Methods for preparing nucleic acids for sequencing

Abstract

The technical aspects disclosed herein relate to methods for preparing and analyzing nucleic acids. In some embodiments, methods are provided for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing).

Description

[0001] Related applications

[0002] Pursuant to 35 U.SC §119, this application claims the benefit of U.S. Provisional Application US61 / 931,959, filed January 27, 2014, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The techniques described in this article relate to methods for preparing and analyzing nucleic acids. Background Technology

[0004] Compared to whole-genome, whole-exome, and whole-transcriptome sequencing, target enrichment prior to next-generation sequencing is more cost-effective and therefore more suitable for widespread implementation in research findings and clinical applications. For example, the high coverage depth provided by target enrichment methods enables allele counting with a wide dynamic range (in gene expression and copy number assessment) and the detection of low-frequency mutations (for evaluating key features of somatic mutations in cancer). Examples of enrichment protocols currently used for next-generation sequencing include hybridization-based capture analyses (TruSeq Capture, Illumina; SureSelect Hybrid Capture, Agilent) and polymerase chain reaction (PCR)-based analyses (HaloPlex, Agilent; AmpliSeq, Ion Torrent; TruSeq amplicon, Illumina; emulsion / digital PCR, Raindance). Hybridization-based methods capture not only the target sequence covered by the captured probe but also nearby off-target bases that consume sequencing power. In addition, these methods are time-consuming, labor-intensive, and have low specificity. Summary of the Invention

[0005] The aspects of the techniques disclosed herein relate to methods for preparing and analyzing nucleic acids. In some embodiments, methods are provided for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing). In some embodiments, the techniques described herein pertain to methods for determining the nucleotide sequence of nucleic acids. In some embodiments, the methods disclosed herein involve enriching target nucleic acids prior to sequencing.

[0006] The aspects of the technology disclosed herein relate to methods for determining nucleotide sequences contiguous to a known target nucleotide sequence. In some embodiments, the method involves: (a) contacting a target nucleic acid molecule containing a known target nucleotide sequence with an initiating target-specific primer under hybridization conditions; (b) performing a template-dependent extension reaction initiated by the initiating target-specific primer using the target nucleic acid molecule as a template; (c) contacting the product of step (b) with a set of tailed random primers under hybridization conditions; (d) performing a template-dependent extension reaction initiated by the tailed random primers using a portion of the target nucleic acid molecule downstream of the hybridization site as a template; (e) amplifying the tailed random primer sequence and a portion of the target nucleic acid molecule with a first tail primer and a first target-specific primer; (f) amplifying a portion of the amplicon generated in step (e) with a second tail primer and a second target-specific primer; and (g) sequencing the amplified portion from step (f) using a first sequencing primer and a second sequencing primer. In some embodiments, the tailed random primer group comprises a single-stranded oligonucleotide molecule having the same 5' nucleic acid sequence as the first sequencing primer and a 3' nucleic acid sequence containing about 6 to about 12 random nucleotides. In some embodiments, the first target-specific primer comprises a nucleic acid sequence capable of specifically annealing to a known target nucleotide sequence of the target nucleic acid at an annealing temperature. In some embodiments, the second target-specific primer comprises a 3' portion and a 5' portion, the 3' portion comprising a nucleic acid sequence capable of specifically annealing to a portion of the known target nucleotide sequence contained in the amplicon produced by step (e), the 5' portion comprising the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer. In some embodiments, the first tail primer comprises the same nucleic acid sequence as the tailed random primer. In some embodiments, the second tail primer comprises a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer.

[0007] In some embodiments, the method involves: (a) contacting a target nucleic acid molecule containing a known target nucleotide sequence with a set of tailed random primers under hybridization conditions; (b) performing a template-dependent extension reaction initiated by the hybridized tailed random primers and using a portion of the target nucleic acid molecule downstream of the hybridization site as a template; (c) contacting the product of step (b) with an initiating target-specific primer under hybridization conditions; (d) performing a template-dependent extension reaction initiated by the hybridized initiating target-specific primer and using the target nucleic acid molecule as a template; (e) amplifying a portion of the tailed random primer sequence and the target nucleic acid molecule with a first tailed primer and a first target-specific primer; (f) amplifying a portion of the amplicon generated in step (e) with a second tailed primer and a second target-specific primer; and (g) sequencing the amplified portion from step (f) using a first sequencing primer and a second sequencing primer. In some embodiments, the tailed random primer group comprises a single-stranded oligonucleotide molecule having the same 5' nucleic acid sequence as the first sequencing primer and a 3' nucleic acid sequence containing about 6 to about 12 random nucleotides. In some embodiments, the first target-specific primer comprises a nucleic acid sequence capable of specifically annealing to a known target nucleotide sequence of the target nucleic acid at an annealing temperature. In some embodiments, the second target-specific primer comprises a 3' portion and a 5' portion, the 3' portion comprising a nucleic acid sequence capable of specifically annealing to a portion of the known target nucleotide sequence contained in the amplicon produced by step (c), the 5' portion comprising the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer. In some embodiments, the first tail primer comprises the same nucleic acid sequence as the tailed random primer. In some embodiments, the second tail primer comprises a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer.

[0008] In some embodiments, the method further involves contacting the sample and / or product with RNase after extension of the initiating target-specific primer. In some embodiments, the tailed random primer may form a hairpin loop structure. In some embodiments, the initiating target-specific primer is the same as the first target-specific primer. In some embodiments, the tailed random primer further includes a barcode portion containing 6-12 random nucleotides between the 5' nucleic acid sequence identical to the first sequencing primer and the 3' nucleic acid sequence containing 6-12 random nucleotides.

[0009] In some embodiments, the method involves: (a) contacting a target nucleic acid molecule containing a known target nucleotide sequence with a set of tailed random primers under hybridization conditions; (b) performing a template-dependent extension reaction initiated by the hybridized tailed random primers and using a portion of the target nucleic acid molecule downstream of the hybridization site as a template; (c) amplifying the tailed random primer sequence and a portion of the target nucleic acid molecule with a first tail primer and a first target-specific primer; (d) amplifying a portion of the amplicon generated in step (c) with a second tail primer and a second target-specific primer; and (e) sequencing the amplified portion from step (d) using a first sequencing primer and a second sequencing primer. In some embodiments, the set of tailed random primers comprises a single-stranded oligonucleotide molecule having the same 5' nucleic acid sequence as the first sequencing primer, an intermediate barcode portion containing 6-12 random nucleotides, and a 3' nucleic acid sequence containing about 6-12 random nucleotides. In some embodiments, the first target-specific primer comprises a nucleic acid sequence capable of specifically annealing to a known target nucleotide sequence of the target nucleic acid at an annealing temperature. In some embodiments, the second target-specific primer comprises a 3' portion and a 5' portion, the 3' portion comprising a nucleic acid sequence capable of specifically annealing to a portion of the known target nucleotide sequence contained in the amplicon produced by step (c), the 5' portion comprising the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer. In some embodiments, the first tail primer comprises the same nucleic acid sequence as the tailed random primer. In some embodiments, the second tail primer comprises a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer. In some embodiments, each tailed random primer further comprises a spacer region nucleic acid sequence between the same 5' nucleic acid sequence as the first sequencing primer and a 3' nucleic acid sequence containing about 6 to about 12 random nucleotides. In certain embodiments, after the extension step, unhybridized primers are removed from the reaction. In some embodiments, the second-tailed primer is nested relative to the first-tailed primer by at least three nucleotides. In a particular embodiment, the first target-specific primer further includes a 5' tag sequence portion comprising a nucleic acid sequence with high GC content that is substantially not complementary to or identical to any other portion of any primer. In some embodiments, the second-tailed primer is identical to the full-length first sequencing primer. In a particular embodiment, the portion of the target-specific primer specifically annealed to a known target will be specifically annealed in PCR buffer at a temperature of approximately 65°C. In some embodiments, the sample comprises genomic DNA. In some embodiments, the sample comprises RNA, and the method further includes a first step of subjecting the sample to a reverse transcriptase protocol.In certain embodiments, the nucleic acids present in the sample are not cleaved or digested. In some embodiments, the sample comprises single-stranded gDNA or single-stranded cDNA. In certain embodiments, the reverse transcriptase protocol includes the use of random hexamers. In some embodiments, the gene rearrangement includes a known target sequence. In certain embodiments, the gene rearrangement is present in nucleic acids selected from the group consisting of genomic DNA, RNA, and cDNA. In some embodiments, the gene rearrangement includes oncogenes. In certain embodiments, the gene rearrangement includes fusion oncogenes. In some embodiments, the nucleic acid product is sequenced using next-generation sequencing. In certain embodiments, the next-generation sequencing method includes methods selected from the group consisting of Ion Torrent, Illumina, SOLiD, 454, massively parallel signature sequencing, solid-phase reversible dye-terminated sequencing, and DNA nanosphere sequencing. In certain embodiments, the first sequencing primer and the second sequencing primer are compatible with the selected next-generation sequencing method. In some embodiments, the method includes contacting the sample or a separate portion of the sample with multiple sets of first target-specific primers and second target-specific primers. In certain embodiments, the method includes contacting a single reaction mixture containing the sample with multiple sets of first target-specific primers and second target-specific primers. In some embodiments, the multiple sets of first target-specific primers and second target-specific primers are specifically annealed to a known target nucleotide sequence contained in a single gene. In certain embodiments, at least two sets of first target-specific primers and second target-specific primers are specifically annealed to different portions of a known target nucleotide sequence. In some embodiments, at least two sets of first target-specific primers and second target-specific primers are specifically annealed to different portions of a single gene containing a known target nucleotide sequence. In certain embodiments, at least two sets of first target-specific primers and second target-specific primers are specifically annealed to different exons of a gene containing a known target nucleotide sequence. In some embodiments, multiple first target-specific primers contain the same 5' tag sequence portion. In certain embodiments, each tailed random primer in a tailed random primer group further contains the same sample barcoding portion. In some embodiments, multiple samples are each contacted with a separate set of tailed random primers, each having a different sample barcode portion; and the samples are pooled after step (b). In certain embodiments, each amplification step includes a PCR amplification protocol cycle set of 5 to 20 cycles in length. In some embodiments, target-specific primers and tail primers are designed to specifically anneal to their complementary sequences at an annealing temperature of approximately 61°C to 72°C.In some embodiments, target-specific primers and tail primers are designed to specifically anneal to their complementary sequences at an annealing temperature of approximately 65°C. In particular embodiments, the target nucleic acid molecule is derived from a sample, optionally a biological sample obtained from a subject. In some embodiments, the sample is obtained from a subject requiring treatment for a disease associated with genetic alteration. In particular embodiments, the disease is cancer. In some embodiments, the sample comprises a tumor cell population. In particular embodiments, the sample is a tumor biopsy. In some embodiments, the cancer is lung cancer. In particular embodiments, the disease-related gene contains a known target sequence. In some embodiments, the gene rearrangement product in the sample contains a known target sequence. In particular embodiments, the gene rearrangement product is an oncogene.

[0010] The aspects of the technology disclosed herein relate to methods for preparing nucleic acids for analysis. In some embodiments, the method involves: (a) contacting a nucleic acid template containing a first strand of a target nucleic acid with a complementary target-specific primer containing a target-specific hybridization sequence, under conditions promoting template-specific hybridization and extension of a target-specific primer; and (b) contacting a nucleic acid template containing a second strand complementary to the first strand of the target nucleic acid with a plurality of different primers sharing a common sequence, the common sequence being the 5' end of the different hybridization sequences, under conditions promoting template-specific hybridization and extension of at least one of a plurality of different primers, wherein an extension product is generated, the extension product simultaneously containing a sequence characteristic of the target-specific primer and a sequence characteristic of at least one of the plurality of different primers. In some embodiments, the target nucleic acid is ribonucleic acid. In a particular embodiment, the target nucleic acid is deoxyribonucleic acid. In some embodiments, steps (a) and (b) are performed sequentially. In a particular embodiment, the nucleic acid template in step (a) contains an extension product generated from the extension and hybridization of at least one of the plurality of different primers in step (b). In some embodiments, the nucleic acid template in step (b) comprises an extension product generated from the extension and hybridization of the target-specific primer in step (a). In certain embodiments, the target nucleic acid is a messenger RNA encoding a chromosomal segment containing a gene rearrangement. In some embodiments, the target nucleic acid is a chromosomal segment containing a portion of a gene rearrangement. In certain embodiments, the gene rearrangement is an inversion, deletion, or translocation. In some embodiments, the method further involves amplifying the extension product. In certain embodiments, the method further involves contacting the extension product or amplified extension product with the fixed oligonucleotide under conditions where hybridization occurs between the extension product and the fixed oligonucleotide. In certain embodiments, the target nucleic acid comprises a target portion having a known sequence and a flanking portion having an unknown sequence. In some embodiments, the different hybridization sequences are complementary to the flanking portions. In certain embodiments, the target-specific hybridization sequence is complementary to the target portion. In some embodiments, the target-specific primer further comprises: the 5' end of the target-specific hybridization sequence; at least one of an index sequence, a barcode sequence, and a adapter sequence. In certain embodiments, the common sequence comprises at least one of an index sequence, a barcode sequence, and a adapter sequence. In some implementations, the adapter sequence is a cleavable adapter sequence used to immobilize oligonucleotides in a flow cell. Attached Figure Description

[0011] Figure 1A and Figure 1B Non-limiting implementations of the workflow described herein for amplifying and sequencing target nucleic acids with an unknown 3' fusion partner on the flank are described.

[0012] Figure 2A and Figure 2B Non-limiting embodiments of the workflow described herein for amplifying and sequencing target nucleic acids with unknown 5' fusion partners on their flanks are described.

