Methods and systems for sequencing nucleic acid molecules

Abstract

Provided herein are methods of targeted genomic sequencing requiring only a single amplification step. The Single Amplification Sequencing (SAS) assay provided herein amplies and sequences targeted genomic regions at reduced time and cost. The assay provides robust coverage and accurate drug resistance profiles for multiple first- and second-line Tuberculosis drugs, showcasing its clinical utility for rapid antimicrobial resistance (AMR) detection. SAS significantly simplifies targeted Next Generation Sequencing (tNGS) workflows and holds significant promise for revolutionizing precision diagnostics across a wide array of clinical and research settings.

Description

Atty. Dkt. No.: 114198-0820METHODS AND SYSTEMS FOR SEQUENCING NUCLEIC ACID MOLECULESCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 738,279, filed December 23, 2024, the entire contents of which are incorporated herein by reference in their entireties.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under AI176401 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] Historically, most drug resistance testing (DST) for Mycobacterium tuberculosis (Mtb) was performed through growing Mtb in culture and assessing growth of Mtb in the presence of antibiotics (i.e., phenotypic DST, pDST). However, Mtb is notoriously slow-growing, requiring 10-14 weeks on solid media (e.g., Lowenstein-Jensen [LJ]) and roughly 3-5 weeks on MGIT960. Additionally, pDST requires viable bacteria, which in turn means specialized reagents or cold-chain requirements and expensive, Biosafety Level 3 (BSL3) laboratories staffed with skilled laboratorians to conduct these tests. These factors have hindered the adoption of universal DST in many high-burden countries and have contributed to prolonging initiation of appropriate antimicrobial therapy. Thus, a need exists in the art for a quick, cost-effective reliable assay for Mtb. This disclosure satisfies this need and provides related advantages as well.SUMMARY OF THE DISCLOSURE

[0004] This disclosure provides a single amplification sequencing (SAS) assay that removes the need for an intermediate culture step, enabling the use and analysis of genomic DNA from sputum (directly from patients) for detection of, for example, antibiotic drug resistance, collectively reducing laboratory costs and the need for advanced training and infrastructure. Other rapid molecular diagnostics have surfaced to also address the issues with pDST, such as 13 WHO-endorsed molecular assays for the rapid detection of resistance to INH and / or RIF; GeneXpert MTB / XDR and Hain MTBDRsl for rapid molecular detection of resistance -1- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820to FLQ and / or injectable drugs, and GenoScreen Deeplex Myc-TB which also includes modern drug regimens of BDQ. CFZ, and LZD. What makes Applicant’s assay stand apart from all other rapid TB molecular diagnostics, including other tNGS platforms like Deeplex Myc-TB, is the removal of the library preparation steps needed for sequencing. Library preparation, the process of preparing a sample to interact with the NGS device, requires numerous enzymatic steps and significant hands-on time by experienced laboratorians. The novelty of Applicant’s assay lies in the simplification of the NGS library preparation into one PCR step, removing several enzymatic steps. In certain exemplary embodiments, Applicant’s assay amplifies and barcodes Mtb drug resistance-associated genomic regions in a novel single multiplex PCR and creates sequencer-ready libraries with sample-specific barcodes.

[0005] Applicant’s “Targeted Next Generation Sequencing Assay” (tNGS assay) can, in one aspect, generate NGS data to allow for drug resistance characterization of Mycobacterium tuberculosis from a clinical sample. The disclosed methods also provide automated, Single Amplification Sequencing (ASAS) assays that can generate interpreted drug resistance results for clinicians. In another aspect, the targeted NGS assay component results in barcoded library after a single PCR. In one aspect, Applicant’s assay targets specific gene regions intuberculosis (Mtb) that are associated with drug resistance and amplifies these regions and adds library specific pieces onto the amplicons without requiring other enzymatic steps and can be done on DNA extracted from clinical samples without an intermediate culture step. This can provide a comprehensive drug resistance profile to first-line drugs, second-line injectable drugs (SLID), new and repurposed drugs, and legacy anti-TB drugs (i.e., isoniazid (INH), rifampicin (RIF), fluoroquinolones (FLQ), kanamycin (KAN), amikacin (AMK), pyrazidamine (PZA), bedaquiline (BDQ), clofazimine (CFZ), linezolid (LZD), delamanid (DLM), ethionamide (ETH), and streptomycin (STM)). In another aspect, this translates to querying 18 drug resistance regions for 10 anti -tubercular drugs, with the addition of a speciation marker, and 8 lineage markers, totaling to 27 gene regions of interest.

[0006] Historically, most drug resistance testing (DST) has been done through growing Mtb in culture and assessing growth of Mtb in the presence of antibiotics (i.e., phenotypic DST, pDST). However, Mtb is notoriously slow-growing, requiring 10-14 weeks on solid media (e.g., Lowenstein-Jensen [LJ]) and roughly 3-5 weeks on MGIT960. Additionally, pDST requires viable bacteria, which in turn means specialized reagents or cold-chain requirements -2- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820and expensive, Biosafety Level 3 (BSL3) laboratories staffed with skilled laboratorians to conduct these tests. These factors have hindered the adoption of universal DST in many high-burden countries and have contributed to prolonging initiation of appropriate antimicrobial therapy. Applicant’s assay removes the need for an intermediate culture step, enabling the use of DNA from sputum (directly from patients) for detection of drug resistance, collectively reducing laboratory costs and the need for advanced training and infrastructure.

[0007] Many other rapid molecular diagnostics have surfaced to also address the issues with pDST, such as 13 WHO-endorsed molecular assays for the rapid detection of resistance to INH and / or RIF; GeneXpert MTB / XDR and Hain MTBDRsl for rapid molecular detection of resistance to FLQ and / or injectable drugs, and GenoScreen Deeplex Myc-TB which also includes modern drug regimens of BDQ, CFZ, and LZD. In contrast to these prior art methods, Applicant’s assay stands apart from all other rapid TB molecular diagnostics, including other tNGS platforms like Deeplex Myc-TB, because of the removal of the library preparation steps needed for sequencing. Library preparation, the process of preparing a sample to interact with the NGS device, requires numerous enzymatic steps and significant hands-on time by experienced laboratorians. The novelty of Applicant’s assay lies in the simplification of the NGS library preparation into one PCR step, removing several enzymatic steps. Applicant’s assay amplifies and barcodes Mtb drug resistance-associated genomic regions in a novel single multiplex PCR and creates sequencer-ready libraries with sample specific barcode

[0008] The novelty of Applicant’s assay also lies in the simultaneous amplification and indexing of targeted regions of Mtb DNA in a single PCR run. As shown in FIG. 1, the assay can utilize long primers consisting of Mtb specific gene sequences, sample-specific index barcodes and sequencing adapter sequences, which may be an Illumina® adapter sequence or functional equivalent. When the long primers bind to the input Mtb DNA, it only amplifies genomic regions of interest and produces sequence-ready libraries. Long primers (also referred to herein as ‘ultra-long primers’), are primers having a total length of at least about 80 nucleotides, in some embodiments about 80 to about 100 nucleotides. Using a tiled amplicon approach, Applicant’s assay allows Applicant to sequence large genomic regions, including whole genes. Since only one PCR amplification is needed, this removes several enzymatic steps, reduces laboratorian hands-on time and library preparation costs.-3- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0009] Thus, Applicant provides herein a Single Amplification Sequencing (SAS) approach that facilitates the simultaneous amplification, indexing, and library preparation of targeted genomic regions in a single polymerase chain reaction (PCR) step.

[0010] In method embodiments, genomic DNA present in at least one biological sample, such as sputum or other clinical specimens, without an intermediate culture step, is contacted with a plurality of oligonucleotide primers in a multiplex configuration. Each oligonucleotide primer comprises:(i) a target-specific gene sequence configured to hybridize to a genomic region of interest; (ii) a sample-specific index barcode sequence for identifying sequences from individual samples in pooled runs; and(iii) a sequencing adapter sequence compatible with a high-throughput sequencing platform.

