia Virus (MMLV), is used to polymerize a DNA complement of the hybridized nucleic acid (Figure IF). The MMLV enzyme adds a poly-C residue at the 3' end of its polymerized DNA molecules (Figure 1G), allowing an oligo containing a 3' poly-G residue to perform a template switch and add a P5 Illumina universal sequence to the grafted molecule (Figure 1H-I). This results in a nucleic acid that is grafted to the sequencing flow cell, and contains the required P5 and P7 Illumina sequences required for clonal amplification and sequencing. Because these P5 and P7 sequences are added to nucleic acids of interest on the sequencing flow cell, there is no need to add these sequences during library preparation.

To test this method, the inventors designed 35 DNA oligonucleotides specific to various regions of the Zika Virus (ZIKV) genome (Figure 2). These oligonucleotides also contain the complement of a flow cell grafted Ilumina oligo, P7. Using these oligonucleotides, a DNA polymerase was used to append Zika-specific probes to the P7 grafted oligonucleotides. 11 oligonucleotides (ten DNA and one RNA) were designed that complement the appended ZIKV-specific probes. These oligonucleotides also contained generic reporter sequences, which were expect to read on an Illumina Hiseq 2500 sequencer. The 11 ZIKV reporter molecules were added to the an Illumina Sequencing flow cell, hybridized them to 11 out of 35 grafted sequencing probes, extended the grafted probes using the MMLV enzyme, and used the template switching method to append the P5 sequence to the grafted constructs. Upon sequence analysis, all 11 of the reporters were detected at high abundance and relatively even sequence coverage (Table 1). Table 1.

ZIKA_2_Reporter 897,445

ZIKA_3_Reporter 790,055

ZIKA_4_Reporter 1,325,715

ZIKA_5_Reporter 792,413

ZIKA_6_Reporter 730,122

ZIKA_7_Reporter 780,170

ZIKA_8_Reporter 492,982

ZIKA_9_Reporter 1,086,768

ZIKA lO Reporter 614,907

ZIKA_1 l_Reporter (RNA) 4,042,684

Reads aligning to Reporters 11,876,824

Total Reads (PF) 46,316,975

To further test this method the 35 ZIKV-specific sequences were appended to the Illumina flow-cell grafted P7 sequence as described herein. For this test, PCR amplified ZIKV products were used. Importantly, these ZIKV PCR products did not contain P5 or P7 Illumina sequences that are normally required for Illumina sequencing. Using the system described herein, the inventors hybridized the PCR products to the P7/Zika grafted oligonucleotides, extended the grafted oligonucleotides, added P5 universal sequence via template switching, amplified, and sequenced using the Illumina Hiseq 2500 instrument. By using the method disclosed herein, nearly full sequence coverage of the ZIKV genome (Figure 3) was obtained. To the inventors' knowledge, this is the first use of the Illumina Sequencing system, or any clonal HTP sequencing system, where the prerequisite, manual library preparation (addition of P5 and P7 sequences to nucleic acid of interest) is completely omitted.

While the disclosure herein describes the use of the Illumina sequencing instruments, Illumina flow cell, and/or the Illumina P5 and P7 primer sequences, it would be readily apparent to a skilled artisan in the art that the methods and apparatus disclosed herein can be used generally for preparing polynucleotides immobilized on a solid surface, which may be helpful for sequencing purposes, irrespective of the type of sequencing instrument or flow cell or primer sequences used. The present disclosure is in no way limited to Illumina sequencing instruments, or Illumina primers, or Illumina flow cells. Example 2

Utility

This technology disclosed herein simplifies the HTP sequencing process by incorporating sequence library preparation on to a sequencing chip/flowcell. Reactions conducted on a chip are amenable to automation which is likely to allow untrained personnel, outside of a laboratory setting, to perform HTP sequencing. The technology, methods and apparatuses disclosed herein also reduces the cost and time needed for HTP sequencing.

Example 3

Prep Free sequencing Methods: DNA Amplicon Sequencing directly on a

Hiseq flowcell surface

In one embodiment, primal scheme software (http://primal.zibraproject.org/) was used to design 35 oligonucleotides spaced at roughly 320 nucleotide intervals in reverse complementation to the Zika virus (ZIKV) genome. These oligonucleotide sequences were appended to the reverse complement sequence of Illumina's P7 grafted oligo and were ordered from Integrated DNA Technologies. In order to inject fluid onto the Hiseq flowcell (Illumina), a P200 pipette tip (Rainin) was cut a quarter inch from its point and a rubber o- ring was placed around the cleaved tip to create a plunger like device. This modified pipette tip was used to inject reagents onto the Hiseq flowcell. The flowcell was washed with lOOul of buffer HT1 (Illumina). The flowcell was placed on a thermal cycler block and heated to 96^°C. A 100 mixture of the 35 equimolarly pooled ZIKV oligonucleotides (20nM concentration) were dispensed onto the flowcell and were hybridized to flowcell grafted P7 oligonucleotides. The flowcell was slowly cooled to 40^°C at a rate of 0.05^°C per second. 75 of PR2 reagent (Illumina) was used to wash away un-hybridized oligonucleotides. 75 of amplification premix 2 (Illumina) was dispensed on to the flowcell followed by 75 of amplification mix 2 (Illumina). Amplification mix 2 comprised a DNA polymerase which extended the ZIKV specific probes on to the sequencing flowcell. The oligonucleotides were washed with 75 μΐ. of 0. IN NaOH and then with 75 μΐ. of PR2 buffer.