[0013] Figure 3 Non-limiting embodiments of the workflow described herein for amplifying and sequencing target nucleic acids with unknown 3' fusion partners on the flanks using oligonucleotides containing hairpins are described. Detailed Implementation

[0014] The aspects of the technology disclosed herein relate to methods for preparing and analyzing nucleic acids. In some embodiments, the methods provided herein are used to determine unknown nucleotide sequences adjacent to (adjacent to) a known target nucleotide sequence. Conventional sequencing methods randomly generate sequence information (e.g., “shotgun” sequencing) or generate sequence information between two known sequences for primer design. In contrast, in some embodiments, the methods described herein enable the determination of upstream or downstream nucleotide sequences of a single region of a known sequence (e.g., sequencing) with high specificity and high sensitivity. Thus, in some embodiments, the methods provided herein are used to determine the sequence of fusions (e.g., fusion mRNAs) resulting from gene arrangement (e.g., rearrangements causing cancer or other conditions). In some embodiments, the methods provided herein for preparing nucleic acids for analysis (e.g., for sequencing) involve: a first round of extension using target-specific primers targeting a known sequence of a target nucleic acid (e.g., a known sequence of a first gene), followed by a second round of extension involving a heterologous population using tailing primers (e.g., tailing random primers), the heterologous population of which includes tailing primers having hybridization sequences complementary to unknown sequences adjacent to known sequences in the target nucleic acid. In some implementations, the tail region of the tail primer contains a barcode sequence or index sequence that promotes multiplex amplification and enrichment of the target nucleic acid.

[0015] In some aspects of the technology disclosed herein, a method for preparing nucleic acids for analysis is provided, the method involving: (a) contacting a nucleic acid template containing a first strand of a target nucleic acid with a target-specific primer containing a complementary target-specific hybridization sequence under conditions promoting template-specific hybridization and extension of a target-specific primer; and (b) contacting a nucleic acid template containing a second strand complementary to the first strand of the target nucleic acid with a plurality of different primers sharing a common sequence, the common sequence being the 5' end of the different hybridization sequences, under conditions promoting template-specific hybridization and extension of at least one of a plurality of different primers, wherein an extension product is generated, the extension product simultaneously containing a sequence characterized by the target-specific primer and a sequence characterized by at least one of the plurality of different primers. In some embodiments, steps (a) and (b) are performed sequentially. In some embodiments, the nucleic acid template in step (a) contains an extension product generated from the extension and hybridization of at least one of the plurality of different primers in step (b). In some embodiments, the nucleic acid template in step (b) contains an extension product generated from the extension and hybridization of the target-specific primer in step (a).

[0016] In some embodiments, methods are provided for preparing nucleic acids having a target region at the 5' end of an adjacent region (e.g., an adjacent region of an unknown sequence). In some embodiments, the methods provided herein can be performed using one or more rounds of PCR.

[0017] For example, Figures 1A-1B A schematic diagram of an exemplary method for amplifying a target nucleic acid having a known target region at the 5' end of an adjacent region (e.g., for sequencing purposes of the adjacent region). In step 101, a starting RNA is obtained or provided in a sample and used as a template. The RNA template is exposed to a plurality of tailing primers (e.g., random tailing primers) containing a common sequence, which is the 5' end of a different hybridization sequence and shared across the entire group of tailing primers. In some embodiments, at least one primer hybridizes to the RNA molecule and initiates a reverse transcriptase reaction to produce a complementary DNA strand. In step 102, the unhybridized oligonucleotides are degraded (e.g., enzymatic degradation, such as degradation by an exonuclease). In step 102, the RNA template is degraded from the complementary DNA strand.

[0018] In some embodiments, a tailing primer is provided that hybridizes to a polyA tail of the RNA molecule. In some embodiments, a primer sequence is provided that hybridizes to a polyA tail comprising polydTs (e.g., stretches of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dTs located at the 3' end). In some embodiments, a plurality of tailing primers are provided, each tailing primer further comprising a barcode sequence or index sequence.

[0019] It should be understood that the RNA template in the methods disclosed herein can be degraded by any suitable method, including, for example, enzymatic degradation (e.g., using RNaseH, uracil glycosylation enzymes, etc.), hydrolysis (e.g., exposing the RNA to relatively high pH conditions (e.g., pH 10, pH 11, pH 12)), etc. In some embodiments, hydrolyzing RNA by exposure to relatively high pH is advantageous because it is relatively inexpensive (compared to some other methods, such as some enzymatic methods) and because it simultaneously destroys RNA and DNA:RNA hybrids. In some embodiments, RNA is degraded by hydrolysis induced by exposure to relatively high pH conditions at relatively high temperatures (e.g., temperatures above 60°C (e.g., 60°C to 95°C)). In some embodiments, using relatively high temperatures is advantageous because the heat inactivates enzymes (e.g., RT enzymes) used in the preceding preparation steps. In some embodiments, the starting nucleic acid can be DNA obtained or provided in the sample and used as a template. In such embodiments, steps 101 and 102 can be omitted.

[0020] In step 103, the DNA molecule produced by reverse transcription is contacted using one or more initiation target-specific primers, which may be the same as or different from the first target-specific primer. In step 104, the initiation target-specific primer hybridizes to a portion of the target nucleic acid (“target sequence”) to initiate an extension reaction using the DNA molecule as a template to produce a complementary DNA strand. In step 105, the extension product is purified. However, in some embodiments, the DNA produced in step 104 can be directly amplified, for example, by PCR, without purification.

[0021] In step 106, the DNA molecule is contacted using a first target-specific primer and a first tail primer. The first target-specific primer hybridizes to a portion of the target nucleic acid. In some embodiments, pools of different first target-specific primers can be used to hybridize to different portions of the target nucleic acid. In some embodiments, using different target-specific primers can be advantageous because it allows for the generation of different extension products with overlapping but interleaved sequences relative to the target nucleic acid. In some embodiments, the different extension products can be sequenced to generate overlapping sequence reads. In some embodiments, the overlapping sequence reads can be evaluated to assess the accuracy of the sequence information, the fidelity of nucleic acid amplification, and / or the increased confidence in detecting mutations (e.g., detecting the location of chromosomal rearrangements (e.g., fusion breakpoints)). In some embodiments, pools of different first target-specific primers can be used to hybridize to different portions of different target nucleic acids present in the sample. In some embodiments, using pools of different target-specific primers is advantageous because it facilitates parallel processing (e.g., amplification) and analysis of different target nucleic acids. In some embodiments, pools of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 100 or more different first target-specific primers are used. In some embodiments, pools of 2-5, 2-10, 5-10, 5-15, 10-15, 10-20, 10-100, 50-100 or more different first target-specific primers are used.

[0022] exist Figure 1A and Figure 1B In step 106, the first tail primer hybridizes to at least a portion of the DNA molecule provided by the tail portion of the tail primer in step 101. In some embodiments, the first tail primer hybridizes to a common sequence provided by the tails of one or more primers in step 101. In some embodiments, nested target-specific primers (as opposed to the target-specific primer nesting in step 103) are used in step 106. In some embodiments, the first tail primer may include, for example, an additional sequence at the 5' end of the hybridization sequence, which may include a barcode sequence, index sequence, adapter sequence, or sequencing primer site. In step 107, the hybridization of the first target-specific primer and the first tail nucleic acid molecule enables the product to be amplified in a polymerase chain reaction (PCR). In some embodiments, the amplified product is purified in step 108.

[0023] In some embodiments, the ssDNA product of step 103 is directly amplified (e.g., by PCR) without the extension reaction of step 104 and the purification of step 105. Similarly, in some embodiments of any of the methods disclosed herein, the ssDNA product may be directly amplified (e.g., by PCR) to produce dsDNA prior to purification and / or PCR without an extension reaction. In some embodiments, a first-tail primer and a second-tail primer may be added during PCR. In some embodiments, primers (e.g., a first-tail primer, a target-specific primer) are used in the PCR or extension reaction, and then excess primers are removed using a single-stranded nuclease. Subsequent rounds of PCR or extension may be performed using different primers (e.g., a second-tail primer, a nested primer, or a second target-specific primer) to incorporate different sequences into the resulting product.

[0024] In some embodiments, an exonuclease (e.g., ExoI) may be used to degrade single-stranded DNA. In some embodiments, an exonuclease may be used to degrade ssDNA, and according to step 109... A-B and step 110 A-B The amplification product can be processed directly without purification in step 108.

[0025] exist Figure 1A In step 109 A The amplified DNA product from step 107 (e.g., purified in step 108) is contacted with a second target-specific primer and a second tail primer. In some embodiments, the second target-specific primer hybridizes to a sequence present at the 3' end of the template DNA molecule of the first target-specific primer sequence, thereby nesting the reaction. In some embodiments, nesting of the second target-specific primer relative to the first target-specific primer can improve the specificity of the hybridization reaction. In some embodiments, the second target-specific primer may include an additional sequence at the 5' end of the hybridization sequence, said additional sequence may include, for example, a barcode sequence, an index sequence, a adapter sequence, or a sequencing primer site. In step 110 A In this embodiment, the amplified DNA product from step 107 (e.g., purified in step 108) is amplified by PCR, wherein extension is initiated using a second target-specific primer and a second tail primer. In some embodiments, a portion of the amplified product from step 107 is further amplified. In some embodiments, a third primer is used, which hybridizes to the common tail of the second target-specific primer and adds additional sequences such as barcode sequences, adapters, etc.

[0026] In some embodiments, the second target-specific primer comprises a nucleotide sequence at the 5' end of the target-specific sequence, said nucleotide sequence comprising a barcode sequence, index sequence, or adapter sequence. In some embodiments, the second tail primer hybridizes to a sequence present at the 3' end of the template DNA molecule of the first tail primer sequence, thereby nesting the reaction. In such embodiments, a portion of the product from step 106 is amplified. In some embodiments, the second tail primer may comprise an additional sequence at the 5' end of the hybridization sequence, said additional sequence comprising a barcode sequence, index sequence, adapter sequence, or sequencing primer site. In the PCR reaction, the hybridization of the second target-specific primer and the second tail primer enables the exponential amplification of a portion of the target nucleic acid molecule.

[0027] In some embodiments, the first target-specific primer in step 106 may be used in conjunction with a second target-specific primer. In such embodiments, hybridization of the first and second target-specific primers allows the product to be amplified in a polymerase chain reaction (PCR). In some embodiments, steps 108-110 may be omitted. The amplified product is purified in reaction 111 and prepared for analysis. For example, the product purified in step 111 may be sequenced (e.g., using a next-generation sequencing platform). In some embodiments, the first or second target-specific primer may contain an additional sequence at the 5' end of the hybridization sequence, said additional sequence may include a barcode sequence, index sequence, adapter sequence, or sequencing primer site.

[0028] In some implementations, such as Figure 1B As described in step 109 B In step 107, the DNA product (e.g., purified in step 108) is contacted with a second target-specific primer and a second tail primer. The second target-specific primer is further contacted with an additional primer (e.g., a primer having a 3' sequencing adapter / index sequence) that hybridizes to a common sequence of the second target-specific primer. In some embodiments, the additional primer may contain an additional sequence at the 5' end of the hybridization sequence, said additional sequence may contain a barcode sequence, index sequence, adapter sequence, or sequencing primer site. In some embodiments, the additional primer is a universal sequencing adapter / index primer. In some embodiments, the second target-specific primer may be nested relative to the target-specific primer used in step 107. In step 110... BIn this embodiment, the DNA product from step 107 (e.g., purified in step 108) is amplified by PCR, wherein extension is initiated by a second target-specific primer and a second tail primer. In the PCR reaction, hybridization of the second target-specific primer, the additional primer, and the second tail primer allows a portion of the target nucleic acid molecule to be exponentially amplified. In this embodiment, a portion of the amplification product from step 108 is amplified.

[0029] In some embodiments, the first tail primer in step 106 may be used in conjunction with a second target-specific primer. In such embodiments, hybridization of the first tail primer and the second target-specific primer enables the product to be amplified in a polymerase chain reaction (PCR), optionally with an additional primer that hybridizes to a common sequence with the second target-specific primer (e.g., a primer with a 3' sequencing adapter sequence / index sequence). In some embodiments, steps 108-110 may be omitted.

[0030] The product is purified in reaction 111 and prepared for analysis. For example, the product purified in step 111 can be sequenced (e.g., using a next-generation sequencing platform).

[0031] In some embodiments, steps 101-104, 106-107, and 109-110 are performed consecutively in a single reaction tube without any intervening purification steps. In some embodiments, all components involved in steps 101-104, 106-107, and 109-110 are present at the start of the reaction and throughout the reaction. In some embodiments, steps 101-104 are performed consecutively in a single reaction tube. In some embodiments, all components involved in steps 101-104 are present at the start of the reaction and throughout the reaction. In some embodiments, steps 106-107 are performed consecutively in a single reaction tube. In some embodiments, all components involved in steps 106-107 are present at the start of the reaction and throughout the reaction. In some embodiments, step 109 is performed consecutively in a single reaction tube. A -Step 110 A Or step 109 B -Step 110 B In some implementations, step 109 A -Step 110 A Or step 109 B -Step 110 B All the components involved are present at the start of the reaction and throughout the entire reaction.

[0032] In some embodiments, methods are provided for preparing nucleic acids having a target region at the 3' end of an adjacent region (e.g., an adjacent region of an unknown sequence inclusion). For example, Figure 2 illustrates a schematic diagram of an exemplary method for amplifying and sequencing a target nucleic acid having a known target region at the 3' end of an adjacent region. Figures 2A-2B In this process, a starting RNA (e.g., fusion mRNA) is obtained or provided in the sample and used as a template for the processing method. In step 201, the RNA template is exposed to one or more starter target-specific primers that hybridize to one or more target nucleotide sequences and initiate a reverse transcription reaction, thereby generating a complementary DNA molecule using the starter RNA as a template. In some embodiments, the starter target-specific primers hybridize to a poly-A tail of the RNA template. In some embodiments, the primer sequence hybridizing to the poly-A tail contains poly-dT (e.g., extensions of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dTs located at the 3' end). In step 202, the unhybridized primers are degraded (e.g., enzymatic degradation, such as degradation by an exonuclease). In step 202, the RNA template is degraded from the complementary DNA strand (e.g., enzymatic degradation, such as degradation by RNase H).

[0033] In step 203, the DNA molecule generated by reverse transcription is contacted via a heterologous group of tailing primers (e.g., random tailing primers). In some embodiments, the tail portion of each tailing primer is a shared or common sequence that is identical across all primers in the tailing primer group. In some embodiments, at least one primer contains a hybridization sequence that is complementary to and hybridizes to the target nucleic acid template. In step 204, the tailing primer that hybridizes to the template nucleic acid is extended in a template-dependent extension reaction to produce a complementary DNA strand with both the tailing primer sequence and the template sequence incorporated. In step 205, the resulting double-stranded DNA product is purified.