[0011] Each primer has a total length of at least about 75 nucleotides, often at least about 80 nucleotides, and forms a primer pair with at least one other primer to flank the genomic region of interest. Amplification produces a plurality of sequencing-ready amplicons in a single amplification reaction, optionally preceded by contacting DNA from multiple biological samples with primer sets having identical target sequences but unique barcode sequences. The plurality of oligonucleotide primers may target at least one drug-resistance marker, a speciation marker, and / or one or more lineage markers of a microorganism, in certain embodiments Mycobacterium tuberculosis. Exemplary drug-resistance markers include fabGl, gid, glmU, glnH, groEL2, gyrA, atpE, ddn, drrA, eis, ethA, gyrB, hemL, katG, hisD, rpoB, fabGl / inhA, pncA, rplC, rrl, Rv0678, mmpL5, rrs, hsp65, IpqQ, pykA, Rv2515c, pepQ, and rpsL. Amplification can produce at least about 24, 72, 130, or 250 distinct amplicons, or from about 20 to about 250 distinct amplicons, with different primer mixes covering complementary subsets of regions.

[0012] The methods may further include bead-based clean-up agents to remove unincorporated primers and primer dimers, followed by sequencing using targeted NGS (tNGS) on Illumina®, Oxford Nanopore®, Pacific Biosciences®, Ion Torrent®, or other high-throughput sequencing instruments. In certain embodiments, an automated library preparation system performs some or all steps without manual intervention.-4- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0013] The disclosure also covers sets of oligonucleotide primers having the above structural features, arranged in multiplex configurations, optionally divided into multiple primer mixes to amplify all targeted regions when products are combined. Primer sequences in different sets may differ only in their sample-specific index barcode sequences.

[0014] Kits are provided that comprise such primer sets along with amplification reagents (including thermostable DNA polymerases), DNA quantification reagents, sequencing agents, bead-based clean-up reagents, and instructions for performing the claimed methods. Kits may be configured for use with automated platforms.

[0015] Applicant further provides systems comprising hardware, reagents, and (optionally) analysis software modules for performing targeted genomic sequencing in a single amplification step. The system may include primer sets as above, a thermal cycler programmed for the SAS protocol, bead-based clean-up agents, a sequencing instrument, optionally an automated library preparation system, sequencing agents, DNA amplification and quantification reagents, and software for post-sequencing bioinformatics processing including adapter trimming, read mapping, variant calling, and antimicrobial resistance interpretation.BRIEF DESCRIPTION OF THE FIGURES

[0016] FIG. 1 is a schematic of the concept of the Single Amplification Sequencing (SAS) assay. This targeted Next Generation Sequencing (tNGS) assay uses long primers in a single PCR amplification step that produces sequence-ready sample libraries. Colored arrows illustrate Mycobacterium tuberculosis specific primers with sample-specific index barcodes and adapters indicated by gray, red, and yellow sections.

[0017] FIG. 2 is a schematic illustrating a comparison of the total time needed to complete a standard, amplicon-based tNGS library preparation workflow (10.5 hours) versus the SAS tNGS assay approach (5.5 hours) disclosed herein, an almost 50% reduction in library preparation time compared to standard workflows.

[0018] FIG. 3 is a gel image with PCR product generated with the 24- multiplex 1 PCR approach. Ladder used was lOObp DNA ladder, M. tuberculosis DNA used was H37Rv, and negative control (NTC) was molecular grade H2O. The annealing temperature range (65-71 °C) are indicated by yellow box and temperature listed.-5- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0019] FIG. 4 is a gel image with PCR product generated with the 72- multiplex 1 PCR approach. Ladder used was lOObp DNA ladder. M. tuberculosis DNA used was H37Rv at two concentrations, and negative control (NTC) was molecular grade H2O.

[0020] FIG. 5 is a graph showing the mean sequencing coverage achieved from sequencing of 72 amplicon TB SAS assay. Raw sequence aligned withtuberculosis gene targets associated with drug resistance. Figure shows all antimicrobial resistance gene regions that can be included in a TB SAS assay with 250 amplicons. Missing coverage data is the result of current proof-of-concept TB SAS assay including only 72 amplicons.

[0021] FIGS. 6A-6B are gel images showing 5 different barcoded PCR products generated with two-well 65-plex 1 PCR approach. FIG. 6A is a gel image showing results obtained Primer Mix A. FIG. 6B is a gel image showing results obtained Primer Mix B. The ladder used was lOObp DNA ladder. M. tuberculosis DNA used was H37Rv, and negative control (NTC) was molecular grade H20. Numbers indicate barcode associated with the primer mix (01, 02, 03, 04, and 07)- five of the six working barcoded primer mixes. Reverse exposed images (right side) help highlight the banding illustrating successful amplification of libraries.

[0022] FIG. 7 is a graph showing a sequencing read distribution from a single iSeqlOO sequencing run. UPD-# designates the different barcoded sets of primers used to produce libraries using SAS approach. Five samples were pooled together and run on a single run, and then the reads were separated by barcode after sequencing.

[0023] FIGS. 8A-8D are panels and graphs showing median depth of coverage across 130 target regions spread across two primer mixes. “Req A” represents Primer Mix A and “Req B” represents Primer Mix B. FIG. 8A is a graph showing median depth across all target regions in the primer mix across five unique indexes. FIG. 8B is a graph showing the zoomed in median depths (<5000X) from FIG. 8A across indexes. FIG. 8C is a graph showing median depth across all target regions in the primer mix across five unique indexes. FIG. 8D is a graph showing the zoomed in median depths (<5000X) from FIG. 8C across indexes. These figures show coverage across target regions in both primer mixes.-6- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820DETAILED DESCRIPTIONDefinitions

[0024] As used herein, the term “bead-based clean-up agent” refers to any composition or system comprising solid-phase beads, particles, or magnetic beads coated or functionalized to reversibly bind nucleic acids, thereby allowing separation of target nucleic acids from unincorporated primers, primer dimers, and other reaction components. Non-limiting examples include AMPure® XP beads (Beckman Coulter, USA), SPRI beads, or functional equivalents employing silica-coated, carboxylated, or paramagnetic particles in a suitable buffer system.

[0025] As used herein, the term “automated library preparation system” refers to any instrument, robotic platform, or integrated workflow configured to perform one or more library preparation steps (including PCR setup, bead-based clean-up, quantification, normalization, and pooling) without manual intervention by an operator. Non-limiting examples include programmable robotic pipetting systems (e.g., Hamilton Microlab® STAR, Tecan Fluent®) and dedicated NGS prep platforms (e.g., Illumina® NeoPrep™, Agilent Bravo®).

[0026] As used herein, the term “thermostable DNA polymerase” refers to any DNA polymerase enzyme capable of catalyzing template-directed DNA synthesis at elevated temperatures typical of PCR cycling without loss of activity, such as Taq polymerase, Q5®, Phusion®, or functional equivalents.

[0027] As used herein, the term “sequencing agent” refers to any reagent or composition used in a sequencing reaction, including but not limited to modified nucleotides, DNA polymerases or ligases compatible with the sequencing chemistry, buffer compositions, enzyme mixes, or sequencing kits supplied by NGS platform manufacturers (e.g., Illumina® sequencing reagent kits, Oxford Nanopore® sequencing kits).

[0028] As used herein, the term “drug-resistance marker” refers to a genomic sequence region in a microorganism wherein presence of certain mutations correlates with phenotypic resistance to one or more antimicrobial agents.-7- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0029] As used herein, the term “speciation marker” refers to a genomic sequence region useful for identifying the organism at the species level, wherein detection of the marker sequence confirms the presence of the species in a biological sample.

[0030] As used herein, the term “lineage marker” refers to a genomic sequence region comprising phylogenetically informative polymorphisms that allow assignment of an organism to a defined lineage, sub-lineage, clade, or strain group within a species.

[0031] As used herein, the term “sequencer-ready libraries” or “sequencing-ready amplicons” refer to nucleic acid molecules that include sequences required for direct compatibility with a sequencing platform, including complete sequencing adapter sequences and sample-specific index barcodes, and are present at concentrations and yields sufficient for sequencing (e.g., at least 5 ng total yield with >90% adapter incorporation efficiency).Single Amplification Sequencing Methods and Systems

[0032] In one aspect, the present disclosure provides a method for simultaneous amplification and indexing of targeted genomic regions in a single amplification. In some embodiments, the method comprises contacting DNA in a sample obtained from a subject with a plurality of oligonucleotide primers, wherein each of the oligonucleotide primers comprises: (i) a targetspecific gene sequence configured to hybridize to a genomic region of interest; (ii) a samplespecific index barcode sequence; and (iii) a sequencing adapter sequence.