The Hiseq flowcell was washed with 75 μί of HT1 and the temperature of the thermal cycler was raised to 65 ^°C. 60 picograms of sheared denatured human DNA was mixed with 60 picograms of denatured ZIKV PCR amplicons and was inj ected onto the Hiseq flowcell at a concentration of 10 picomolar. The flowcell ports were sealed with tape and the flowcell was left on the thermal cycler for 7 hours. After 7 hours, the temperature was ramped down to 42^° C at a rate of 0.05^°C per second and was washed with 75 μί of HT1. A reverse transcription reaction was prepared with the following master mix: 27ul of water, 8 μΐ. of 5x SMARTScribe buffer (Clonetech), lul of SMARTScribe DTT (Clonetech), 2 of lOmM dNTP (ThermoFisher), 2 of custom template switching oligo (IDT), 2 of RNase inhibitor (Clonetech), and 4 μί of SMARTScribe M-MLV reverse transcriptase (Clonetech). 45 μί of the template switching master mix was injected onto the Hiseq flowcell and the reaction was incubated for 90 minutes at 42^° C then heated to 70^° C for 10 minutes to deactivate the reaction. 7 μί 1 of 0.1 N NaOH was injected onto the Hiseq flowcell followed by 75 μί of Illumina reagent PR2. The flowcell was loaded onto a Hiseq 2500 following a standard sequencing protocol with the cBot template generation option selected and was sequenced to yield 75 nucleotide reads. These sequence reads were aligned to the ZIKV genome using BWA and the results are shown in figure 3.

Example 4

Prep Free sequencing Methods: Viral RNA Sequencing directly on a Miseq

flowcell surface

The same 35 oligonucleotides described above were fixed to Miseq flowcell (Illumina). However, since the Miseq flowcell contains rubber ports, all reagents were dispensed with a normal P200 pipette tip (Rainin). First, the flowcell was washed with 40ul of Illumina buffer HT1. The flowcell was placed on a thermal cycler block and heated to 96^° C. 20 μί of the 35 equimolarly pooled ZIKV oligonucleotides (20nM concentration) were mixed with 20 \L of denatured Illumina phiX control library (lOpM concentration). The mixture was dispensed onto the flowcell and was hybridized to P7 grafted oligonucleotides. The flowcell was slowly cooled to 40^°C at a rate of .05^°C per second. 40 μί of Illumina PR2 reagent was used to wash un-hybridized oligonucleotides. 40 μί of amplification premix (Illumina) was dispensed on to the flowcell followed by 40 μί of amplification mix 2 (Illumina). Amplification mix 2 contains a DNA polymerase which extends the ZIKV specific probes on to the sequencing flowcell. The oligonucleotides were washed with 40ul of 0.1N NaOH and then with 40ul of PR2 buffer. The thermal cycler temperature was ramped down to 42 °C.

6.75 \L of ZIKV RNA containing 125 million ZIKV genome equivalent copies

(determined by qPCR) was mixed with 2 μί of 5x SMARTScribe buffer (Clonetech) and heated to 94^° C for one minute to shear the viral RNA. A reverse transcription reaction was prepared with the following master mix: 8.75 μί of 5x SMARTScribe buffer (Clonetech) and ZIKV RNA mix, 0.25 μΐ. of SMARTScribe DTT (Clonetech), 0.5 \L of lOmM dNTP (ThermoFisher), 0.5 of custom template switching oligo (IDT), 0.5 of RNase inhibitor (Clonetech), and 1 μΐ, of SMARTScribe M-MLV reverse transcriptase (Clonetech). 11.25 μΐ, of the template switching master mix was injected onto the Miseq flowcell and the reaction was incubated for 60 minutes then heated to 70^° C for 10 minutes to deactivate the reaction. 40 μί of 0.1 N NaOH was injected onto the Miseq flowcell followed by 40 μί of Illumina reagent PR2. The flowcell was loaded onto a Miseq using a custom recipe which omits the standard template hybridization step. These sequence reads were aligned to the ZIKV genome using BWA and the results are shown in figure 3.

The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps, some will be specifically included and others specifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are the selection of constituent modules for the inventive compositions, and the diseases and other clinical conditions that may be diagnosed, prognosed or treated therewith. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

In some embodiments, the terms "a," "an," and "the" and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described.