[0034] In step 206, the DNA product purified in step 205 is contacted with a first target-specific primer and a first tail primer. The first target-specific primer hybridizes to the target sequence (region) of the DNA. The first tail primer hybridizes to a portion of the DNA molecule having the tail characteristics of the tail primer of step 203. In some embodiments, the first tail primer hybridizes to a common sequence provided by the tails of one or more primers of step 203. In some embodiments, the first tail primer contains a barcode sequence or an index sequence. In step 207, the hybridization of the first target-specific primer and the first tail primer promotes exponential amplification of a portion of the target nucleic acid molecule in an amplification reaction (e.g., a PCR reaction). In some embodiments, the first tail primer may contain an additional sequence at the 5' end of the hybridization sequence, said additional sequence may contain a barcode sequence, an index sequence, an adapter sequence, or a sequencing primer site. In step 208, the amplification product is purified. In some embodiments, purification in step 208 is omitted. For example, in some implementations, primers (e.g., a second target-specific primer and a second tail primer) are used in the PCR or extension reaction, and then excess primers are removed using a single-stranded DNA nuclease. Subsequent rounds of PCR or extension can be performed using different primers to add different sequences to the resulting product.

[0035] In some implementations, such as in Figure 2A Step 209 A As described, the DNA molecules produced in step 207 (e.g., purified in step 208) are contacted with a second target-specific primer and a second tail primer. In some embodiments, the second target-specific primer is nested relative to the target-specific primer used in step 207. In some embodiments, the DNA molecules from step 209 are contacted with a second target-specific primer and a second tail primer. A At least a portion of the product is amplified.

[0036] In some embodiments, the second target-specific primer comprises a nucleotide sequence at the 5' end of a target-specific sequence, said nucleotide sequence comprising a barcode sequence, an index sequence, or a linker sequence. In some embodiments, the second tail primer hybridizes to a sequence present at the 3' end of the template DNA molecule of the first tail primer sequence, thereby nesting the reaction. In some embodiments, the reaction proceeds from step 209... A At least a portion of the product is amplified.

[0037] In some embodiments, the first tail primer in step 206 may be used in conjunction with a second target-specific primer. In such embodiments, hybridization of the first tail primer and the second target-specific primer allows the product to be amplified in a polymerase chain reaction (PCR). In such embodiments, steps 208-210 may be omitted.

[0038] In some implementations, such as in Figure 2B Step 209 B As described, the DNA molecule produced in step 207 (e.g., purified in step 208) is contacted with a second target-specific primer and a second tail primer, wherein the second target-specific primer hybridizes to the target sequence present in the template DNA molecule at the 3' end of the first target-specific primer sequence, thereby nesting the reaction. In some embodiments, the reaction proceeds from step 209... B At least a portion of the product is amplified.

[0039] In some embodiments, the second target-specific primer is further contacted with an additional primer (e.g., a primer having a 3' sequencing adapter / index sequence), which hybridizes with a common sequence of the second target-specific primer. In some embodiments, the additional primer may contain an additional sequence at the 5' end of the hybridization sequence, which may include a barcode sequence, index sequence, adapter sequence, or sequencing primer site. In some embodiments, the additional primer is a universal sequencing adapter / index primer. In such embodiments, the hybridization of the second target-specific primer, the additional primer, and the second tail primer enables a portion of the target nucleic acid molecule to be exponentially amplified in the PCR reaction.

[0040] In some embodiments, the second target-specific primer comprises a nucleotide sequence at the 5' end of a target-specific sequence, said nucleotide sequence comprising a barcode sequence, an index sequence, or a linker sequence. In some embodiments, the second tail primer hybridizes to a sequence present at the 3' end of the template DNA molecule of the first tail primer sequence, thereby nesting the reaction. In some embodiments, the reaction proceeds from step 209... B At least a portion of the product is amplified.

[0041] In some embodiments, the second tail primer may include an additional sequence at the 5' end of the hybridization sequence, said additional sequence may include a barcode sequence, index sequence, adapter sequence, or sequencing primer site. In step 210, the hybridization of the second target-specific primer and the second tail primer promotes the exponential amplification of a portion of the target nucleic acid molecule in the amplification reaction (e.g., PCR reaction). The amplification product from step 210 is purified in reaction 211 and prepared for analysis. For example, the product purified in step 211 may be sequenced (e.g., using a next-generation sequencing platform).

[0042] In some embodiments, steps 201-204 are performed consecutively in a single reaction tube. In some embodiments, all components involved in steps 201-204 are present at the start of the reaction and throughout the reaction. In some embodiments, steps 206-207 are performed consecutively in a single reaction tube. In some embodiments, all components involved in steps 206-207 are present at the start of the reaction and throughout the reaction. In some embodiments, step 209 is performed consecutively in a single reaction tube. A - Step 210 or Step 209 B - Step 210. In some embodiments, step 209 A - Step 210 or Step 209 B - All components involved in step 210 are present at the start of the reaction and throughout the entire reaction.

[0043] In some embodiments, the methods provided herein involve using random sequences as molecular barcodes. In some embodiments, molecular barcodes are incorporated into primers (e.g., RT primers, target-specific primers, extension sequence primers), such that individual molecules generated by the primers each acquire a unique barcode tag. Therefore, in some embodiments, molecular barcode tags enable the determination of whether a sequenced molecule is unique. In some embodiments, molecular barcodes can be used to eliminate sequencing errors, increase the confidence required for fusions or other mutations, and improve the detection limit.

[0044] In some embodiments, a method is provided for preparing nucleic acids using hairpin-containing oligonucleotides, said nucleic acids having a target region at the 5' end of an adjacent region (e.g., an adjacent region of an unknown sequence). In some embodiments, the oligonucleotide may have a non-hairpin structure; for example, the oligonucleotide may be linear. Figure 3A schematic diagram of an exemplary method for amplifying a target nucleic acid having a known target region at the 5' end of an adjacent region (for sequencing purposes of the adjacent region) is shown. In step 301, a starting RNA is obtained or provided in a sample and used as a template for the processing method. The RNA template is exposed to a plurality of hairpin primers (e.g., random primers with hairpin tails) containing hairpin sequences, the hairpin sequences being the 5' ends of different hybridization sequences and shared across the entire primer group. The hairpin sequence contains two complementary common sequences flanking a molecular barcode sequence (MBC). The complementary common sequence bases pair to form a stem-loop hairpin structure and protect the MBC sequence. In some embodiments, the plurality of primers are in another structure (a non-hairpin structure) and contain two complementary common sequences flanking a molecular barcode sequence (MBC). In some embodiments, at least one primer hybridizes to the RNA molecule and initiates a reverse transcriptase reaction to produce a complementary DNA strand. In some embodiments, the primers hybridize to a poly-A tail of the RNA molecule. In some embodiments, the primer sequence hybridizing to the polyA-tail contains polydT (e.g., extensions of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dTs located at the 3' end). In step 302, any unhybridized oligonucleotides are enzymatically degraded (e.g., by exonuclease degradation). Similarly, in step 302, the RNA template is enzymatically degraded from the complementary DNA strand (e.g., by RNase H degradation).

[0045] In step 303, the DNA molecule generated by reverse transcription is contacted with one or more initiation target-specific primers, which may be the same as or different from the first target-specific primer. In step 304, the first target-specific primer hybridizes to a portion of the target nucleic acid primer to initiate an extension reaction that uses the DNA molecule as a template to generate a complementary DNA strand. In some embodiments, the synthesis of the complementary DNA strand may reduce or eliminate hairpin formation of complementary common sequences. In step 305, the extension product is purified.

[0046] In step 306, the DNA molecule is contacted using a first target-specific primer and a tailing primer. The first target-specific primer hybridizes to a portion of the target nucleic acid. The first tailing primer hybridizes to a portion of the DNA molecule provided by forming the common sequence involved in step 301 via hairpin formation. In some embodiments, nested target-specific primers are used in step 306 (e.g., nested relative to the target-specific primers in step 303). In some embodiments, the first tailing primer may contain an additional sequence at the 5' end of the hybridization sequence, said additional sequence may include, for example, a barcode sequence, an index sequence, an adapter sequence, or a sequencing primer site. In step 307, hybridization of each of the first target-specific primers and the tailing primers allows a portion of the target nucleic acid molecule to be amplified in a polymerase chain reaction (PCR). In some embodiments, the amplification product is purified in step 308.

[0047] In step 309, the amplified DNA product (e.g., the amplified DNA product purified in step 308) is contacted with a second target-specific primer and a common sequence primer. In some embodiments, the second target-specific primer hybridizes to a sequence present at the 3' end of the template DNA molecule of the first target-specific primer sequence, thereby nesting the reaction. In some embodiments, the common sequence primer hybridizes to a sequence provided by the first tail primer in step 306. In some embodiments, in step 310, the DNA product purified in step 308 is amplified by PCR, wherein the extension is initiated by the second target-specific primer and the common sequence primer. In some embodiments, the amplification product from step 308 may be amplified.

[0048] In some embodiments, the second target-specific primer comprises a nucleotide sequence at the 5' end of the target-specific sequence, said nucleotide sequence comprising a barcode sequence, index sequence, or adapter sequence. In some embodiments, the second tail primer hybridizes to a sequence present at the 3' end of the template DNA molecule of the first tail primer sequence to allow the reaction to be nested. In such embodiments, a portion of the product from step 308 is amplified. In some embodiments, the common sequence primer may comprise an additional sequence at the 5' end of the hybridization sequence, said additional sequence comprising a barcode sequence, index sequence, adapter sequence, or sequencing primer site. Hybridization of the second target-specific primer and the common sequence primer allows a portion of the target nucleic acid molecule to be exponentially amplified in the PCR reaction. In some embodiments, the product for analysis is purified in reaction 311. For example, the product purified in step 311 may be sequenced (e.g., using a next-generation sequencing platform).

[0049] In some embodiments, all components involved in steps 301-311 are present at the start of the reaction and throughout the reaction. In some embodiments, steps 301-304, 306-307, and 309-310 are performed consecutively in a single reaction tube without any intervening purification steps. In some embodiments, steps 301-304 are performed consecutively in a single reaction tube. In some embodiments, all components involved in steps 301-304 are present at the start of the reaction and throughout the reaction. In some embodiments, steps 306-307 are performed consecutively in a single reaction tube. In some embodiments, all components involved in steps 306-307 are present at the start of the reaction and throughout the reaction. In some embodiments, steps 309-310 are performed consecutively in a single reaction tube. In some embodiments, all components involved in steps 309-310 are present at the start of the reaction and throughout the reaction.

[0050] In some embodiments, this document provides methods relating to determining nucleotide sequences adjacent to (near) a known target nucleotide sequence. In some embodiments, the method includes contacting a target nucleic acid molecule containing the known target nucleotide sequence with an initiating target-specific primer under suitable hybridization conditions. In some embodiments, the method further includes holding the target nucleic acid molecule under conditions promoting hybridization with initiating target-specific primer extension (e.g., using the target nucleic acid molecule as a template) to produce a first extension product. In some embodiments, the method further includes contacting the extension product with a tailed random primer group under suitable hybridization conditions. In some embodiments, the method further includes holding the extension product under conditions promoting hybridization with tailed random primer extension (using a portion of the target nucleic acid molecule downstream of the hybridization site as a template) to produce a second extension product. In some embodiments, the method further includes amplifying a portion of the target nucleic acid molecule and the tailed random primer sequence with a first tail primer and a first target-specific primer to produce a first amplicon. In some embodiments, the method further includes amplifying a portion of the amplicon with a second tail primer and a second target-specific primer to produce a second amplicon.

[0051] In some embodiments, one or more target-specific primers used in the method may be nested relative to one or more other target-specific primers. For example, in some embodiments, a second target-specific primer is inside a first target-specific primer. In some embodiments, the target-specific primers are identical. In some embodiments, the target-specific primers are nested but overlapped relative to a target complement. In some embodiments, the target-specific primers are nested and non-overlapping. In some embodiments, a combination of identical and nested target-specific primers is used in the same or different amplification steps. In some embodiments, primer nesting increases target specificity. In some embodiments, the method further includes sequencing a second amplicon using a first sequencing primer and a second sequencing primer. In some embodiments, the tailed random primer group comprises a single-stranded oligonucleotide molecule having a 5' nucleotide sequence (identical to the first sequencing primer) and a 3' nucleotide containing random nucleotides (e.g., about 6 to about 12 random nucleotides). In some embodiments, the first target-specific primer comprises a nucleic acid sequence capable of specifically annealing to a target nucleic acid at an appropriate annealing temperature. In some embodiments, the second target-specific primer comprises a 3' portion and a 5' portion, the 3' portion comprising a nucleic acid sequence capable of specifically annealing to a portion of a known target nucleotide sequence contained in the first amplicon, and the 5' portion comprising the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer. In some embodiments, the first tail primer comprises a nucleic acid sequence identical to the common sequence of the tail of the tailed random primer. In some embodiments, the common sequence on the tailed random primer is an exact match to the common sequence on the first tail primer. In some embodiments, the second tail primer comprises a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer.

[0052] As used herein, the term "target nucleic acid" refers to a nucleic acid molecule of interest (e.g., the nucleic acid to be analyzed). In some embodiments, the target nucleic acid comprises both a target nucleotide sequence (e.g., a known or predetermined nucleotide sequence) and an adjacent nucleotide sequence to be determined (which may be referred to as an unknown sequence). The target nucleic acid may have any suitable length. In some embodiments, the target nucleic acid is double-stranded. In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is genomic DNA or chromosomal DNA (gDNA). In some embodiments, the target nucleic acid may be complementary DNA (cDNA). In some embodiments, the target nucleic acid is single-stranded. In some embodiments, the target nucleic acid may be RNA, such as mRNA, rRNA, tRNA, long non-coding RNA, or microRNA.

[0053] As used herein, the term "known target nucleotide sequence" refers to a sequence (e.g., the identity and sequence of nucleotide bases of a nucleic acid) that is part of a known target nucleic acid. For example, in some embodiments, a known target nucleotide sequence is a nucleotide sequence of a known nucleic acid or a nucleotide sequence of a nucleic acid that has been determined prior to interrogation of adjacent unknown sequences. A known target nucleotide sequence can have any suitable length.