[0033] In some embodiments, a target-specific gene sequence can be gene sequence that is complementary to a gene sequence in an organism, to which an oligonucleotide primer comprising the target-specific gene sequence is capable of hybridizing. In some embodiments, a target-specific gene sequence is a gene sequence from a microorganism, such as Mycobacterium tuberculosis (Mtb). Non-limiting examples of target-specific gene sequences are shown in Table 1.Table 1. Exemplary target-specific gene sequences in Mycobacterium tuberculosis (Mtb)-8- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0034] Non-limiting examples of sample-specific index barcode sequences are shown in Table 2Table 2. Exemplary sample-specific index barcode sequences

[0035] Non-limiting examples of adapter sequences are shown in Table 3.Table 3. Exemplary sequencing adapter sequences

[0036] Non-limiting examples of oligonucleotide primer sequences are shown in Table 4. Sample-specific index barcode sequences are shown in underline.Table 4. Exemplary oligonucleotide primer sequences-9- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0037] The methods described herein are not limited to the detection of gene sequences from microorganisms. For example, in some embodiments, the methods described herein address -10- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820critical gaps in current sequencing methodologies, with broad applications across precision diagnostics, infectious disease monitoring, oncology, and genetic testing for newborn screening. While embodiments herein exemplify Mycobacterium tuberculosis (Mtb) drugresistance detection, the methods, primer designs, and workflows described can be adapted for use with other microorganisms (bacterial, viral, fungal, or parasitic) and host genomic targets, by selecting target-specific gene sequences relevant to the organism or condition of interest. The SAS assay architecture is compatible with sequencing on Illumina® platforms and with other NGS technologies such as Oxford Nanopore®, Pacific Biosciences®, BGISEQ®, and Ion Torrent® systems, by substituting sequencing adapter sequences appropriate for the chosen platform.

[0038] In some embodiments, each of the oligonucleotide primers has a length of at least 75 nucleotides. In some embodiments, each of the oligonucleotide primers has a length of at least 80 nucleotides. In some embodiments, each of the oligonucleotide primers has a length of at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or at least 100 nucleotides. In some embodiments, each of the oligonucleotide primers has a length of about 80 to about 100 oligonucleotides. In some embodiments, each of the oligonucleotide primers has a length of about 84 to about 99 oligonucleotides.

[0039] In some embodiments, each of the oligonucleotide primers forms a primer pair with at least one other oligonucleotide primer of the plurality of oligonucleotide primers, wherein the oligonucleotide primers of each primer pair flank a genomic region of interest in the DNA. Non-limiting examples of primer pairs include SEQ ID NO: 15 and SEQ ID NO: 20, SEQ ID NO: 16 and SEQ ID NO: 21, SEQ ID NO: 17 and SEQ ID NO: 22, SEQ ID NO: 18 and SEQ ID NO: 23, and SEQ ID NO: 19 and SEQ ID NO: 24.

[0040] In some embodiments, the method comprises amplifying the DNA, thereby producing a plurality of amplicons, wherein amplifying comprises a single polymerase chain reaction (PCR) run.

[0041] In some embodiments, the method comprises sequencing the amplicons of the plurality of amplicons. In one aspect, the present disclosure provides a method for-11- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820simultaneous amplification and indexing of targeted genomic regions in a single amplification reaction, comprising: (a) contacting genomic DNA present in at least one biological sample with a plurality of oligonucleotide primers, each oligonucleotide primer of the plurality comprising: (i) a target-specific gene sequence configured to hybridize to a genomic region of interest; (ii) a sample-specific index barcode sequence; and (iii) a sequencing adapter sequence, wherein each oligonucleotide primer has a total length of at least about 80 nucleotides, and wherein at least one of the oligonucleotide primers is capable of forming a primer pair with at least one other oligonucleotide primer of said plurality to form a primer pair that flanks the genomic region of interest; (b) amplifying said genomic DNA using said plurality of oligonucleotide primers so as to produce an amplification product comprising a plurality of sequencing-ready amplicons in said single amplification reaction; and (c) sequencing the plurality of amplicons.

[0042] In some embodiments, the amplifying produces at least about 24 distinct types of amplicons. In some embodiments, the amplifying produces at least about 72 distinct types of amplicons. In some embodiments, the amplifying produces at least about 250 distinct types of amplicons. In some embodiments, the amplifying produces from about 20 to about 250 distinct types of amplicons. In one aspect, the number of amplicons is set a priori, using specific primer pairs, in order to work to and ensure that all targets to be sequenced are amplified. For example, in a DR TB assay, one may create amplicons from all the genes and gene regions associated with the phenotypic expression of resistance. When applied in oncology and cancer, fewer amplicons are likely required to capture all the information for a certain cancer.

[0043] In some embodiments, (a) the genomic regions of interest are present in a single gene; or (b) the genomic regions of interest are present in different genes.

[0044] The DNA sample can be a sample obtained from a subject, such as a human subject. In some embodiments, the DNA sample is a sputum sample. In some embodiments, the biological sample is isolated from a subject or an in vitro cell or tissue culture. In some embodiments, the biological sample is isolated from a bacterial culture. In some embodiments, the method does not comprise culturing the biological sample.-12- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0045] In some embodiments, sequencing comprises Next Generation Sequencing (NGS). In some embodiments, sequencing comprises targeted NGS (tNGS).

[0046] In some embodiments, the plurality of oligonucleotide primers target one or more of: (a) a plurality of drug resistance markers of a microorganism; (b) a speciation marker of a microorganism; and (c) a lineage marker of a microorganism. In some embodiments, the microorganism is Mycobacterium tuberculosis (Mtb), and wherein the plurality of drug resistance markers are selected from the group consisting of fabGl, gid, glmU, glnH, groEL2, gyrA, atpE, ddn, drrA, eis, ethA, gyrB, hemL, katG, hisD, rpoB, fabGl / inhA, pncA, rplC, rrl,RvO678, mmpL5, rrs, hsp65, glnH, IpqQ, pykA, Rv2515c, pepQ, and rpsL. In some embodiments, the method further comprises detecting antimicrobial resistance (AMR) Mycobacterium tuberculosis (Mtb) when one or more of the drug resistance markers are detected, wherein the method does not comprise culturing the sample.

[0047] In some embodiments, the method comprises, before sequencing the amplicons, removing unincorporated primers and primer dimers from the amplification product. In some embodiments, wherein removing unincorporated primers and primer dimers comprises contacting the sample with a bead-based clean-up agent.

[0048] In some embodiments, contacting comprises contacting DNA in at least 2, at least 3, at least 4, or at least 5 biological samples with a plurality of oligonucleotide primers.Accordingly, in some embodiments, contacting comprises contacting 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more biological samples with a plurality of oligonucleotide primers. In some embodiments, each biological sample is contacted with a different primer set, each primer set comprising primers having identical sample-specific index barcode sequences, the barcode sequences of each primer set being different from the barcode sequences of primers of any other primer set. In some embodiments, each of the different primer sets comprise oligonucleotide primers comprising identical target-specific gene sequences and identical sequencing adaptor sequences. In some embodiments, the plurality of oligonucleotide primers is divided into two separate multiplex primer mixes, each multiplex primer mix being used in a separate amplification reaction, the amplification products from the two reactions being combined prior to sequencing. In some embodiments, each multiplex primer mix amplifies a different subset of the genomic regions of interest, and the combined amplification products-13- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820collectively provide coverage of at least 130 distinct amplicon types. The method can be performed using an automated library preparation system.

[0049] In one aspect, the present disclosure provides a set of oligonucleotide primers, wherein at least one of the oligonucleotide primers of the set comprises: (i) a target-specific gene sequence configured to hybridize to a genomic region of interest; (ii) a sample-specific index barcode sequence; and (iii) a sequencing adapter sequence, wherein the at least one oligonucleotide primer has a total length of at least about 80 nucleotides, and the oligonucleotide primer is capable of forming a primer pair with at least one other oligonucleotide primer of the set, such that the oligonucleotide primers of the primer pair flank the genomic region of interest. In some embodiments, the oligonucleotide primers are arranged in a multiplex configuration to collectively hybridize to at least 24, at least 72, at least 130, or at least 250 distinct genomic regions of interest. In some embodiments, the oligonucleotide primers are divided into at least two multiplex primer mixes, each primer mix being intended for use in a separate PCR reaction, the products of said separate reactions being combinable to provide coverage of all targeted genomic regions. In some embodiments, the target-specific gene sequences, sample-specific index barcode sequences, and adapter sequences are identical between primer mixes, except that the sample-specific index barcode sequences differ between primer mixes allocated to different biological samples.