Claims

CLAIMS What is claimed is:

1. A method of generating a template library for a polynucleotide sequencing reaction, comprising:

immobilizing a first primer sequence on a solid surface;

appending a probe to the first primer sequence;

hybridizing to the probe a polynucleotide that is complementary in sequence to the target polynucleotide to be sequenced;

polymerizing the target polynucleotide; and

attaching a second primer sequence to the target polynucleotide.

2. The method of claim 1, wherein the first primer sequence comprises a P7 primer sequence.

3. The method of claim 1 , wherein the second primer sequence comprises a P5 primer sequence.

4. The method of claim 1 , wherein the solid surface comprises a flow cell surface.

5. The method of claim 4, wherein the flow cell comprises a Hiseq flowcell.

6. The method of claim 4, wherein the flow cell comprises a Miseq flowcell.

7. The method of claim 1, wherein the polynucleotide complementary in sequence to the target polynucleotide is a RNA.

8. The method of claim 1, wherein the target polynucleotide is a cDNA.

9. The method of claim 1 , wherein the polymerization step comprises reacting with a reaction mixture having nucleotides.

10. The method of claim 9, wherein the nucleotides comprise a mixture of standard nucleotide triphosphates and modified dideoxynucleotides with an azido modification.

11. The method of claim 10, wherein the polymerization reaction is terminated when a modified dideoxynucleotides having an azido modification is encountered, and the grafted polynucleotide comprises a terminal 3 '-azido group.

12. The method of claim 1 1, wherein the attaching step comprises attachment by click chemistry ligation, and wherein the second primer sequence comprises a P5 primer sequence having a 5' alkyne group.

13. The method of claim 1 , wherein the polymerization step further comprises attaching poly-C residues at the 3 ' end of the polymerized DNA molecule.

14. The method of claim 1, wherein the second primer sequence is attached to the target polynucleotide by using template switching, Tn5 transposition, ligation, DNA/RNA nanoballs/rolling circle amplification, bridge amplification, template walking, emulsion PCR on beads, and/or click reaction.

15. The method of claim 1 , wherein the probe is specific to the polynucleotide being sequenced.

16. The method of claim 1, wherein the probe is a fragment of a genome sequence of an organism.

17. The method of claim 16, wherein the organism is a human.

18. The method of claim 16, wherein the organism is a bacteria or virus.

19. The method of claim 1 , wherein the probe comprises a polynucleotide comprising a random sequence that complements a variety of nucleotide sequences in a target polynucleotide.

20. The method of claim 1, wherein the polymerization step further comprises polymerization by an enzyme.

21. The method of claim 20, wherein the enzyme comprises a DNA polymerase.

22. The method of claim 20, wherein the enzyme comprises a reverse transcriptase enzyme.

23. The method of claim 22, wherein the reverse transcriptase enzyme is Moloney Murine Lukemia Virus (MMLV).

24. The method of claim 1 , further comprising sequencing the population of target nucleotides by high throughput sequencing.

25. An apparatus for sequencing a target polynucleotide, comprising:

a solid surface comprising target polynucleotides immobilized on the surface according to claim 1 ;

an amplifier adapted to amplify the target polynucleotide sequences; and a polynucleotide sequencer adapted to sequentially and accurately sequence the amplified target polynucleotides.

26. The apparatus of claim 25, wherein the amplifier uses clonal amplification.

27. The apparatus of claim 25, wherein the polynucleotide sequencer uses single- molecule real time sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, sequencing by ligation, Nanopore sequencing, chain termination, shotgun sequencing and/or bridge PCR.

28. The apparatus of claim 25, wherein the surface comprises the surface of a flow cell.

29. The apparatus of claim 25, wherein the surface comprises the surface of a bead.

30. The apparatus of claim 25, wherein the target polynucleotide is a cDNA.

31. A method of preparing a target oligonucleotide for sequencing, comprising:

fragmenting the target oligonucleotide to a desired length to form target fragments; and

preparing the target oligonucleotide for sequencing by attaching adapters to the ends of the target fragments according to the method of claim 1.

32. The method of claim 31, wherein the desired length of the target fragments is between 100 and 1000 nucleotides.

33. A method of diagnosing a disease and/or diagnosing susceptibility to a disease in a subj ect, comprising:

providing a nucleic acid sample of the subject;

sequencing the nucleic acid sample according to the method of claim; and diagnosing a disease and/or diagnosing susceptibility to a disease based on the presence or absence of biomarkers in the sequenced nucleic acid sample.

34. The method of claim 33, further comprising treating the subject who is diagnosed with a disease and/or susceptible to the disease.

35. The method of claim 33, wherein the disease is a caused by a pathogen.

36. The method of claim 33, wherein the nucleic acid comprises a DNA and/or RNA.