[0054] In some embodiments, the target nucleotide sequence (e.g., a known target nucleotide sequence) has a length of 10 or more nucleotides, 30 or more nucleotides, 40 or more nucleotides, 50 or more nucleotides, 100 or more nucleotides, 200 or more nucleotides, 300 or more nucleotides, 400 or more nucleotides, or 500 or more nucleotides. In some embodiments, the length of the target nucleotide sequence (e.g., a known target nucleotide sequence) ranges from 10 to 100 nucleotides, 10 to 500 nucleotides, 10 to 1000 nucleotides, 100 to 500 nucleotides, 100 to 1000 nucleotides, 500 to 1000 nucleotides, or 500 to 5000 nucleotides.

[0055] In some embodiments, this document provides methods for determining the sequence of a neighboring (or adjacent) portion of a nucleic acid. As used herein, the term "nearby nucleotide sequence" refers to a nucleotide sequence of a nucleic acid molecule (e.g., a target nucleic acid) immediately upstream or downstream of another nucleotide sequence (e.g., a known nucleotide sequence). In some embodiments, the nucleotide sequence adjacent to the known target nucleotide sequence may have any suitable length. In some embodiments, the nucleotide sequence adjacent to the known target nucleotide sequence comprises nucleotide sequences less than 1 kb, such as nucleotide sequences less than 1 kb, nucleotide sequences less than 750 bp, nucleotide sequences less than 500 bp, nucleotide sequences less than 400 bp, nucleotide sequences less than 300 bp, nucleotide sequences less than 200 bp, and nucleotide sequences less than 100 bp. In some embodiments, in cases where a sample contains different target nucleic acids containing a known target nucleotide sequence (e.g., a known target nucleotide sequence occurring multiple times in its genome or on a separate non-identical chromosome), multiple sequences containing "nucleotide sequences adjacent to the known target nucleotide sequence" may be present. The term “determine nucleotide sequence” as used in this article refers to determining the types and relative positions of nucleotide bases in a nucleic acid.

[0056] In some embodiments of the methods disclosed herein, one or more tailed random primers are hybridized to a nucleic acid template (e.g., a template containing the target nucleic acid strand). In some embodiments, the target nucleic acid is present in or obtained from a sample containing a variety of nucleic acids, one or more of which do not contain the target nucleic acid. In some embodiments, one or more primers (e.g., one or more tailed random primers) hybridize substantially to all nucleic acids in the sample. In some embodiments, one or more primers (e.g., one or more tailed random primers) hybridize to nucleic acids containing the target nucleic acid and to nucleic acids that do not contain the target nucleotide sequence.

[0057] Some aspects of the methods disclosed herein involve contacting a nucleic acid template with multiple different primers that share a common sequence, which is the 5' end (or upstream) of different hybridization sequences. In some embodiments, the multiple different primers may be referred to as different primer groups. In some embodiments, the common sequence may be referred to as a tail, and in this respect, the primers are referred to as "tailed primers". In some embodiments, the different hybridization sequences of the group comprise nucleotide sequences that occur randomly or pseudo-randomly within the group. In some embodiments, the nucleotide sequences that occur randomly within the group do not contain an identifiable pattern, such that for each nucleotide in each sequence within the group, the nucleotide has an equal probability of containing a base complementary to A, T, G, or C. In such embodiments, it should be understood that each nucleotide containing a base complementary to A, T, G, or C can be a naturally occurring nucleotide, a non-naturally occurring nucleotide, or a modified nucleotide.

[0058] As used herein, "common sequence" or "shared sequence" refers to the nucleotide sequence present in each nucleic acid of a nucleic acid group. In some embodiments, the length of the common sequence ranges from approximately 4 to 75, 4 to 50, 4 to 30, or 4 to 20 nucleotides. In some embodiments, the length of the common sequence is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 nucleotides. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 nucleotides.

[0059] As used herein, the term "tailed random primer" refers to a single-stranded nucleic acid molecule having a 5' nucleotide sequence (e.g., a 5' nucleotide sequence that is the same as or complementary to the first sequencing primer) and a 3' nucleic acid sequence, wherein the 3' nucleotide comprises random nucleotides (e.g., about 3 to about 15 random nucleotides, about 6 to about 12 random nucleotides). In some embodiments, the length of the 3' nucleotide sequence comprising random nucleotides is at least 6 nucleotides, for example, 6 or more nucleotides, 7 or more nucleotides, 8 or more nucleotides, 9 or more nucleotides, 10 or more nucleotides, 11 or more nucleotides, 12 or more nucleotides, 13 or more nucleotides, 14 or more nucleotides, 15 or more nucleotides, 20 or more nucleotides, or 25 or more nucleotides. In some embodiments, the 3' nucleotide sequence comprising random nucleotides is 3-6 nucleotides long, 3-9 nucleotides long, 3-12 nucleotides long, 5-9 nucleotides long, 6-12 nucleotides long, 3-25 nucleotides long, 6-15 nucleotides long, or 6-25 nucleotides long. In some embodiments, the tailed random primer may further include a spacer between the 5' nucleotide sequence and the 3' nucleotide sequence comprising about 6 to about 12 random nucleotides. In some embodiments, the spacer is a molecular barcode, for example, a molecular barcode that independently tags the template nucleic acid (e.g., template RNA). In some embodiments, the spacer may be 3-6 nucleotides long, 3-12 nucleotides long, 3-25 nucleotides long, 3-45 nucleotides long, 6-12 nucleotides long, 8-16 nucleotides long, 6-25 nucleotides long, or 6-45 nucleotides long. In some embodiments, for primer groups, the spacer is composed of random nucleotides (e.g., NNNNNNNNN, where each N is independently selected from A, G, C, and T). In some embodiments, two complementary common regions are located on the flanks of the spacer (e.g., molecular barcode (MBC)). In some embodiments, the complementary common regions base pair to form the stem of a hairpin with a loop portion containing the MBC (e.g., in...). Figure 3(As described in [the document]). In some embodiments, in the case of 5' end fusion, this hairpin configuration protects the MBC from annealing to other targets of the RT reaction that inhibit the elongation reaction. In some embodiments, the tailed random primer group may comprise individual primers with varying 3' sequences. In some embodiments, the tailed random primer group may comprise individual primers with the same 5' nucleotide sequence, for example, all of which are compatible with the same sequencing primer. In some embodiments, the tailed random primer group may comprise individual primers with varying 5' nucleotide sequences, for example, the first individual primer is compatible with the first sequencing primer, and the second individual primer is compatible with the second sequencing primer.

[0060] As used herein, a "hybridization sequence" refers to a nucleic acid sequence (e.g., a portion of a primer) that is sufficiently complementary to the sequence of another nucleic acid (e.g., a template molecule, a target sequence) to enable hybridization between nucleic acids. In some embodiments, the length of the hybridization sequence is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides. In some embodiments, the length of the hybridization sequence ranges from 5 to 50 nucleotides, 5 to 40 nucleotides, 5 to 35 nucleotides, 5 to 30 nucleotides, 5 to 25 nucleotides, 5 to 20 nucleotides, 5 to 15 nucleotides, 5 to 10 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides, or 10 to 20 nucleotides.

[0061] In some embodiments, the methods disclosed herein include an extension scheme or extension step. In such embodiments, extension may be performed using a primer-hybridized nucleic acid molecule as a template, from one or more hybridized tailed random primers. The extension step is described herein. In some embodiments, one or more tailed random primers may hybridize substantially to all nucleic acids in a sample, many of which may not contain a known target nucleotide sequence. Therefore, in some embodiments, extension of the random primers may occur due to the use of a template that does not contain a known target nucleotide sequence for hybridization.

[0062] In some embodiments, the methods described herein may relate to a polymerase chain reaction (PCR) amplification protocol involving one or more amplification cycles. As used herein, the term "amplification protocol" refers to a procedure for specifically amplifying a nucleic acid of interest (increasing the abundance of the nucleic acid of interest). In some embodiments, exponential amplification occurs when a previous polymerase extension product serves as a template for subsequent multiple rounds of extension. In some embodiments, a PCR amplification protocol according to the methods disclosed herein may include at least one, and in some cases at least five or more, repeating cycles. In some embodiments, each repeating cycle includes the following steps: 1) strand separation (e.g., thermal denaturation); 2) annealing of oligonucleotide primers to the template molecule; and 3) nucleic acid polymerase extension of the annealed primers. It should be understood that any suitable conditions and times involved in each of these steps may be used. In some embodiments, the selected conditions and times may depend on the length, sequence contents, melting temperature, secondary structure characteristics, or other factors associated with the nucleic acid template and / or primers used in the reaction. In some embodiments, the amplification protocol according to the methods described herein is performed in a thermal cycler, many of which are commercially available.

[0063] In some embodiments, the nucleic acid extension reaction involves the use of a nucleic acid polymerase. As used herein, the phrase "nucleic acid polymerase" refers to an enzyme that catalyzes the template-dependent polymerization of nucleoside triphosphates to form a primer extension product complementary to the template nucleic acid sequence. The nucleic acid polymerase is synthesized by annealing the 3' end of the primer and proceeding toward the 5' end of the template. Numerous nucleic acid polymerases are known and commercially available in the art. A group of nucleic acid polymerases are thermostable, meaning that the polymerase retains its function after being subjected to temperatures sufficient to denature the complementary nucleic acid annealing strand (e.g., 94°C) or sometimes higher. Non-limiting examples of amplification protocols involve using a polymerase (e.g., PhoenixTaq, VeraSeq) under the following conditions: 98°C for 30 seconds, followed by 14–22 cycles of melting at 98°C for 10 seconds, then annealing at 68°C for 30 seconds, followed by extension at 72°C for 3 minutes, and then holding the reaction at 4°C. However, other suitable reaction conditions may be used. In some embodiments, the annealing / extending temperature can be adjusted to induce differences in salt concentration (e.g., 3°C or more corresponds to higher salt concentrations). In some embodiments, slowing down the ramp rate (e.g., from 98°C to 65°C) improves primer performance and coverage uniformity in highly reused samples.

[0064] In some embodiments, a nucleic acid polymerase is used under conditions where the enzyme performs template-dependent elongation. In some embodiments, the nucleic acid polymerase is DNA polymerase I, Taq polymerase, Pheonix Taq polymerase, Phusion polymerase, T4 polymerase, T7 polymerase, Klenow fragment, Klenow exo-, phi29 polymerase, AMV reverse transcriptase, M-MuLV reverse transcriptase, HIV-1 reverse transcriptase, VeraSeq Ultra polymerase, VeraSeq HF 2.0 polymerase, EnzScript, or another suitable polymerase. In some embodiments, the nucleic acid polymerase is not a reverse transcriptase. In some embodiments, the nucleic acid polymerase acts on a DNA template. In some embodiments, the nucleic acid polymerase acts on an RNA template. In some embodiments, the elongation reaction involves reverse transcription against RNA to produce complementary DNA molecules (RNA-dependent DNA polymerase activity). In some embodiments, the reverse transcriptase is mouse molony murine leukemia virus (M-MLV) polymerase, AMV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, or another suitable reverse transcriptase.

[0065] In some embodiments, the nucleic acid amplification reaction involves a cycle including a chain separation step, which typically involves heating the reaction mixture. As used herein, the term "chain separation" or "chain separation" refers to processing a nucleic acid sample to separate complementary double-stranded molecules into two single strands that can be annealed to oligonucleotide primers. In some embodiments, chain separation according to the methods described herein involves heating the nucleic acid sample to above its melting temperature (T0). m This is achieved by heating to 94°C for samples containing nucleic acid molecules in a reaction formulation suitable for a nucleic acid polymerase, thereby achieving chain separation. In some embodiments, a suitable reaction formulation comprises one or more salts (e.g., 1 mM-100 mM KCl, 0.1 mM-10 mM MgCl2), at least one buffer (e.g., 1 mM-20 mM Tris-HCl), and a carrier (e.g., 0.01%-0.5% BSA). Non-limiting examples of suitable buffers include 50 mM KCl, 10 mM Tris-HCl (pH 8.8 @ 25°C), 0.5 mM-3 mM MgCl2, and 0.1% BSA.

[0066] In some embodiments, nucleic acid amplification involves annealing primers to a nucleic acid template having a strand characteristic of the target nucleic acid. In some embodiments, the strand of the target nucleic acid may be used as the template nucleic acid.

[0067] As used herein, the term "annealing" refers to the formation of one or more complementary base pairs between two nucleic acids. In some embodiments, annealing involves hybridization of two complementary or substantially complementary nucleic acid strands together. In some embodiments, in the context of an extension reaction, annealing involves hybridizing a primer to a template to form a primer extension substrate for template-dependent polymerase. In some embodiments, annealing conditions (e.g., between the primer and the nucleic acid template) may vary based on the primer length and sequence. In some embodiments, annealing conditions are based on the primer's T... m (For example, the calculated T) m In some implementations, the annealing step of the extension scheme involves lowering the temperature to primer T after the chain separation step. m (For example, the calculated T) m The temperature is maintained for a sufficiently long time to allow for annealing. In some implementations, various algorithms (e.g., OLIGO) can be used. TM (Molecular Biology Insights Inc. Colorado) primer design software and VENTRO NTI TM Primer design software (Invitrogen, Inc., California) and any of the programs available on the Internet, including Primer3, Oligo Calculator, and NetPrimer (Premier Biosoft; Palo Alto, CA; and those freely available on the World Wide Web (e.g., at premierbiosoft.com / netprimer / netprlaunch / Help / xnetprlaunch.html)), to determine T m In some implementations, the T of the primer m The following formula can be used for calculation, which is used by the NetPrimer software and described in more detail in Frieir et al., PNAS 198683:9373-9377, which is incorporated herein by reference in its entirety.