[0050] In some embodiments, the target-specific gene sequences, sample-specific index barcode sequences, and adapter sequences are identical between primer mixes, except that the sample-specific index barcode sequences differ between primer mixes allocated to different biological samples. In some embodiments, the oligonucleotide primers hybridize to: (a) one or more drug-resistance markers of Mycobacterium tuberculosis,' (b) a speciation marker of Mycobacterium tuberculosis,' and / or (c) one or more lineage markers of Mycobacterium tuberculosis. For example, in some embodiments, the drug-resistance markers comprise at least one marker selected from the group consisting of fabGl, gid, glmU, glnH, groEL2, gyrA, atpE, ddn, drrA, eis, ethA, gyrB, hemL, katG, hisD, rpoB, fabGl / inhA, pncA, rplC, rrl, Rv0678, mmpL5, rrs, hsp65, IpqQ, pykA, Rv2515c, pepQ, and rpsL.

[0051] Disclosed herein are kits and articles of manufacture containing the set of oligonucleotide primers disclosed herein. The articles of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include,-14- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820for example, bottles, vials, syringes, pouches, test tubes, or any suitable container. Additional examples of packaging materials include, but are not limited to, bottles, tubes, bags, containers, and any packaging material suitable for practicing methods described herein. It may further include other materials such as other buffers, diluents, and / or filters. In some embodiments, kit components are provided as concentrates (including lyophilized compositions), which are further diluted prior to use or provided at the concentration of use.

[0052] In some embodiments, a kit includes labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[0053] In some embodiments, the kit comprises at least two sets of oligonucleotide primers as described herein, each set having sample-specific index barcode sequences that are identical within the set but different from those in any other set. In some embodiments, the kit further comprises one or more of: (a) a DNA amplification reagent; (b) a DNA quantification reagent; and (c) a sequencing agent. In some embodiments, the DNA amplification reagent comprises a thermostable DNA polymerase. In some embodiments, the kit further comprises a bead-based reagent for removal of unincorporated primers and primer dimers. In some embodiments, the kit is configured for use with an automated library preparation system. In some embodiments, the kit further comprises instructions for using the kit and / or performing methods described herein.

[0054] In some embodiments, the present disclosure provides a system for performing targeted genomic sequencing in a single amplification step. The system can be configured to implement any of the methods described herein, for example by providing all necessary hardware, reagents, and software in an integrated setup.

[0055] The system can comprise oligonucleotide primer sets, such as one or more sets of oligonucleotide primers as described herein, wherein each primer comprises: (i) a targetspecific gene sequence configured to hybridize to a genomic region of interest; (ii) a samplespecific index barcode sequence; and (iii) a sequencing adapter sequence compatible with a high-throughput sequencing platform. The primers may be provided in lyophilized form or in liquid working stocks and can be arranged in multiplex configurations for single-reaction amplification of multiple regions.-15- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0056] The system can comprise a thermal cycler, such as a programmable thermal cycler configured to execute a polymerase chain reaction (PCR) protocol suitable for the SAS assay. Non-limiting examples include benchtop PCR instruments and high-throughput thermocyclers integrated into automated library preparation systems. The thermal cycler can be pre-programmed with cycling conditions described herein (see Table 5) or with equivalent optimized conditions.

[0057] The system can comprise a bead-based clean-up agent, such as a magnetic bead-based nucleic acid purification reagent, such as AMPure® XP or functional equivalents, configured to selectively bind amplicons and remove unincorporated primers, primer dimers, and other undesired PCR products. The bead-based clean-up can be performed manually or automatically within the system workflow.

[0058] The system can comprise a high-throughput sequencing platform, such as an instrument suitable for sequencing the amplicons produced by the SAS assay. Non-limiting examples include Illumina® sequencing instruments (iSeq, MiSeq, NextSeq, NovaSeq), Oxford Nanopore Technologies® platforms (MinlON, GridlON, PromethlON), Pacific Biosciences® instruments (Sequel, Revio), BGISEQ® instruments, and Ion Torrent® platforms. The sequencing adapter sequences on the primers are selected for compatibility with the intended platform, and functional equivalents may be used for non-Illumina® instruments.

[0059] The system can comprise, optionally, an automated library preparation system, such as a robotic pipetting system or an integrated NGS prep platform. Suitable examples include Hamilton Microlab® STAR, Tecan Fluent®, Agilent Bravo® NGS workstation, Illumina® NeoPrep™, or equivalent systems. The automated library preparation system can be configured to perform one or more workflow steps without manual intervention, including PCR setup, magnetic bead-based clean-up, quantification, normalization, and pooling of libraries prior to sequencing.

[0060] In some embodiments, the system comprises a sequencing agent, such as a reagent used to perform sequencing on the chosen platform, such as nucleotide mixes, platformspecific enzymes, buffer compositions, or manufacturer-supplied sequencing reagent kits (e.g., Illumina® sequencing reagent kits).-16- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0061] In some embodiments, the system comprises a DNA amplification reagent, such as a thermostable DNA polymerase (e.g., Taq®, Q5®, Phusion®, and equivalents) and associated buffers and dNTPs required for the SAS amplification reaction.

[0062] In some embodiments, the system comprises a DNA quantification reagent, such as a composition suitable for determining nucleic acid concentration post-amplification, such as qPCR, fluorescent dye-based assays (e.g., Qubit®, PicoGreen®) or microfluidic electrophoresis systems (e.g., Agilent Bioanalyzer®).

[0063] In some embodiments, the system comprises an analysis software module. In some embodiments, the system further comprises a computer-readable medium storing instructions that, when executed by a processor, cause the system to perform bioinformatic processing of raw sequencing reads. Such processing can include adapter trimming, read mapping to a target reference genome, variant calling, and interpretation according to a curated mutation catalog.ExamplesExample 1. Single Amplification Sequencing (SAS) Platform

[0064] Applicant has developed a novel, targeted Next Generation Sequencing (tNGS) assay called Single Amplification Sequencing (SAS), a transformative technology poised to redefine library preparation workflows for tNGS. Unlike traditional approaches that rely on complex, multi-step processes, SAS achieves simultaneous amplification and indexing of targeted genomic regions in a single PCR step. This innovation reduces the cost-of-goods (COG) by an order of magnitude, overall assay time by half, and substantially reduces opportunities for laboratorian error typically associated with library preparation, while eliminating the risk of cross-contamination.

[0065] By simplifying workflows, SAS not only improves operational efficiency but also democratizes access to NGS technology by enabling its use in settings with minimal infrastructure or technical expertise. The SAS assay is designed to address critical gaps in current sequencing methodologies, with broad applications across precision diagnostics, infectious disease monitoring, oncology, and genetic testing for newborn screening. Its ability to streamline tNGS workflows makes it particularly well-suited for both high-resource laboratories aiming to improve throughput and automation and low-resource environments -17- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820seeking affordable and accessible sequencing solutions. SAS brings NGS technology closer to point-of-need applications, enabling real-time, high-resolution genomic analysis for a wide range of clinical and research purposes.

[0066] While SAS has the potential to enhance any amplicon-based tNGS application, Applicant has demonstrated its use in the rapid detection and diagnosis of antimicrobial resistance (AMR) in Mycobacterium tuberculosis (Mtb). The SAS assay targets key genomic regions associated with tuberculosis (TB) drug resistance and eliminates the need for intermediate culture steps, reducing the turnaround time for AMR diagnostics from weeks to hours. By leveraging a highly multiplexed amplicon sequencing approach, the assay delivers comprehensive resistance profiles for first- and second-line drugs, as well as emerging therapies. This capability enables clinicians to make faster, evidence-based treatment decisions while improving patient outcomes and global TB control efforts. Through its novel design and novel time and reagent efficiency, SAS addresses major bottlenecks in current sequencing workflows for transformative advancements in genomic medicine and precision diagnostics.Methods

[0067] The novelty of the Single Amplification Sequencing (SAS) assay lies in its ability to perform simultaneous amplification and indexing of targeted genomic regions in a single PCR step (FIG. 1). This approach eliminates the need for multiple, labor-intensive enzymatic reactions typically required for library preparation, significantly reducing hands-on time, cost, and potential for cross-contamination and other sources of human / operator error. The SAS assay is designed for scalability, adaptability, and ease of use, making it suitable for both manual workflows and high-throughput automated systems.Workflow Overview

[0068] The SAS assay begins with a single PCR amplification step using proprietary long primers. These primers are specifically designed with three functional regions:1. Species / target-specific gene sequences to target genomic regions of interest.2. Sample-specific index barcodes for multiplexing.3. Illumina® sequencing adapters for direct sequencing compatibility.-18- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0069] As these primers bind to the input target DNA, they amplify the targeted regions of interest while simultaneously adding sequencing-ready adapters and sample-specific index barcodes. The assay uses a multiple amplicon approach, enabling sequencing of large genomic regions, including entire genes, with high accuracy and coverage. Following PCR, samples undergo a bead-based clean-up to remove unincorporated primers and primer dimers. Libraries are then quantified for normalization and pooled for sequencing on an Illumina® sequencing instrument. While the current proof-of-concept assay is designed for use on the Illumina® iSeq 100 instrument, it is compatible with a range of Illumina® sequencing platforms and potentially other NGS technologies as well.Proof-of-Concept Application for TB AMR Detection

[0070] To validate the SAS technology, Applicant developed a proof-of-concept assay for the detection of antimicrobial resistance (AMR) in Mycobacterium tuberculosis (Mtb). The TB SAS assay targets 27 key genomic regions, including:• 18 drug resistance markers for first-line drugs, second-line injectable drugs (SLID), new and repurposed drugs, and legacy anti-TB therapies. These include isoniazid (INH), rifampicin (RIF), fluoroquinolones (FLQ), kanamycin (KAN), amikacin (AMK), pyrazinamide (PZA), bedaquiline (BDQ), clofazimine (CFZ), linezolid (LZD), delamanid (DLM), ethionamide (ETH), and streptomycin (STM).• A speciation marker to confirm the presence of Mtb.• Eight lineage markers for epidemiological and phylogenetic insights.

[0071] The targeted approach allows the rapid and accurate identification of genetic variants directly from clinical samples, bypassing the need for an intermediate culture step. This reduces the diagnostic turnaround time for TB AMR detection from weeks to hours.PCR Parameters and Multiplexing

[0072] The proof-of-concept assay is optimized to amplify 130 amplicons in a single PCR run, with plans to expand to a 250-amplicon multiplex for comprehensive TB AMR profiling and complete gene coverage. The thermal cycler program includes initial denaturation, cycling through denaturation, annealing, and extension steps, followed by final extension and cooling (Table 5). After PCR, amplicon products (generated libraries) were cleaned with a-19- 4900-9818-5091.1Atty. Dkt. No.: 114198-08200.8x SPRIselect bead clean up (Beckman Coulter, USA) then normalized and pooled for sequencing.Sequencing

[0073] After PCR amplification (library generation) and library pooling, samples are sequenced on the Illumina® iSeqlOO platform. The raw sequencing data undergoes proprietary bioinformatics processing, including alignment of the reads to a target-specific reference genome and detection of variants within the targeted regions.Sequence Data Analysis

[0074] Raw reads were processed through a proprietary in-house bioinformatics pipeline:1. Adapter and primer sequences were trimmed using pTrimmer.2. Reads were mapped to a custom, target-specific Mtb reference genome based on the H37Rv strain using bwa-mem.3. Variants were identified using Mutect2 and VarScan2.4. Variants were interpreted in accordance with the publicly-available 2023 TB Mutation Catalog (also known as WHO 2023 mutation catalogue).Automation and Scalability

[0075] The SAS assay is designed and optimized for automation, with library normalization and pooling processes being adaptable to high-throughput workflows. This scalability ensures the assay can meet the demands of both centralized diagnostic laboratories and decentralized point-of-need applications.Table 5. Multiplex Amplicon 1-PCR - thermal cycler Program:-20- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820ResultsAmplicon Results

[0076] Applicant designed the ultra-long primers to target multiple amplicons in the Mtb genome in a single PCR, demonstrating the effectiveness of the SAS assay. As a proof-of-concept, a subset of 24 extended primers was first tested as a small multiplex. FIG. 3 shows the gel results from this 24-plex TB SAS run under varying annealing temperatures. The No Template Control (NTC), which contained no Mycobacterium tuberculosis (Mtb) DNA, produced non-specific amplicons (-100-150 bp), while reactions with the reference Mtb DNA (H37Rv strain) generated specific PCR products at the expected size (-300 bp). These results confirm the successful amplification of the TB SAS-specific library.

[0077] Notably, higher annealing temperatures yielded cleaner, more distinct bands, with 71 °C identified as the optimal annealing temperature for the current version of the TB SAS assay.

[0078] FIG. 4 presents results from a 72-plex run as a single PCR, highlighting the effectiveness of a larger multiplex and the effect of different Mtb DNA dilutions. The gel image shows robust PCR amplification across all conditions, demonstrating the assay’s capability to generate a library with 72 high-quality amplicons even at lower DNA concentrations.Sequencing Results

[0079] The amplicons generated from the 72-plex TB SAS assay were sequenced on an Illumina® iSeq instrument. Sequencing yielded a high number of reads, with a mean depth exceeding 12,499X across the targeted regions (FIG. 5). Alignment of sequencing data to the Mtb reference confirmed successful amplification and sequencing of the targeted genomic regions.

[0080] The sequencing data allowed genotypic drug susceptibility testing for anti-TB drugs included in the 72-plex TB SAS assay. This 72-plex does not cover all regions planned, and is currently being expanded to include 250 amplicons. The 72-plex data was run through the-21- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820full bioinformatics pipeline and results were examined based on all targets (250 amplicons), not just the targets currently in the assay (Table 6). For some drugs, such as delamanid (DLM), ethionamide (ETH), and isoniazid (INH), certain genomic regions showed no coverage due to the current proof-of-concept multiplex containing only 72 amplicons which are not associated with those drugs. These gaps are expected to be resolved in the final assay, which will include 130 amplicons for comprehensive coverage of all relevant targets.

[0081] FIG. 5 illustrates the mean sequencing coverage achieved across the 72 targeted regions, demonstrating sufficient depth for variant detection in most gene regions. Missing coverage data corresponds to targets not yet included in the proof-of-concept assay but planned for future iterations.Interpretation of Results

[0082] The results demonstrate the effectiveness of the SAS assay in amplifying and sequencing targeted regions associated with Mtb drug resistance. The ability to generate sequencing-ready libraries in a single PCR step, coupled with high sequencing depth and specificity, validates the SAS technology as a robust library preparation technology suitable for AMR detection. The assay's performance at varying annealing temperatures and DNA concentrations underscores its versatility and potential scalability for clinical and research applications.

[0083] Table 6. Sequencing depth report and genotypic drug susceptibility testing results from sequencing of Mtb amplicons generated from 72-multiplex TB SAS assay demonstrating proof-of-concept that the SAS approach works and can be used for AMR applications. The 72-multiplex does not contain all targets requited for all drugs listed. S = susceptible to drug, “!” no / low coverage.-22- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820Experimental Discussion

[0084] The findings from Applicant’s studies underscore the transformative potential of the Single Amplification Sequencing (SAS) assay. By successfully demonstrating the ability to amplify and sequence targeted genomic regions of Mtb in a single PCR step, Applicant have validated its capacity to deliver high-quality libraries with minimal hands-on time and cost. The 72-multiplex assay provided robust coverage and accurate drug resistance profiles for multiple first- and second-line TB drugs, showcasing its clinical utility for rapid AMR detection. Future iterations of the assay aim to expand to 250 amplicons, enabling comprehensive TB genotyping and broader diagnostic applications. SAS represents a breakthrough in simplifying tNGS workflows and holds significant promise for revolutionizing precision diagnostics across a wide array of clinical and research settings.Example 2. Sample Multiplexing Using Single Amplification Sequencing (SAS) Platform

[0085] Applicant has designed a two-well approach with 65-plex reactions to be run in parallel, producing an end library with 130 amplicons. These two reactions collectively capture all necessary target regions to fully cover the aforementioned 27 key gene target -23- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820regions. Each reaction has a specific multiple primer mix that is used, with all other components being identical, including the PCR cycling parameters in Table 5. FIG. 6 presents results from the two-well reaction set-up covering 130 amplicons run as a single PCR, highlighting the ability to cover all regions needed for comprehensive drug resistance detection in the Mtb assay. The two-wells were differentiated by Primer Mix A and Primer Mix B, each containing specific primers chosen to perform well in multiplex. Additionally, FIG. 6 demonstrates successful development of 6 different barcoded primer sets, all 6 barcoded sets successfully generate bands representing amplicons ~300bp in the presence of Mtb DNA (H37Rv). The sample-specific index barcodes allowed for sample specific libraries to be generated after the single PCR, which libraries can then be sequenced together and the sequence data be separated by sample in the analysis process.