[0068] T m =ΔH / (ΔS+R*In(C / 4))+16.6log([K + ] / (1+0.7[K + ]))-273.15

[0069] Where ΔH is the enthalpy of helical formation; ΔS is the entropy of helical formation; R is the molar gas constant (1.987 cal / ℃*mol); C is the nucleic acid concentration; and [K + [This represents the salt concentration.] For most amplification protocols, the annealing temperature is chosen to be lower than the predicted T.m Approximately 5°C, although temperatures closer to T can be used. m and higher than T m Temperature (e.g., below predicted T) m Temperatures between 1°C and 5°C or higher than predicted T m Temperatures between 1°C and 5°C, for example, below the predicted T m Temperatures exceeding 5°C (e.g., 6°C, 8°C, 10°C, or lower) are also acceptable. In some embodiments, the closer the annealing temperature is to T... m The more specific the annealing, the better. In some embodiments, the time used for primer annealing during the extension reaction (e.g., in the context of a PCR amplification protocol) is determined at least in part based on the volume of the reaction (e.g., larger volumes involve longer times). In some embodiments, the time used for primer annealing during the extension reaction (e.g., in the context of a PCR amplification protocol) is determined at least in part based on the concentrations of the primers and template (e.g., higher relative concentrations of primers and template involve less time compared to lower relative concentrations). In some embodiments, depending on the volume and relative primer / template concentrations, the primer annealing step in the extension reaction (e.g., in the context of a PCR amplification protocol) can range from 1 second to 5 minutes, 10 seconds to 2 minutes, or 30 seconds to 2 minutes. As used herein, “substantially annealed” refers to the degree to which complementary base pairs formed between two nucleic acids, when used in the context of a PCR amplification protocol, reach a level sufficient to produce a specific amplified product at a detectable level.

[0070] As used herein, the term "polymerase extension" refers to the template-dependent addition of at least one complementary nucleotide to the 3' end of a primer annealed to a nucleic acid template by a nucleic acid polymerase. In some embodiments, polymerase extension adds more than one nucleotide, for example, up to and including nucleotides corresponding to the full length of the template. In some embodiments, the conditions for polymerase extension are based at least in part on the type of polymerase used. In some embodiments, the temperature used for polymerase extension is based on the known activity properties of the enzyme. In some embodiments, it is acceptable to use a lower extension temperature where the annealing temperature is below the enzyme's optimal temperature. In some embodiments, the enzyme can retain at least partial activity below its optimal extension temperature. In some embodiments, polymerase extension is performed at 65°C–75°C or 68°C–72°C (e.g., polymerase extension performed with a thermostable polymerase) (e.g., Taq polymerase and its variants). In some embodiments, the methods provided herein involve polymerase extension of primers annealed to a nucleic acid template in each cycle of a PCR amplification protocol. In some embodiments, polymerase extension is performed using a polymerase with relatively strong strand displacement activity. In some implementations, polymerases with strong strand displacement activity are used to prepare nucleic acids for the purpose of detecting fusions (e.g., 5' end fusions).

[0071] In some embodiments, primer extension is performed under conditions that allow for annealing of oligonucleotide primers. As used herein, the term "conditions that allow for annealing of oligonucleotides to produce an extension product" refers to the group of conditions including, for example, temperature, salt and cofactor concentrations, pH, and enzyme concentrations, under which a nucleic acid polymerase catalyzes primer extension. In some embodiments, these conditions are at least partially based on the nucleic acid polymerase used. In some embodiments, the polymerase can carry out the primer extension reaction in a suitable reaction formulation. In some embodiments, a suitable reaction formulation comprises one or more salts (e.g., 1 mM–100 mM KCl, 0.1 mM–10 mM MgCl2), at least one buffer (e.g., 1 mM–20 mM Tris-HCl), a carrier (e.g., 0.01%–0.5% BSA), and one or more NTPs (e.g., 10 μM–200 μM each of dATP, dTTP, dCTP, and dGTP). The non-limiting group of conditions is 50 mM KCl, 10 mM Tris-HCl (pH 8.8 @ 25 °C), 0.5 mM-3 mM MgCl2, 200 μM each of dNTPs, and 0.1% BSA, at 72 °C, with a polymerase (e.g., Taq polymerase) catalyzing primer extension under these conditions. In some embodiments, the initiation and extension conditions may include the presence of one, two, three, or four different deoxyribonucleoside triphosphates (e.g., selected from dATP, dTTP, dCTP, and dGTP) and a polymerization inducer (e.g., DNA polymerase or reverse transcriptase) in a suitable buffer. In some embodiments, the "buffer" may include a solvent (e.g., an aqueous solvent), plus appropriate cofactors and reagents affecting pH, ionic strength, etc.

[0072] In some embodiments, nucleic acid amplification involves up to 5, 10, 20, 30, 40, or more rounds (cycles) of amplification. In some embodiments, nucleic acid amplification may include a PCR amplification protocol cycle of 5 to 20 cycles. In some embodiments, the amplification step may include a PCR amplification protocol cycle of 10 to 20 cycles. In some embodiments, each amplification step may include a PCR amplification protocol cycle of 12 to 16 cycles. In some embodiments, the annealing temperature may be below 70°C. In some embodiments, the annealing temperature may be below 72°C. In some embodiments, the annealing temperature may be below 65°C. In some embodiments, the annealing temperature may be from about 61°C to about 72°C.

[0073] In various embodiments, the methods and compositions described herein relate to PCR amplification protocols using one or more types of primers described herein. As used herein, "primer" refers to an oligonucleotide capable of specifically annealing to a nucleic acid template and providing a 3' end as a substrate for a template-dependent polymerase to produce an extension product complementary to the template. In some embodiments, primers used in the methods described herein are single-stranded so that the primer and its complement can anneal to form a double strand. Primers according to the methods and compositions described herein may comprise a hybridization sequence (e.g., a sequence annealed to a nucleic acid template) of less than or equal to 300 nucleotides, for example, less than or equal to 300, or 250, or 200, or 150, or 100, or 90, or 80, or 70, or 60, or 50, or 40, or 30 or less, or 20 or less, or 15 or less nucleotides, but at least 6 nucleotides. In some implementations, the hybridization sequence of the primer can be 6-50 nucleotides, 6-35 nucleotides, 6-20 nucleotides, or 10-25 nucleotides in length.

[0074] Any suitable method for synthesizing oligonucleotides and primers can be used. In some embodiments, commercial sources provide oligonucleotide synthesis services suitable for providing primers for the methods and compositions described herein, for example, INVITROGEN. TM Custom DNA Oligos; Life Technologies; Grand Island, NY or custom DNAOligos from IDT; Coralville, IA.

[0075] In some embodiments, after extension from the tailed random primers has occurred, the extension product and template can be amplified in a first amplification step. In some embodiments, amplification may involve a PCR amplification cycle using a first target-specific primer and a first tailed primer. In some embodiments, amplification may amplify at least a portion of the tailed random primer sequence present in the extension product. In some embodiments, amplification may amplify the entire tailed random primer sequence present in the extension product.

[0076] As used in this article, the term "first target-specific primer" refers to a single-stranded oligonucleotide containing a nucleic acid sequence that can specifically anneal to a nucleic acid template (a strand with the characteristics of the target nucleic acid) under suitable annealing conditions.

[0077] In some embodiments, primers (e.g., target-specific primers) may contain a 5' tag sequence portion. In some embodiments, multiple primers present in the reaction (e.g., all first target-specific primers) may contain the same 5' tag sequence portion. In some embodiments, in multiplex PCR reactions, different primer species can interact with each other in an off-target manner, causing primer extension and subsequent amplification by DNA polymerase. In such embodiments, these primer dimers are generally short, and their efficient amplification can dominate the reaction, resulting in poor amplification of the desired target sequence. Therefore, in some embodiments, the inclusion of a 5' tag sequence in the primers (e.g., on target-specific primers) can cause the formation of primer dimers (which contain the same complementary tails at both ends). In some embodiments, in subsequent amplification cycles, such primer dimers will denature into single-stranded DNA primer dimers, each containing a complementary sequence introduced by the 5' tag at both ends. In some embodiments, instead of primer annealing to these single-stranded DNA primer dimers, intramolecular hairpin (pot handle-like structures) formation can occur due to the proximate accessibility of complementary tags on the same primer dimer molecule, rather than intermolecular interactions with new primers on different molecules. Therefore, in some embodiments, these primer dimers may be difficult to amplify efficiently, preventing the primers from being consumed exponentially by dimer amplification; instead, tagged primers can maintain a high and sufficient concentration for specific amplification of the desired target sequence. In some embodiments, primer dimer accumulation may be undesirable in the context of multiplex amplification because primer dimers compete for and consume other reagents in the reaction.

[0078] In some embodiments, the 5' tag sequence may be a GC-rich sequence. In some embodiments, the 5' tag sequence may contain at least 50% GC, at least 55% GC, at least 60% GC, at least 65% GC, at least 70% GC, at least 75% GC, at least 80% GC, or higher GC content. In some embodiments, the tag sequence may contain at least 60% GC content. In some embodiments, the tag sequence may contain at least 65% GC content.

[0079] In some implementations, the target-specific primer (e.g., a second target-specific primer) is a single-stranded oligonucleotide comprising a 3' portion and a 5' portion, wherein the 3' portion comprises a nucleic acid sequence that can specifically anneal to a portion of a known target nucleotide sequence of an amplicon for an amplification reaction, and the 5' portion comprises a tag sequence (e.g., a nucleotide sequence that is the same as or complementary to that of the sequencing primer (e.g., a second sequencing primer).

[0080] In some embodiments, the second target-specific primer of the amplification protocol is nested relative to the first target-specific primer of the amplification protocol. In some embodiments, the second target-specific primer is nested relative to the first target-specific primer by at least 3 nucleotides (e.g., by more than 3, 4, 5, 6, 7, 8, 9, 10, or 15 nucleotides). In some embodiments, all target-specific primers used in the amplification protocol (e.g., the second target-specific primer) contain the same 5' portion. In some embodiments, the 5' portion of the target-specific primer may be configured to suppress the primer dimer described herein.

[0081] In some embodiments, the first and second target-specific primers used in the amplification protocol are substantially complementary to the same strand of the target nucleic acid. In some embodiments, portions of the first and second target-specific primers specifically annealed to the target sequence (e.g., a known target sequence) may contain a total of at least 20 unique bases of the known target nucleotide sequence, for example, more than 20 unique bases, more than 25 unique bases, more than 30 unique bases, more than 35 unique bases, more than 40 unique bases, or more than 50 unique bases. In some embodiments, portions of the first and second target-specific primers specifically annealed to the target sequence (e.g., a known target sequence) may contain a total of at least 30 unique bases of the known target nucleotide sequence.

[0082] The term "first tail primer" as used in this article refers to a nucleic acid molecule containing the same nucleic acid sequence as the tail portion of the tail primer.

[0083] As used herein, the term "second-tailed primer" refers to a nucleic acid molecule containing a portion of the same nucleic acid sequence as the first sequencing primer, adapter, index primer, etc., and optionally nested relative to the first tailed primer. In some embodiments, the second-tailed primer is located outside the first tailed primer to facilitate the addition of appropriate index tags, adapters (e.g., for sequencing platforms), etc. In some embodiments, the second tailed primer is identical to the sequencing primer. In some embodiments, the second tailed primer is complementary to the sequencing primer.

[0084] In some embodiments, the second tail primer is nested relative to the first tail primer. In some embodiments, the second tail primer is not nested relative to the first tail primer. In some embodiments, the tail primers of the amplification protocol are nested relative to each other by at least 3 nucleotides (e.g., by 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides).

[0085] In some embodiments, the first tail primer contains a nucleic acid sequence that is identical or complementary to the extended product of the strand in step (b), which is not contained in the second tail primer and is closer to the 5' end of the tailed random primer than any sequence identical or complementary to the second tail primer. Therefore, in some embodiments, the second tail primer is located outside (at the 5' end) of the region added by the random tail primer, for example, within the 5' tail added by the first tail primer.

[0086] In some embodiments, the first tail primer may contain a nucleic acid sequence that is the same as or complementary to the 5' end nucleotide extension of the tailed random primer (e.g., having about 20 nucleotides), and the second tail primer may contain a nucleic acid sequence that is the same as or complementary to about 30 bases of the tailed random primer, the second tail primer having a 5' nucleotide of at least 3 nucleotides in the 3' direction as the 5' end of the tailed random primer.

[0087] In some implementations, the use of nested tail primers minimizes or eliminates the generation of amplifiable (e.g., in bridge PCR or emulsion PCR) but unsequential final amplicons, a situation that can occur in hemi-nested methods. In some implementations, hemi-nested methods using the same primers as sequencing primers can result in unwanted amplification products being carried over from the first PCR step to the second PCR step, ultimately producing artificial sequencing reads. In some implementations, as described herein, the use of two tail primers can reduce such problems, and in some implementations, eliminate them altogether.

[0088] In some embodiments, during the first PCR amplification cycle of the first amplification step, the first target-specific primer is specifically annealed to the template strand of any nucleic acid containing a known target nucleotide sequence. In some embodiments, depending on the design direction of the first target-specific primer, a sequence upstream or downstream of the known target nucleotide sequence and complementary to the template strand is synthesized. In some embodiments, in the case of forming an extension product containing a hybridization sequence (with which the first target-specific primer forms a complementary base pair), a double-stranded amplification product can be formed comprising: the first target-specific primer (and its complementary sequence), a target nucleotide sequence downstream of the first target-specific primer (and its complementary sequence), and a tailed random primer sequence (and its complementary sequence). In such embodiments, during subsequent PCR amplification cycles, both the first target-specific primer and the first tailed primer are specifically annealed to the appropriate strand of the amplification product, and the sequence between the known nucleotide target sequence and the tailed random primer is amplified.

[0089] In some embodiments of the methods described herein, a portion of the amplification product (amplifier) ​​is amplified in further rounds of amplification. In some embodiments, the further rounds of amplification may involve PCR amplification cycles using a second target-specific primer and a first sequencing primer or a second tail primer. In some embodiments, the PCR amplification cycles may involve using PCR parameters that are the same as or different from those of one or more other (e.g., prior) PCR amplification cycles. In some embodiments, the PCR amplification protocol may have the same or different annealing temperatures, or the same or different extension step durations.

[0090] In some embodiments, the methods described herein enable the determination of nucleotide sequences adjacent to a known target nucleotide sequence in flanking regions on one or both sides of the known target nucleotide sequence. Regardless of whether the target nucleic acid is normally present as a single-stranded or double-stranded nucleic acid, the sequence information can be represented in a 5' to 3' single-stranded format (strand A). In some embodiments, if the 5' end of the known target nucleotide sequence of strand A is to be determined, a gene-specific primer may be complementary to strand A (annealed to strand A). If the 3' end of the known target nucleotide sequence of strand A is to be determined, a gene-specific primer may be identical to strand A, such that the primer annealed to the complementary strand of the double-stranded target nucleic acid.

[0091] In some embodiments, the methods described herein involving the use of first gene-specific primers and second gene-specific primers enable analyses with excellent on-target rates, for example, 70%–90%. In some embodiments, the analyses and methods described herein can have a target specificity of at least 85%.

[0092] In some embodiments, the primers disclosed herein (e.g., target-specific primers, tail primers) are designed such that they specifically anneal to their complementary sequences at an annealing temperature of about 61°C–72°C (e.g., about 61°C–69°C, about 63°C–69°C, about 63°C–67°C, about 64°C–66°C). In some embodiments, the primers disclosed herein are designed such that they specifically anneal to their complementary sequences at an annealing temperature below 72°C. In some embodiments, the primers disclosed herein are designed such that they specifically anneal to their complementary sequences at an annealing temperature below 70°C. In some embodiments, the primers disclosed herein are designed such that they specifically anneal to their complementary sequences at an annealing temperature below 68°C. In some embodiments, the primers disclosed herein are designed such that they specifically anneal to their complementary sequences at an annealing temperature of about 65°C.

[0093] In some embodiments, the portion of the target-specific primer specifically annealed to the known target nucleotide sequence will be specifically annealed at a temperature of approximately 61°C–72°C (e.g., approximately 61°C–69°C, approximately 63°C–69°C, approximately 63°C–67°C, approximately 64°C–66°C). In some embodiments, the portion of the target-specific primer specifically annealed to the known target nucleotide sequence will be specifically annealed in PCR buffer at a temperature of approximately 65°C.

[0094] In some embodiments, the primers described herein do not contain modified bases (e.g., the primers may not contain blocking 3' amine). However, in some embodiments, the primers described herein do not contain modified bases or naturally occurring bases. In some embodiments, the primers may be modified directly or indirectly with a label capable of providing a detectable signal. Non-limiting examples of such labels include radioisotopes, fluorescent molecules, biotin, and others. In some embodiments, the primers disclosed herein may include a biotin adapter or other suitable adapter (e.g., for conjugating the primer to a support). In some embodiments, the primers may contain a target sequence of a nuclease for cleavage with an appropriate enzyme. In other embodiments, the 5' end of the primer may contain a sequence complementary to the nucleic acid bound to the bead or other support (e.g., a flow cell substrate). The primers may or may not contain modified nucleotide bonds.

[0095] In some embodiments of the methods described herein, nucleic acids (e.g., amplified nucleic acids, extension products, target nucleic acids) can be sequenced. In some embodiments, sequencing can be performed using next-generation sequencing. As used herein, “next-generation sequencing” refers to oligonucleotide sequencing technology: said oligonucleotide sequencing technology has the ability to sequence oligonucleotides at speeds higher than that of conventional sequencing methods (e.g., Sanger sequencing) because said next-generation sequencing performs and reads thousands to millions of sequencing reactions in parallel. Non-limiting examples of next-generation sequencing methods / platforms include: massively parallel signature sequencing (Lynx Therapeutics); 454 pyrosequencing (454Life Sciences / RocheDiagnostics); solid-phase reversible dye-terminated sequencing (Solexa / Illumina); SOLiD technology (Applied Biosystems); Ion semiconductor sequencing (ION Torrent); DNA nanosphere sequencing (Complete Genomics); and technologies available from Pacific Biosciences, Intelligen Bio-systems, Oxford Nanopore Technologies, and Helicos Biosciences. In some implementations, sequencing primers may include portions compatible with selected next-generation sequencing methods.The limitations and design parameters of next-generation sequencing technologies and related sequencing primers are well known in the field (see, for example, Shendure et al., “Next-generation DNA sequencing”, Nature, 2008, Vol. 26, No. 10, pp. 1135-1145; Mardis, “The impact of next-generation sequencing technology on genetics”, Trends in Genetics, 2007, Vol. 24, No. 3, pp. 133-141; Su et al., “Next-generation sequencing and its applications in molecular diagnostics”, Expert Rev Mol Diagn, 2011, 11(3):333-43; Zhang et al., “The impact of next-generation sequencing on genomics”, J Genet Genomics, 2011, 38(3):95-109; Nyren, P. et al., Anal Biochem 208:17175 (1993); Bentley, DR, Curr Opin Genet Dev 16:545-52 (2006); Strausberg, RL et al., Drug Disc Today 13:569-77 (2008); U.S. Patent Nos. 7,282,337; 7,279,563; 7,226,720; 7,220,549; 7,169,560; 6,818,395; 6,911,345; U.S. Publications 2006 / 0252077, 2007 / 0070349 and 20070070349, which are incorporated herein by reference in their entirety.

[0096] In some implementations, the sequencing step involves the use of a first sequencing primer and a second sequencing primer. In some implementations, the first and second sequencing primers are selected to be compatible with the next-generation sequencing methods described herein.

[0097] The method of aligning sequencing reads with known sequence databases of genome and / or cDNA sequences is well known in the art, and software used in this process is commercially available. In some embodiments, reads that are not fully mapped to a wild-type sequence database (with sequencing primer nucleotide sequences removed) may be genomic rearrangements or large indel mutations. In some embodiments, reads containing sequences mapped to multiple locations in the genome (with sequencing primer nucleotide sequences removed) may be genomic rearrangements.

[0098] In some embodiments, primers may include additional sequences, such as identifier sequences (e.g., barcode sequences, index sequences), sequencing primer hybridization sequences (e.g., Rd1), and adapter sequences. In some embodiments, the adapter sequences are sequences used with next-generation sequencing systems. In some embodiments, the adapter sequences are P5 and P7 sequences for Illumina-based sequencing technologies. In some embodiments, the adapter sequences are P1 and A sequences compatible with Ion Torrent sequencing technologies.

[0099] In some embodiments, the terms “barcode,” “molecular barcode,” “molecular barcode label,” and “index” used herein are used interchangeably and generally refer to the nucleotide sequence of a nucleic acid used as an identifier (e.g., a source identifier, location identifier, date identifier, or time identifier (e.g., the date or time of sampling or processing) or other identifier for the nucleic acid). In some embodiments, such barcode sequences or index sequences are used to identify different aspects of nucleic acids present in a group of nucleic acids. In some embodiments, barcode sequences or index sequences may provide a source identifier or location identifier to a target nucleic acid. For example, barcode sequences or index sequences may be used to identify a patient from whom nucleic acid was obtained. In some embodiments, barcode sequences or index sequences enable sequencing of multiple samples on a single reaction (e.g., in a single flow cell). In some embodiments, index sequences may be used in directed sequence imaging for the purpose of detecting individual sequencing reactions. In some embodiments, the length of a barcode sequence or index sequence may be 2–25 nucleotides, 2–15 nucleotides, 2–10 nucleotides, or 2–6 nucleotides. In some implementations, the barcode sequence or index sequence may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or at least 25 nucleotides.

[0100] In some embodiments, when using a set of tailed random primers according to the methods described herein, multiple distinguishable amplification products may exist after amplification. In some embodiments, because the tailed random primers hybridize at different locations along the nucleic acid molecules throughout the sample, the target-specific primer set can hybridize (and amplify) through extension products generated by more than one hybridization event. For example, one tailed random primer may hybridize at a first distance (e.g., 100 nucleotides) from the hybridization site of the target-specific primer, and another tailed random primer may hybridize at a second location (e.g., 200 nucleotides) from the hybridization site of the target-specific primer, thereby producing two amplification products (e.g., an amplification product of about 100 bp and an amplification product of about 200 bp). In some embodiments, these multiple amplification products can be sequenced individually. In some embodiments, sequencing of these multiple amplification products is advantageous because it provides multiple overlapping sequence reads that can be compared with each other to detect sequence errors introduced during amplification or sequencing. In some implementations, individual amplification products can be compared, and in cases where the sequences differ at specific bases, errors or artifacts in PCR and / or sequencing may exist.

[0101] In some embodiments, the target nucleic acid and / or its amplification product may be separated from the enzyme, primer, or buffer components before and / or after any suitable step of the method. Any suitable method for isolating nucleic acids may be used. In some embodiments, isolation may include solid-phase reversible immobilization (SPRI) purification. SPRI purification methods are well known in the art, and kits are commercially available, such as AgencourtAMPure XP-PCR purification (catalog number A63880, Beckman Coulter; Brea, CA). In some embodiments, the enzyme may be inactivated by heat treatment.

[0102] In some embodiments, unhybridized primers can be removed from the nucleic acid preparation using appropriate methods (e.g., purification, digestion, etc.). In some embodiments, a nuclease (e.g., exonuclease I) is used to remove primers from the preparation. In some embodiments, such nucleases are inactivated by heating after primer digestion. After nuclease inactivation, a further primer set can be added along with other appropriate components (e.g., enzymes, buffers) for further amplification reactions.

[0103] In some embodiments, the target nucleic acid may be genomic DNA or a portion thereof. In some embodiments, the target nucleic acid may be ribonucleic acid (RNA) (e.g., mRNA) or a portion thereof. In some embodiments, the target nucleic acid may be cDNA or a portion thereof.

[0104] Many sequencing methods suitable for use with the methods described herein provide sequencing runs with optimal read lengths of tens to hundreds of nucleotide bases (e.g., Ion Torrent technology can produce read lengths of 200 bp to 400 bp). The target nucleic acid may be substantially longer than or substantially shorter than this optimal read length. In some embodiments, in order to give the amplified nucleic acid portion a suitable length for use in a particular sequencing technology, the average distance between the known target nucleotide sequence and the end of the target nucleic acid (to which a tailing random primer may be hybridized) should be as close as possible to the optimal read length of the selected technology. In some embodiments, if the optimal read length for a given sequencing technology is 200 bp, then the nucleic acid molecule amplified according to the methods described herein should have an average length of about 800 bp, about 700 bp, about 600 bp, about 500 bp, about 400 bp, about 300 bp, or less than about 200 bp.

[0105] The nucleic acids used in this paper can be cleaved (e.g., before sequencing) (e.g., mechanical cleavage or enzymatic cleavage) to produce fragments of any desired size. Non-limiting examples of mechanical cleavage methods include sonication, nebulization, and AFA available from Covaris (Woburn, MA). TM Shearing techniques. In some implementations, nucleic acids can be mechanically sheared using ultrasound.

[0106] In some embodiments, the target nucleic acid is not cleaved or digested. In some embodiments, the nucleic acid products (e.g., extension products, amplification products) from the preparation steps are not cleaved or enzymatically digested.

[0107] In some embodiments, when the target nucleic acid is RNA, the sample may be subjected to a reverse transcriptase protocol to generate a DNA template, which may then be cleaved. In some embodiments, the target RNA may be cleaved prior to the reverse transcriptase protocol. In some embodiments, samples containing the target RNA may be used in the methods described herein that: use total nucleic acids extracted from fresh or degraded samples; do not require removal of genomic DNA for cDNA sequencing; do not require depletion of ribosomal RNA for cDNA sequencing; do not require mechanical or enzymatic cleavage at any step; and subject the RNA to double-stranded cDNA synthesis using random hexamers.

[0108] In some embodiments, the known target nucleotides may comprise a fusion sequence resulting from a gene rearrangement. In some embodiments, the methods described herein are suitable for determining the presence and / or type of a gene rearrangement. In some embodiments, the type of a portion of the gene rearrangement is previously known (e.g., a portion of the gene rearrangement to be targeted by a gene-specific primer), and the sequences of other portions may be determined using the methods disclosed herein. In some embodiments, the gene rearrangement may involve an oncogene. In some embodiments, the gene rearrangement may comprise a fusion oncogene.

[0109] In some embodiments, the target nucleic acid is present in or obtained from a suitable sample (e.g., a food sample, an environmental sample, a biological sample such as a blood sample, etc.). In some embodiments, the sample is a biological sample obtained from a subject. In some embodiments, the sample may be a diagnostic sample obtained from a subject. In some embodiments, the sample may further comprise proteins, cells, fluids, biofluids, protective agents, and / or other substances. By way of non-limiting examples, the sample may be a cheek swab, blood, serum, plasma, sputum, cerebrospinal fluid, urine, tears, alveolar isolates, pleural fluid, pericardial fluid, cyst fluid, tumor tissue, tissue, biopsy material, saliva, aspirate, or a combination thereof. In some embodiments, the sample may be obtained by excision or biopsy.

[0110] In some implementations, the sample may be obtained from a subject who requires treatment for a disease associated with a genetic alteration (e.g., cancer or a hereditary disease). In some implementations, the known target sequence is present in the disease-related gene.

[0111] In some embodiments, the sample is obtained from a subject requiring cancer treatment. In some embodiments, the sample comprises a population of tumor cells, for example, a population of at least one type of tumor cell. In some embodiments, the sample comprises tumor biopsy material, including but not limited to untreated biopsy tissue or treated biopsy tissue (e.g., formalin-fixed biopsy tissue and / or paraffin-embedded biopsy tissue).

[0112] In some embodiments, the sample is collected fresh. In some embodiments, the sample is stored prior to use in the methods and compositions described herein. In some embodiments, the sample is an untreated sample. As used herein, an "untreated sample" means a biological sample that has not undergone any prior sample pretreatment other than dilution in solution and / or suspension in solution. In some embodiments, the sample is obtained from a subject and preserved or processed prior to use in the methods and compositions described herein. By way of non-limiting examples, the sample may be embedded in paraffin, refrigerated, or frozen. Frozen samples may be thawed prior to determining the presence of nucleic acids according to the methods and compositions described herein. In some embodiments, the sample may be a processed or treated sample. Exemplary methods for processing or manipulating samples include, but are not limited to: centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, contact with a protective agent (e.g., an anticoagulant or a nuclease inhibitor), and any combination thereof. In some embodiments, the sample may be treated with chemical and / or biological reagents. Chemical and / or biological reagents may be used to protect and / or maintain the stability of the sample or the nucleic acids contained in the sample during processing and / or storage. Alternatively or additionally, chemical and / or biological reagents may be used to release nucleic acids from other components of the sample. By way of non-limiting example, blood samples may be treated with an anticoagulant prior to use in the methods and compositions described herein. Suitable methods and procedures for sample processing, preservation, or handling for nucleic acid analysis may be used in the methods disclosed herein. In some embodiments, the sample may be a clear fluid sample, for example, obtained by centrifugation. In some embodiments, the sample may be clarified by low-speed centrifugation (e.g., below 3000 × g) and collection of the supernatant containing the clear fluid sample.