[0086] Applicant ran a single iSeqlOO sequencing run with 5 different sample-specific index barcoded samples pooled in equal molar concentrations. The sequencing of libraries prepped with the two-well 65-plex TB SAS assay produced successful sequencing reads across the different barcoded sets (FIG. 7, Table 7). Six sample-specific index barcoded sets of primers were prepared that have all successfully generated sequencing reads of the targeted genomic regions, illustrating how this approach can be expanded for high throughput settings, allowing for multiple samples to be combined onto one sequencing run.Table 7. Sequencing data from libraries prepped with the two-well 65-multiplex TB SAS assay

[0087] The two-well approach covers all genomic targets needed for comprehensive drug resistance detection. The percentage of sequencing reads were well dispersed across all tested sample-specific index barcodes and had similar depth distribution across target amplicon-24- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820regions illustrating the ability to run different libraries on the same sequencing run using this SAS approach (FIG. 8).

[0088] The proof-of-concept assay generates large numbers of reads across 130 amplicons using the SAS approach as indicated by both the depth of coverage and breadth of coverage (Table 8). Depth of coverage being defined as the number of reads per nucleotide position within the targets, and breadth of coverage being defined as the proportion of targeted region bases showing sequencing reads. The variability in coverage results in some targets not reaching our preset thresholds, but upon completion of amplification balancing, all targets will produce enough reads to give a comprehensive drug resistance profileTable 8. Resulting sequencing depth and breadth of coverage of exemplary sample prepped with the two-well 65-multiplex TB SAS assay.>Experimental Discussion

[0089] The findings from our proof-of-concept studies underscore the transformative potential of the Single Amplification Sequencing (SAS) assay. By successfully demonstrating our ability to amplify and sequence targeted genomic regions of Mtb in a single PCR step, we have validated the assay’s capacity to deliver high-quality libraries in only half the time needed for legacy approaches, as well as substantial reductions in hands-on time and cost. The current two-well, 65-multiplex assay provided robust coverage and accurate drug resistance profiles for multiple first- and second-line TB drugs, new and repurposed drugs, and legacy anti-TB therapies, in addition to a speciation marker, and eight lineage markers. This showcases the SAS approach and utility for rapid AMR detection based on tNGS. In addition, we have now also shown that multiple sample-specific index barcodes can be designed and used with the SAS approach. Future iterations of the assay aim to improve balance of target amplification within multiplexes, enabling more efficient sequencing data distribution. SAS represents a breakthrough in simplifying tNGS workflows and holds significant promise for revolutionizing precision diagnostics across a wide array of clinical and research settings.-25- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0090] While current demonstration includes up to 130 amplicons, the design principles disclosed herein (primer length, multiplex layout, targeted region selection) enable expansion to 250 or more distinct types of amplicons, for example by inclusion of additional primer pairs targeting further genomic regions.Example 3. Automated SAS Workflow

[0091] In some embodiments, the SAS assay is performed entirely using an automated library preparation system. For example, a robotic pipetting platform can be programmed to dispense PCR master mix and long primers into reaction wells containing DNA samples, run the PCR thermocycling program via an integrated thermal cycler module, perform beadbased clean-up steps using magnetic separation, quantify the resulting libraries using a platebased fluorometer, normalize library concentrations, and pool libraries for sequencing without human intervention. The combined libraries may be directly loaded onto an NGS instrument (e.g., Illumina® iSeqlOO), with the sequencing agent supplied according to the instrument manufacturer’s standard protocols. This embodiment illustrates full compatibility of the SAS assay with automated, high-throughput workflows.

[0092] Traditional targeted next-generation sequencing (tNGS) workflows require multiple enzymatic steps and manual interventions to prepare sequencing libraries from clinical samples. For slow-growing pathogens such as Mycobacterium tuberculosis, drug resistance testing often relies on culture-based phenotypic DST (pDST), delaying actionable results by weeks. This culture step also necessitates biosafety level 3 (BSL-3) facilities, skilled personnel, and costly infrastructure. Existing molecular diagnostics either cover limited drug resistance markers, require culture, or retain complex library preparation steps that consume time, reagents, and labor. There is therefore a need for a rapid, comprehensive, and cost-effective sequencing method that eliminates the intermediate culture step, simplifies library preparation to reduce turnaround, and remains accurate and scalable across clinical settings, including low-resource environments.

[0093] In sum, Applicant’s disclosure provides a Single Amplification Sequencing (SAS) assay in which targeted genomic regions are both amplified and indexed in a single PCR reaction using uniquely designed ultra-long oligonucleotide primers. These primers integrate: (i) pathogen or target-specific gene sequences; (ii) sample-specific index barcode sequences;-26- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820and (iii) sequencing adapter sequences. Multiplex configurations enable simultaneous amplification of dozens to hundreds of targeted regions, including drug-resistance markers, speciation markers, and lineage markers, directly from DNA extracted from clinical samples such as sputum, bypassing culture. Post-PCR clean-up with bead-based agents yields sequencing-ready amplicons compatible with Illumina® and other NGS platforms. The SAS architecture reduces library preparation to one enzymatic step, minimizes hands-on time, halves total workflow duration, lowers cost-of-goods, and is compatible with manual or fully automated library preparation systems. Kits and systems containing primer sets, reagents, clean-up agents, thermal cyclers, sequencing instruments, and analysis software implement the approach end-to-end, from sample to clinically interpretable result.

[0094] Applicant’s studies demonstrated that the SAS approach, despite compressing all library preparation into a single PCR, produced high-quality, sequencing-ready libraries with robust coverage and depth across targeted M. tuberculosis genomic regions. For example, a 72-plex SAS run achieved mean sequencing depths exceeding 12,000*, allowing confident variant calling for most included drug-resistance genes, even from low DNA input concentrations. A two-well 65-plex configuration successfully generated libraries covering 130 distinct amplicons for comprehensive detection, with multiple sample-specific index barcodes enabling pooled sequencing of different samples in one run withoutcross-contamination or read distribution bias. These results were achieved in only 5.5 hours of library preparation time, nearly half that of standard amplicon-based workflows, and without the variability or contamination risks linked to multi-step enzymatic preparations. The combination of high multiplexing capacity, speed, minimal infrastructure needs, scalability, and accuracy was unexpected given the conventional necessity for several separate library prep steps, and positions SAS as a transformative advance in targeted sequencing workflows.Equivalents

[0095] It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.-27- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0096] Unless otherwise defined, all 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. All nucleotide sequences provided herein are presented in the 5' to 3' direction.

[0097] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0098] The terms “substantially” and “about” are used herein to describe and account for small variations. When used in conjunction with an event or circumstance, the terms can refer to instances in which the event or circumstance occurs precisely as well as instances in which the event or circumstance occurs to a close approximation. When used in conjunction with a numerical value, the terms can refer to a range of variation of less than or equal to ±10% of that numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ± 1 %, less than or equal to ±0.5%, less than or equal to ±0.1 %, or less than or equal to ±0.05%. When referring to a first numerical value as “substantially” or “about” the same as a second numerical value, the terms can refer to the first numerical value being within a range of variation of less than or equal to ±10% of the second numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ± 1 %, less than or equal to ±0.5%, less than or equal to ±0.1 %, or less than or equal to ±0.05%.

[0099] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.-28- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0100] Clauses

[0101] Clause 1. A method for simultaneous amplification and indexing of a plurality of targeted genomic regions in a single amplification reaction, comprising: (a) contacting genomic DNA present in at least one biological sample with a plurality of oligonucleotide primers, each oligonucleotide primer of the plurality comprising: (i) a target-specific gene sequence configured to hybridize to a genomic region of interest; (ii) a sample-specific index barcode sequence; and (iii) a sequencing adapter sequence, wherein each oligonucleotide primer has a total length of at least about 80 nucleotides, and wherein at least one of the oligonucleotide primers is capable of forming a primer pair with at least one other oligonucleotide primer of said plurality to form a primer pair that flanks the genomic region of interest; (b) amplifying said genomic DNA using said plurality of oligonucleotide primers so as to produce an amplification product comprising a plurality of sequencing-ready amplicons in said single amplification reaction; and (c) sequencing the plurality of amplicons.