[0113] In some embodiments, nucleic acids present in a sample may be isolated, enriched, or purified prior to use in the methods and compositions described herein. Suitable methods for isolating, enriching, or purifying nucleic acids from a sample may be used. For example, kits for isolating genomic DNA from various sample types are commercially available (e.g., catalog numbers 51104, 51304, 56504, and 56404; Qiagen; Germantown, MD). In some embodiments, the methods described herein relate to enriching target nucleic acids, for example, prior to sequencing of the target nucleic acid. In some embodiments, the sequence at one end of the target nucleic acid to be enriched is unknown prior to sequencing. In some embodiments, the methods described herein relate to enriching specific nucleotide sequences prior to determining the nucleotide sequence using next-generation sequencing technology. In some embodiments, the methods for enriching specific nucleotide sequences do not include hybridization enrichment.

[0114] The methods described herein can be used in multiplex form. In embodiments of the methods described herein, multiplex application may include identifying nucleotide sequences adjacent to one or more known target nucleotide sequences. As used herein, “multiplex amplification” refers to a process involving the simultaneous amplification of more than one target nucleic acid in a single reaction vessel. In some embodiments, the method involves subsequently determining the sequence of the multiplex amplification product using one or more sets of primers. Multiplex can refer to the detection of approximately 2 to 1,000 different target sequences in a single reaction. As used herein, multiplex refers to the detection of any range between 2 and 1,000 (e.g., 5-500, 25-1000, or 10-100) different target sequences in a single reaction. When applied to PCR, the term “multiplex” means the presence of primers specific for at least two different target sequences in the same PCR reaction.

[0115] In some embodiments, multiple primers (e.g., multiple first target-specific primers and second target-specific primers) can be used to amplify target nucleic acids in a sample or a single portion of a sample. In some embodiments, multiple primers (e.g., multiple first target-specific primers and second target-specific primers) may be present in a single reaction mixture, for example, multiple amplification products may be generated in the same reaction mixture. In some embodiments, multiple primers (e.g., multiple sets of first target-specific primers and second target-specific primers) are specifically annealed to a known target sequence contained in a single gene. In some embodiments, at least two sets of primers (e.g., at least two sets of first target-specific primers and second target-specific primers) are specifically annealed to different portions of a known target sequence. In some embodiments, at least two sets of primers (e.g., at least two sets of first target-specific primers and second target-specific primers) are specifically annealed to different exons of a gene containing a known target sequence. In some implementations, multiple primers (e.g., a first target-specific primer) may contain the same 5' tag sequence portion.

[0116] In embodiments of the methods described herein, multiple applications may include identifying nucleotide sequences in multiple samples adjacent to one or more known target nucleotide sequences within a single sequencing reaction or sequencing run. In some embodiments, the multiple samples may have different origins, e.g., from different tissues and / or different subjects. In such embodiments, primers (e.g., tailed random primers) may further include a barcode portion. In some embodiments, primers with unique barcode portions (e.g., tailed random primers) may be added to each sample and ligated to the nucleic acids therein; the samples may then be merged. In such embodiments, each sequencing read resulting from the amplification products will contain a barcode identifying a sample containing template nucleic acids derived from those template nucleic acids.

[0117] In some embodiments of the methods described herein, identifying sequences adjacent to a known oligonucleotide target sequence can provide information relevant to disease treatment. Therefore, in some embodiments, the methods disclosed herein can be used to aid in the treatment of diseases. In some embodiments, the sample may be from a subject requiring treatment for a disease associated with a genetic alteration. In some embodiments, the known target sequence may be a sequence of a disease-associated gene (e.g., an oncogene). In some embodiments, the sequence adjacent to the known oligonucleotide target sequence and / or the known oligonucleotide target sequence may contain disease-associated mutations or genetic abnormalities, such as SNPs, insertions, deletions, and / or gene rearrangements. In some embodiments, the sequence adjacent to the known target sequence and / or the known target sequence present in the sample contains a sequence of a gene rearrangement product. In some embodiments, the gene rearrangement may be an oncogene, such as a fusion oncogene.

[0118] Some cancer treatments are particularly effective against tumors containing specific oncogenes. For example, a therapeutic agent targeting the action or expression of a given fusion oncogene may be effective against tumors containing that fusion oncogene, but ineffective against tumors lacking that fusion oncogene. The methods described herein facilitate the identification of specific sequences revealing oncogene status (e.g., mutations, SNPs, and / or rearrangements). In some embodiments, when the sequences of flanking regions are known, the methods described herein can further enable the identification of specific sequences. For example, the methods described herein can determine the presence and type of gene rearrangements involving known genes (e.g., oncogenes), wherein the precise location and / or rearrangement partner are unknown prior to performing the methods described herein.

[0119] In some embodiments, the techniques described herein relate to methods of treating cancer. Therefore, in some embodiments, the methods provided herein may involve: detecting the presence of one or more oncogene rearrangements in a tumor sample obtained from a subject requiring cancer treatment; and administering cancer treatment effective against tumors having any detected oncogene rearrangements. In some embodiments, the techniques described herein relate to methods of determining whether a subject requiring cancer treatment will respond to a given treatment. Therefore, in some embodiments, the methods provided herein may involve: detecting the presence of oncogene rearrangements in a tumor sample obtained from a subject; wherein, if an oncogene rearrangement is detected, the subject is determined to be responsive to treatment targeting the oncogene rearrangement product.

[0120] In some implementations, the subject requires treatment for lung cancer. In some implementations, for example, when the sample is obtained from a subject requiring treatment for lung cancer, the known target sequence may comprise sequences from genes selected from the group consisting of ALK, ROS1, and RET. Therefore, in some implementations, gene rearrangements result in fusions involving ALK, ROS1, or RET. Non-limiting examples of gene rearrangements involving ALK, ROS1, or RET are described, for example, in Soda et al., Nature 2007 448561-6; Rikova et al., Cell 2007 131:1190-1203; Kohno et al., Nature Medicine 2012 18:375-7; and Takouchi et al., Nature Medicine 2012 18:378-81, which are incorporated herein by reference in their entirety. However, it should be understood that the precise location of the gene rearrangement and the type of second gene involved in the rearrangement may be unknown beforehand. Therefore, in the method described herein, the presence and type of such rearrangements can be detected without knowing the location of the rearrangement or the type of second gene involved in the gene rearrangement.

[0121] In some implementations, the known target sequence may comprise a sequence from a gene selected from the group consisting of ALK, ROS1, and RET.

[0122] In some embodiments, the presence of ALK gene rearrangements in a sample obtained from a tumor of a subject may indicate that the tumor is susceptible to treatment with a therapeutic agent selected from the group consisting of: ALK inhibitors, crizotinib (PF-02341066), AP26113, LDK378, 3-39, AF802, IPI-504, ASP3026, AP-26113, X-396, GSK-1838705A, CH5424802, diamino inhibitors and aminopyrimidine inhibitors of ALK kinase activity (e.g., NVP-TAE684 and PF-02341066) (see, for example, Galkin et al., Proc Natl Acad Sci USA, 2007, 104:270-275; Zou et al., Cancer Res, 2007, 67:4408-4417; Hallberg and Palmer F1000 Med Reports). 20113:21; and Sakamoto et al., Cancer Cell 2011 19:679-690); and molecules disclosed in WO 04 / 079326. All of the above references are incorporated herein by reference in their entirety. ALK inhibitors may include any agent that reduces the expression and / or kinase activity of ALK or a portion thereof, including, for example, oligonucleotides, small molecules and / or peptides that reduce the expression and / or activity of ALK or a portion thereof. As used herein, “anaplastic lymphoma kinase” or “ALK” refers to the transmembrane tyrosine kinase commonly involved in neuronal regulation in its wild-type form. The nucleotide sequences of the ALK gene and mRNA are known in many species, including humans (e.g., SEQ ID NO:2 (mRNA), NCBI Gene ID:238).

[0123] In some embodiments, the presence of ROS1 gene rearrangements in a sample obtained from a tumor of a subject may indicate that the tumor is susceptible to treatment with a therapeutic agent selected from the group consisting of ROS1 inhibitors and ALK inhibitors as described above (e.g., crizotinib). ROS1 inhibitors may include any agent that reduces the expression and / or kinase activity of ROS1 or a portion thereof, including, for example, oligonucleotides, small molecules, and / or peptides that reduce the expression and / or activity of ROS1 or a portion thereof. As used herein, “c-ros oncogene 1” or “ROS1” (also referred to in the art as ROS-1) refers to a transmembrane tyrosine kinase of the sevenless subfamily that interacts with PTPN6. The nucleotide sequences of the ROS1 gene and mRNA are known in many species, including humans (e.g., SEQ ID NO:1 (mRNA), NCBI Gene ID:238).

[0124] In some embodiments, the presence of RET gene rearrangements in a sample obtained from a tumor of a subject may indicate that the tumor is susceptible to treatment with a therapeutic agent selected from the group consisting of: RET inhibitors, DP-2490, DP-3636, SU5416, BAY 43-9006, BAY 73-4506 (regorafenib), ZD6474, NVP-AST487, sorafenib, RPI-1, XL184, vandetanib, sunitinib, imatinib, pazopanib, axitinib, motracene, gefitinib, and solanine A (see, for example, Samadi et al., Surgery 2010148:1228-36; Cuccuru et al., JNCI 200413:1006-1014; Akeno-Stuart et al., Cancer Research 2007 67:6956; Grazma et al., J ClinOncol 2010 28:15s 5559; Mologni et al., J Mol Endocrinol 2006 37:199-212; Calmomagno et al., Journal NCI 2006 98:326-334; Mologni. Curr Med Chem 2011 18:162-175; and compounds disclosed in WO06 / 034833, U.S. Patent Publication 2011 / 0201598, and U.S. Patent 8,067,434. All of the foregoing references are incorporated herein by reference in their entirety. RET inhibitors may include any agent that reduces the expression and / or kinase activity of RET or a moiety thereof, including, for example, oligonucleotides, small molecules, and / or peptides that reduce the expression and / or activity of RET or a moiety thereof. As used in this article, "rearranged during transfection" or "RET" refers to the receptor tyrosine kinase of the cadherin superfamily, which is involved in neural crest development and recognizes neurotrophic factor family signaling molecules derived from glial cell lines. The nucleotide sequences of RET genes and mRNAs are known in many species, including humans (e.g., SEQ ID NO:3-SEQ ID NO:4 (mRNA), NCBI Gene ID:5979).

[0125] Further non-limiting examples of the application of the methods described herein include the detection of hematological malignancy markers and panels (e.g., markers and panels for detecting genomic rearrangements in lymphoma and leukemia), the detection of sarcoma-associated genomic rearrangements and panels, and the detection of IGH / TCR gene rearrangements and panels for lymphoma testing.

[0126] In some embodiments, the methods described herein involve treating a subject who has or has been diagnosed with, for example, cancer, with a cancer therapeutic agent. Subjects with cancer can be identified by a physician using methods currently used to diagnose cancer. For example, symptoms and / or complications characteristic of these conditions and helpful in diagnosis of lung cancer are well known in the art, including but not limited to: weak breathing, enlarged lymph nodes above the clavicle, abnormal lung sounds, dullness on tapped chest, and chest pain. Tests that may help in the diagnosis of, for example, lung cancer include, but are not limited to, X-rays, blood tests for high levels of specific substances (e.g., calcium), CT scans, and tumor biopsies. A family history of lung cancer or exposure to lung cancer risk factors (e.g., smoking or exposure to smoke and / or air pollution) may also help determine whether a subject is likely to have lung cancer or help in the diagnosis of lung cancer.

[0127] Cancer can include, but is not limited to: malignant tumors (carcinoma), including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, leukemia, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's lymphoma and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, basal cell carcinoma, biliary tract cancer, bladder cancer, brain cancer (including glioblastoma and medulloblastoma); breast cancer, cervical cancer, choriocarcinoma; colon cancer, colorectal cancer, endometrial carcinoma, endometrial cancer. Cancer); esophageal cancer, stomach cancer; various head and neck cancers, intraepithelial neoplasia (including Bowen's disease and Paget's disease); hematologic malignancies (including acute lymphoblastic leukemia and acute myeloid leukemia); Kaposi's sarcoma, hairy cell leukemia; chronic myeloid leukemia, AIDS-related leukemia and adult T-cell leukemia lymphoma; renal cell carcinoma (such as renal cell carcinoma), T-cell acute lymphoblastic leukemia / lymphoma, lymphoma (including Hodgkin's disease and lymphocytic lymphoma); liver cancer (such as hepatocellular carcinoma and hepatocellular carcinoma), Merkel cell malignancies, melanoma, multiple myeloma; neurological disorders; and other cancers. Transblastoma; oral cancer (including squamous cell carcinoma); ovarian cancer (including ovarian cancer arising from epithelial cells), sarcoma (including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma); pancreatic cancer; skin cancer (including melanoma, stromal cell, germ cell, and mesenchymal cell cancer); prostate cancer, rectal cancer; vulvar cancer, kidney cancer (including adenocarcinoma); testicular cancer (including embryonic tumors (e.g., seminoma), non-seminomatous tumors (teratoma, choriocarcinoma), stromal tumors, and germ cell tumors); thyroid cancer (including thyroid adenocarcinoma and medullary thyroid carcinoma); esophageal cancer, salivary gland malignancies, and Wilm's tumor. In some embodiments, the cancer may be lung cancer.