[0102] Clause 2. The method of clause 1, wherein each oligonucleotide primer has a total length of at least about 75 nucleotides.

[0103] Clause 3. The method of clause 1, wherein the amplifying produces at least about 24 distinct types of amplicons.

[0104] Clause 4. The method of clause 1, wherein the amplifying produces at least about 72 distinct types of amplicons.

[0105] Clause 5. The method of clause 1, wherein the amplifying produces at least about 250 distinct types of amplicons.

[0106] Clause 6. The method of any one of clauses 1-5, wherein the one or more genomic regions of interest are: (a) present in a single gene; or (b) present in different genes.

[0107] Clause 7. The method of any one of clauses 1-6, wherein the biological sample is isolated from a subject or an in vitro cell or tissue culture.

[0108] Clause 8. The method of any one of clauses 1-6, wherein the biological sample is isolated from a bacterial culture.

[0109] Clause 9. The method of any one of clauses 1-8, wherein the biological sample is a sputum sample.-29- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0110] Clause 10. The method of any one of clauses 1-9, wherein the sequencing is performed using Next Generation Sequencing (NGS).[OHl] Clause 11. The method of clause 10, wherein sequencing comprises targeted NGS (tNGS).

[0112] Clause 12. The method of any one of clauses 1-11, wherein the plurality of oligonucleotide primers target one or more of: (a) a plurality of drug resistance markers of a microorganism; (b) a speciation marker of a microorganism; and (c) a lineage marker of a microorganism.

[0113] Clause 13. The method of clause 12, wherein the microorganism is Mycobacterium tuberculosis (Mtb), and wherein the plurality of drug resistance markers are selected from the group consisting of fabGl, gid, glmU, glnH, groEL2, gyrA, atpE, ddn, drrA, eis, ethA, gyrB, hemL, katG, hisD, rpoB, fabGl / inhA, pncA, rplC, rrl,RvO678, mmpL5, rrs, hsp65, glnH, IpqQ, pykA, Rv2515c, pepQ, and rpsL.

[0114] Clause 14. The method of any one of clauses 1-13, wherein the method does not comprise culturing the biological sample.

[0115] Clause 15. The method of any one of clauses 1-14, further comprising before sequencing the amplicons, removing unincorporated primers and primer dimers from the amplification product.

[0116] Clause 16. The method of clause 15, wherein removing unincorporated primers and primer dimers comprises contacting the sample with a bead-based clean-up agent.

[0117] Clause 17. The method of any one of clauses 1-16, wherein contacting comprises contacting DNA in at least 2, at least 3, at least 4, or at least 5 biological samples with a plurality of oligonucleotide primers.

[0118] Clause 18. The method of clause 17, wherein each biological sample is contacted with a different primer set, each primer set comprising primers having identical sample-specific index barcode sequences, the barcode sequences of each primer set being different from the barcode sequences of primers of any other primer set.4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0119] Clause 19. The method of clause 18, wherein each of the different primer sets comprise oligonucleotide primers comprising identical target-specific gene sequences and identical sequencing adaptor sequences.

[0120] Clause 20. The method of any one of clauses 1-19, wherein the plurality of oligonucleotide primers is divided into two separate multiplex primer mixes, each multiplex primer mix being used in a separate amplification reaction, the amplification products from the two reactions being combined prior to sequencing.

[0121] Clause 21. The method of clause 20, wherein each multiplex primer mix amplifies a different subset of the genomic regions of interest, and the combined amplification products collectively provide coverage of at least 130 distinct amplicon types.

[0122] Clause 22. The method of any one of clauses 1-21, wherein the method is performed using an automated library preparation system.

[0123] Clause 23. A set of oligonucleotide primers, wherein at least one of the oligonucleotide primers of the set comprises: (i) a target-specific gene sequence configured to hybridize to a genomic region of interest; (ii) a sample-specific index barcode sequence; and (iii) a sequencing adapter sequence, wherein the at least one oligonucleotide primer has a total length of at least about 80 nucleotides, and the oligonucleotide primer is capable of forming a primer pair with at least one other oligonucleotide primer of the set, such that the oligonucleotide primers of the primer pair flank the genomic region of interest.

[0124] Clause 24. The set of clause 23, wherein the oligonucleotide primers are arranged in a multiplex configuration to collectively hybridize to at least 24, at least 72, at least 130, or at least 250 distinct genomic regions of interest.

[0125] Clause 25. The set of clause 23 or 24, wherein the oligonucleotide primers are divided into at least two multiplex primer mixes, each primer mix being intended for use in a separate PCR reaction, the products of said separate reactions being combinable to provide coverage of all targeted genomic regions.

[0126] Clause 26. The set of any one of clauses 23-25, wherein the target-specific gene sequences, sample-specific index barcode sequences, and adapter sequences are identical-31- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820between primer mixes, except that the sample-specific index barcode sequences differ between primer mixes allocated to different biological samples.

[0127] Clause 27. The set of any one of clauses 23-26, wherein the oligonucleotide primers hybridize to: (a) one or more drug-resistance markers of Mycobacterium tuberculosis; (b) a speciation marker of Mycobacterium tuberculosis; and / or (c) one or more lineage markers of Mycobacterium tuberculosis.

[0128] Clause 28. The set of clause 27, wherein the drug-resistance markers comprise at least one marker selected from the group consisting of fabGl, gid, glmU, glnH, groEL2, gyrA, atpE, ddn, drrA, eis, ethA, gyrB, hemL, katG, hisD, rpoB, fabGl / inhA, pncA, rplC, rrl, Rv0678, mmpL5, rrs, hsp65, IpqQ, pykA, Rv2515c, pepQ, and rpsL.

[0129] Clause 29. A kit comprising the set of oligonucleotide primers of any one of clauses 23-28.

[0130] Clause 30. The kit of clause 29, comprising at least two sets of oligonucleotide primers as defined in any one of claims 23-28, each set having sample-specific index barcode sequences that are identical within the set but different from those in any other set.

[0131] Clause 31. The kit of any one of clauses 23-26, further comprising one or more of (a) a DNA amplification reagent; (b) a DNA quantification reagent; and (c) a sequencing agent.

[0132] Clause 32. The kit of clause 31, wherein said DNA amplification reagent comprises a thermostable DNA polymerase.

[0133] Clause 33. The kit of any one of clauses 29-32, wherein the kit further comprises a bead-based reagent for removal of unincorporated primers and primer dimers.

[0134] Clause 34. The kit of any one of clauses 29-33, wherein the kit is configured for use with an automated library preparation system.

[0135] Clause 35. The kit of any one of clauses 29-34, further comprising instructions for performing the method of any one of claims 1-22.

[0136] Clause 36. A system for performing targeted genomic sequencing in a single amplification step, comprising one or more of (a) a set of oligonucleotide primers, wherein each oligonucleotide primer comprises: (i) a target-specific gene sequence configured to hybridize to a genomic region of interest; (ii) a sample-specific index barcode sequence; and -32- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820(iii) a sequencing adapter sequence compatible with a high-throughput sequencing platform; (b) a thermal cycler programmed to execute a polymerase chain reaction protocol that produces sequencing-ready amplicons in a single amplification reaction; (c) a bead-based clean-up agent configured to remove unincorporated primers and primer dimers; (d) a high-throughput sequencing instrument; and (e) optionally, an automated library preparation system configured to perform one or more steps of the method without manual intervention.

[0137] Clause 37. The system of clause 36, further comprising a sequencing agent configured for use with said high-throughput sequencing instrument.

[0138] Clause 38. The system of clause 36 or 37, wherein the automated library preparation system comprises a robotic pipetting platform or an integrated NGS library prep platform.

[0139] Clause 39. The system of any one of clauses 36-38, wherein the high-throughput sequencing instrument comprises an Illumina® sequencing platform.

[0140] Clause 40. The system of any one of clauses 36-39, wherein the genomic regions of interest comprise at least one drug-resistance marker, at least one speciation marker, or at least one lineage marker of a microorganism.

[0141] Clause 41. The system of any one of clauses 36-40, wherein the genomic regions of interest comprise a set of clinically relevant genomic regions of a host organism. 42. has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0142] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0143] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.-33- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820

[0144] Other aspects are set forth within the following claims.4900-9818-5091.1

Claims

Atty. Dkt. No.: 114198-0820WHAT IS CLAIMED IS:

1. A method for simultaneous amplification and indexing of a plurality of targeted genomic regions in a single amplification reaction, comprising:(a) contacting genomic DNA present in at least one biological sample with a plurality of oligonucleotide primers, each oligonucleotide primer of the plurality comprising:(i) a target-specific gene sequence configured to hybridize to a genomic region of interest;(ii) a sample-specific index barcode sequence; and(iii) a sequencing adapter sequence,wherein each oligonucleotide primer has a total length of at least about 80 nucleotides, andwherein at least one of the oligonucleotide primers is capable of forming a primer pair with at least one other oligonucleotide primer of said plurality to form a primer pair that flanks the genomic region of interest;(b) amplifying said genomic DNA using said plurality of oligonucleotide primers so as to produce an amplification product comprising a plurality of sequencing-ready amplicons in said single amplification reaction; and(c) sequencing the plurality of amplicons.

2. The method of claim 1, wherein each oligonucleotide primer has a total length of at least about 75 nucleotides.

3. The method of claim 1, wherein the amplifying produces at least about 24 distinct types of amplicons.

4. The method of claim 1, wherein the amplifying produces at least about 72 distinct types of amplicons.

5. The method of claim 1, wherein the amplifying produces at least about 250 distinct types of amplicons.-35- 4900-9818-5091.1Atty. Dkt. No.: 114198-08206. The method of any one of claims 1-5, wherein the one or more genomic regions of interest are:(a) present in a single gene; or(b) present in different genes.

7. The method of any one of claims 1-6, wherein the biological sample is isolated from a subject or an in vitro cell or tissue culture.

8. The method of any one of claims 1-6, wherein the biological sample is isolated from a bacterial culture.

9. The method of any one of claims 1-8, wherein the biological sample is a sputum sample.

10. The method of any one of claims 1-9, wherein the sequencing is performed using Next Generation Sequencing (NGS).

11. The method of claim 10, wherein sequencing comprises targeted NGS (tNGS).

12. The method of any one of claims 1-11, wherein the plurality of oligonucleotide primers target one or more of:(a) a plurality of drug resistance markers of a microorganism;(b) a speciation marker of a microorganism; and(c) a lineage marker of a microorganism.

13. The method of claim 12, wherein the microorganism is Mycobacterium tuberculosis (Mtb), and wherein the plurality of drug resistance markers are selected from the group consisting of fabGl, gid, glmU, glnH, groEL2, gyrA, atpE, ddn, drrA, eis, ethA, gyrB, hemL, katG, hisD, rpoB, fabGl / inhA, pncA, rplC, rrl,RvO678, mmpL5, rrs, hsp65, glnH, IpqQ, pykA, Rv2515c, pepQ, and rpsL.

14. The method of any one of claims 1-13, wherein the method does not comprise culturing the biological sample.-36- 4900-9818-5091.1Atty. Dkt. No.: 114198-082015. The method of any one of claims 1-14, further comprising before sequencing the amplicons, removing unincorporated primers and primer dimers from the amplification product.

16. The method of claim 15, wherein removing unincorporated primers and primer dimers comprises contacting the sample with a bead-based clean-up agent.

17. The method of any one of claims 1-16, wherein contacting comprises contacting DNA in at least 2, at least 3, at least 4, or at least 5 biological samples with a plurality of oligonucleotide primers.

18. The method of claim 17, wherein each biological sample is contacted with a different primer set, each primer set comprising primers having identical sample-specific index barcode sequences, the barcode sequences of each primer set being different from the barcode sequences of primers of any other primer set.

19. The method of claim 18, wherein each of the different primer sets comprise oligonucleotide primers comprising identical target-specific gene sequences and identical sequencing adaptor sequences.

20. The method of any one of claims 1-19, wherein the plurality of oligonucleotide primers is divided into two separate multiplex primer mixes, each multiplex primer mix being used in a separate amplification reaction, the amplification products from the two reactions being combined prior to sequencing.

21. The method of claim 20, wherein each multiplex primer mix amplifies a different subset of the genomic regions of interest, and the combined amplification products collectively provide coverage of at least 130 distinct amplicon types.

22. The method of any one of claims 1-21, wherein the method is performed using an automated library preparation system.

23. A set of oligonucleotide primers, wherein at least one of the oligonucleotide primers of the set comprises:(i) a target-specific gene sequence configured to hybridize to a genomic region of interest;(ii) a sample-specific index barcode sequence; and-37- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820(iii) a sequencing adapter sequence,wherein the at least one oligonucleotide primer has a total length of at least about 80 nucleotides, and the oligonucleotide primer is capable of forming a primer pair with at least one other oligonucleotide primer of the set, such that the oligonucleotide primers of the primer pair flank the genomic region of interest.

24. The set of claim 23, wherein the oligonucleotide primers are arranged in a multiplex configuration to collectively hybridize to at least 24, at least 72, at least 130, or at least 250 distinct genomic regions of interest.

25. The set of claim 23 or 24, wherein the oligonucleotide primers are divided into at least two multiplex primer mixes, each primer mix being intended for use in a separate PCR reaction, the products of said separate reactions being combinable to provide coverage of all targeted genomic regions.

26. The set of any one of claims 23-25, wherein the target-specific gene sequences, sample-specific index barcode sequences, and adapter sequences are identical between primer mixes, except that the sample-specific index barcode sequences differ between primer mixes allocated to different biological samples.

27. The set of any one of claims 23-26, wherein the oligonucleotide primers hybridize to:(a) one or more drug-resistance markers of Mycobacterium tuberculosis,' (b) a speciation marker of Mycobacterium tuberculosis,' and / or(c) one or more lineage markers of Mycobacterium tuberculosis.

28. The set of claim 27, wherein the drug-resistance markers comprise at least one marker selected from the group consisting of fabGl, gid, glmU, glnH, groEL2, gyrA, atpE, ddn, drrA, eis, ethA, gyrB, hemL, katG, hisD, rpoB, fabGl / inhA, pncA, rplC, rrl, Rv0678, mmpL5, rrs, hsp65, IpqQ, pykA, Rv2515c, pepQ, and rpsL.

29. A kit comprising the set of oligonucleotide primers of any one of claims 23-28.

30. The kit of claim 29, comprising at least two sets of oligonucleotide primers as defined in any one of claims 23-28, each set having sample-specific index barcode sequences that are identical within the set but different from those in any other set.

31. The kit of any one of claims 23-26, further comprising one or more of:-38- 4900-9818-5091.1Atty. Dkt. No.: 114198-0820(a) a DNA amplification reagent;(b) a DNA quantification reagent; and(c) a sequencing agent.

32. The kit of claim 31, wherein said DNA amplification reagent comprises a thermostable DNA polymerase.

33. The kit of any one of claims 29-32, wherein the kit further comprises a bead-based reagent for removal of unincorporated primers and primer dimers.

34. The kit of any one of claims 29-33, wherein the kit is configured for use with an automated library preparation system.

35. The kit of any one of claims 29-34, further comprising instructions for performing the method of any one of claims 1-22.

36. A system for performing targeted genomic sequencing in a single amplification step, comprising one or more of(a) a set of oligonucleotide primers, wherein each oligonucleotide primer comprises:(i) a target-specific gene sequence configured to hybridize to a genomic region of interest;(ii) a sample-specific index barcode sequence; and (iii) a sequencing adapter sequence compatible with a high-throughput sequencing platform;(b) a thermal cycler programmed to execute a polymerase chain reaction protocol that produces sequencing-ready amplicons in a single amplification reaction;(c) a bead-based clean-up agent configured to remove unincorporated primers and primer dimers;(d) a high-throughput sequencing instrument; and(e) optionally, an automated library preparation system configured to perform one or more steps of the method without manual intervention.-39- 4900-9818-5091.1Atty. Dkt. No.: 114198-082037. The system of claim 36, further comprising a sequencing agent configured for use with said high-throughput sequencing instrument.

38. The system of claim 36 or 37, wherein the automated library preparation system comprises a robotic pipetting platform or an integrated NGS library prep platform.

39. The system of any one of claims 36-38, wherein the high-throughput sequencing instrument comprises an Illumina® sequencing platform.

40. The system of any one of claims 36-39, wherein the genomic regions of interest comprise at least one drug-resistance marker, at least one speciation marker, or at least one lineage marker of a microorganism.

41. The system of any one of claims 36-40, wherein the genomic regions of interest comprise a set of clinically relevant genomic regions of a host organism.4900-9818-5091.1

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