[0128] In some embodiments, the methods described herein include administering to a subject an effective amount of the composition described herein (e.g., a cancer therapeutic agent) to alleviate cancer symptoms. As used herein, “alleviate cancer symptoms” means to improve any condition or symptom associated with cancer. Such a reduction, measured by any standard technique, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or more, compared to an equivalent untreated control. Various means of administering the composition described herein to a subject are known to those skilled in the art. Such methods may include, but are not limited to, oral administration, parenteral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway (aerosol) administration, pulmonary administration, skin administration, local administration, injection administration, or intratumoral administration. Administration may be local or systemic. The term “effective amount” as used herein refers to the amount of therapeutic agent required to alleviate at least one or more symptoms of a disease or disorder, and relates to an amount of pharmaceutical composition sufficient to provide the desired effect. Therefore, the term “therapeuticly effective amount” refers to an amount sufficient to produce a specific anticancer effect when administered to a typical subject. The term "effective amount" as used herein can also encompass, in different contexts, amounts sufficient to delay the progression of disease symptoms, amounts sufficient to alter the course of disease symptoms (e.g., but not limited to, slowing the progression of disease symptoms), or amounts sufficient to reverse disease symptoms. Therefore, specifying a precise "effective amount" is generally impractical. However, for any given situation, an appropriate "effective amount" can be determined by a person skilled in the art using only routine experiments. The effect of any particular dose can be monitored by appropriate bioassays. Dosage can be determined by a physician and adjusted as appropriate to accommodate observed therapeutic effects.

[0129] Non-limiting examples of cancer treatments may include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents, such as thiotepa and... Cyclophosphamides; alkyl sulfonates, such as busulfan, indomethacin, and piperosulfan; aziridines, such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including hexamethylmelamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adolaxine, calcexin, and pyrazin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin Ting; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard, such as chlorambucil, naphthylmustine, cholophosphamide, estradiol, ifosfamide, dichloromethyldiethylamine, nitrogen mustard oxide hydrochloride, melphalan, neonitrogen mustard, benzylmustine, prednimustine, ethoprofen. Phosphoramides, uracil mustard; nitrosoureas, such as carmustine, chloramphenicol, formustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1 and calicheamicin Ω1 (see, for example, Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates, such as clophosphonates; esperamicin; and neomycin chromophores and related chromogens (ene diyne antibiotic chromophores), aclacinomysins, actinomycins, authramycin, diazoserine, bleomycin, cactinomycin, carabicin, caminomycin, chromomycinis, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine. Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxy-doxorubicin), epirubicin, isorubicin, idarubicin, mesorubicin (e.g., mitomycin C), mycophenolate mofetil, nogamycin, oligomycin, pepromycin, potfiromycin, puromycin, triamcinolone acetonide, rhodopsin, streptozotocin, streptozotocin, tuberculin, ubenimex, fenestrated statin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as bifida. Acids, methotrexate, pteroxetine, and trimethyltraxa; purine analogs, such as fludarabine, 6-mercaptopurine, thioimidazoline, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azouridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, and fluorouridine; androgens, such as capprotestone, drotalbutone propionate, cyclothionol, meandrolone, and testrolide; anti-adrenergic agents, such as aminoglutethimide, mitotane, and trilosterone; folic acid supplements, such as folinic acid. acid); acetylphosphamide lactone; aldophosphamide glycoside; aminolevulinic acid; enuramicin; acridine; bestrabucil; edatraxate; defofamine; dimethicone; elformithine; elformithine; epothilone; epothilone; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and anthraquinone; mitoxanthraquinone; mitoxanthraquinone; mopidanmol; nitraerine; pentostatin; methamidophos; pirarubicin; loxoanthraquinone; podophylloic acid; 2-ethylhydrazide; procarbazine; Polysaccharide complexes (JHS Natural Products, Eugene, Oreg.); acrylamide; rhizobium; sizofuran; spirogermanium; Alternaria ketoacid; triamine quinone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); ethyl carbamate; vinblastine; dacarbazine; mannitol nitrogen mustard; dibromomannitol; dibromoeutherol; piperobromane; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, for example, Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ) Cremophor-free, paclitaxel albumin-engineered nanoparticle formulations (American Pharmaceutical Partners, Schaumberg, Ill.) and doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine RTM; vinorelbine; norsoxamine; teniposide; edaraxacum; daunorubicin; aminopterin; capecitabine; ibandronate; irinotecan (Camptosar, CPT- 11) (including irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; compressoritine; leucovorin (LV); oxaliplatin, including oxaliplatin regimens (FOLFOX); lapatinib (Tykerb.RTM); PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib) Inhibitors of VEGF-A (which reduce cell proliferation), and pharmaceutically acceptable salts, acids, or derivatives of any of the above substances. Furthermore, treatment may include the use of radiation or radiation therapy. Additionally, treatment may include surgical intervention.

[0130] In some embodiments, the methods described herein can be applied to resequencing, for example, to confirm particularly relevant, low-quality, and / or complex sequences obtained from non-directional sequencing of large numbers of nucleic acids. By way of non-limiting examples, the methods described herein enable: directed and / or targeted resequencing of disease genomes (e.g., 10-100 genes); resequencing to confirm variants obtained in large-scale sequencing projects; whole exome resequencing; and / or targeted resequencing for detecting single nucleotide variants, polynucleotide variants, insertions, deletions, copy number changes, and methylation status.

[0131] In some embodiments, the methods described herein enable microbiota sequencing, ancient sample sequencing, and / or genotyping of novel variant viruses.

[0132] For convenience, the meanings of some terms and phrases used in the specification, embodiments, and appended claims are provided below. Unless otherwise stated or implied in the context, the following terms and phrases include the meanings provided below. These definitions are provided to aid in the description of specific embodiments, and since the scope of the invention is limited only by the claims, it is not intended to limit the claimed invention. 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 pertains. In the event of any apparent inconsistency between the use of terminology in the art and the definitions provided herein, the definitions provided herein shall prevail.

[0133] For convenience, certain terms used in the specification, embodiments and appended claims are collected herein.

[0134] As used herein, the terms “decrease,” “reduced / reduction,” or “inhibit” generally mean a statistically significant reduction. However, to avoid ambiguity, “reduced / reduction,” “decrease,” or “inhibit” means a reduction of at least 10% compared to a reference level, for example, a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a reduction of 100% (e.g., compared to a missing or undetectable level in a reference sample), or any amount between 10% and 100% compared to a reference level. In the case of markers or symptoms, this means a statistically significant reduction in such levels. For example, the reduction may be at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably reduced to a level considered to be within the normal range for individuals without such disorder.

[0135] The terms “increased / increase,” “enhance,” or “activate” as used in this article generally mean a statistically significant increase. For the avoidance of ambiguity, the terms “increased / increase,” “enhance,” or “activate” mean an increase of at least 10% compared to the reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including an increase of 100%, or any amount between 10% and 100% compared to the reference level; or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to the reference level, or any amount between 2 times and 10 times, or a larger amount.

[0136] As used herein, the term "subject" refers to a person or animal. Generally, the animal is a vertebrate, such as a primate, rodent, domestic animal, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic animals and game animals include cows, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cats), canine species (e.g., dogs, foxes, wolves), bird species (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments, the subject is a mammal, such as a primate, like a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0137] Preferably, the subject is a mammal. The mammal may be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Mammals other than humans may be advantageous as subjects representing, for example, animal models of lung cancer. The subject may be male or female.

[0138] Subjects may be those who have been previously diagnosed with or identified as suffering from or suffering from a condition (e.g., cancer) or one or more complications associated with such a condition, and optionally have received treatment for the condition or one or more complications associated with such a condition. Alternatively, subjects may be those who have not previously been diagnosed with the condition (e.g., cancer) or one or more complications associated with such a condition. For example, subjects may be those exhibiting one or more risk factors for the condition or one or more complications associated with such a condition, or subjects who do not exhibit risk factors.

[0139] For the treatment of a specific condition, "subjects in need" may be subjects who have the condition, have been diagnosed with the condition, or are at risk of developing the condition.

[0140] As used herein, “disease associated with genetic alteration” refers to any disease caused at least in part by alterations (e.g., deletions, insertions, SNPs, gene rearrangements) in a subject’s genetic material relative to a healthy wild-type subject. A disease can be caused at least in part by said alterations in a subject’s genetic material if the alteration increases a subject’s risk of developing the disease, increases a subject’s susceptibility to the disease (including infectious diseases or diseases with infectious components), causes the production of disease-related molecules, or makes cells diseased or abnormal (e.g., loss of cell cycle regulation in cancer cells). Diseases can be associated with a variety of genetic alterations, such as cancer.

[0141] As used herein, the term "nucleic acid" refers to any molecule, preferably a polymer, incorporating units of ribonucleic acid, deoxyribonucleic acid, or similar molecules. The nucleic acid may be single-stranded or double-stranded. A single-stranded nucleic acid may be the nucleic acid of one strand of denatured double-stranded DNA. Alternatively, a single-stranded nucleic acid may be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the template nucleic acid is DNA. In another aspect, the template is RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.

[0142] In the context of nucleic acids, the terms "isolated" or "partially purified" as used herein refer to nucleic acids isolated from at least one other component (e.g., nucleic acid or polypeptide) present with nucleic acids as seen in their natural sources; and / or nucleic acids isolated from at least one other component (e.g., nucleic acid or polypeptide) present with nucleic acids when expressed by cells. Chemically synthesized nucleic acids or nucleic acids synthesized using in vitro transcription / translation are considered "isolated".

[0143] As used herein, the term "complementary" refers to the ability of a nucleotide to form hydrogen-bonded base pairings. In some embodiments, complementarity refers to a preference for hydrogen-bonded base pairings between the nucleotide bases G, A, T, C, and U, such that when two given polynucleotide or polynucleotide sequences anneal to each other, A pairs with T and G pairs with C in DNA, and G pairs with C and A pairs with U in RNA. As used herein, "substantially complementary" means that a nucleic acid molecule or a portion thereof (e.g., a primer) is at least 90% complementary to a second nucleotide sequence over its entire length, for example, 90%, 95%, 98%, 99%, or 100%. As used herein, "substantially identical" means that a nucleic acid molecule or a portion thereof is at least 90% identical to a second nucleotide sequence over its entire length, for example, 90%, 95%, 98%, 99%, or 100%.

[0144] In the case of specific primers for the target nucleic acid, the level of complementarity between the "specific" primers used in this paper and the target results in an annealing temperature at which the primers anneal to the target nucleic acid and mediate the amplification of the target nucleic acid, without annealing to or mediating the amplification of non-target sequences present in the sample.

[0145] As used herein, "amplified product" or "amplicon" refers to an oligonucleotide produced by an amplification reaction, which is a copy of a portion of a specific target nucleic acid template strand and / or its complementary sequence, corresponding nucleotideally to the template nucleic acid sequence and / or its complementary sequence. The amplified product may further contain primer-specific sequences and sequences flanking the primer-specific sequence (which is a portion of the target nucleic acid and / or its complementary sequence). While it can refer to a single strand, the amplified product described herein will generally be double-stranded DNA.

[0146] As used in this article, a “portion” of a nucleic acid molecule refers to a neighborhood of nucleotides contained in that molecule. A portion may contain all or only a subset of the nucleotides contained in that molecule. A portion may be double-stranded or single-stranded.

[0147] As used herein, the terms “treat / treatment / treating” or “amelioration” refer to a therapeutic action aimed at reversing, reducing, improving, inhibiting, slowing, or stopping the progression or severity of a condition associated with a disease or disorder (e.g., lung cancer). The term “treatment” includes reducing or alleviating at least one adverse effect or symptom of a condition, a disease, or a disorder associated with it. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if disease progression is reduced or stopped. That is, “treatment” includes not only improvement in symptoms or markers but also cessation or at least delay of symptom progression or worsening compared to what would have been expected without treatment. Beneficial or desired clinical outcomes include, but are not limited to: reduction of one or more symptoms, reduction of disease severity, stabilization (i.e., no worsening) of the disease state, delay or slowing of disease progression, improvement or palliation of the disease state, remission (partial or complete), and / or reduction of mortality, whether the foregoing outcomes are detectable or undetectable. The term "treatment" also includes providing relief from the symptoms or adverse effects of a disease (including palliative treatment).

[0148] The terms “statistically significant” or “significantly” refer to statistical significance and typically mean that the concentration is two standard deviations (2SD) or less below the normal marker concentration.

[0149] Except where indicated in the operational examples or elsewhere, all numerical values ​​representing amounts of components or reaction conditions as used herein should in all cases be understood to be modified by the term "about". The term "about" used in conjunction with percentages may mean ±1%.

[0150] As used herein, the term “comprising or comprise” means that a composition, method and its respective components are necessary for a method or composition, and remains open to any unspecified elements, whether or not they are necessary.

[0151] The term “composed of” refers to the compositions, methods and their respective components described herein, excluding any elements not detailed in the description of the embodiments.

[0152] As used herein, the term "substantially composed of" refers to those elements required for a given implementation. This term allows for the presence of elements that do not substantially affect the basis and novelty of the implementation or the features that function.

[0153] Unless the context explicitly indicates otherwise, the singular terms “a / an” and “the” cover the plural referent. Similarly, unless the context explicitly indicates otherwise, the word “or” is intended to cover “and”. Although methods and materials similar to or equivalent to those described and materials herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation “eg” derives from the Latin word for example (exempli gratia) and is used herein to denote a non-limiting instance. Therefore, the abbreviation “eg” is synonymous with the term “for example”.

[0154] Definitions of commonly used terms in cell biology and molecular biology can be found in: "The Merck Manual of Diagnosis and Therapy", 19th edition, Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al., The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9). Definitions of commonly used terms in molecular biology can also be found in: Benjamin Lewin, Genes X, Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al., Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al.

[0155] Unless otherwise stated, this invention has been carried out using standard procedures, such as those described below: Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2001); and Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995), which are incorporated herein by reference in their entirety.

[0156] Other terms are defined in the description of various aspects of this invention.

[0157] For purposes of description and disclosure, all patents referenced throughout this application and other publications (including references, granted patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference, such as methodologies described in such publications that can be used with the techniques described herein. These publications are provided solely because their publications predate the filing date of this application. In this respect, it should not be construed as an admission that the inventor has no right to advance the disclosure by means of prior inventions or for any other reason. All statements regarding the dates of these documents or descriptions of their contents are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or contents of these documents.

[0158] The description of embodiments of this disclosure is not intended to be exhaustive or to limit the disclosure to the exact forms disclosed. Although specific embodiments and examples of this disclosure have been described herein for illustrative purposes, those skilled in the art will recognize that various equivalent modifications can be made within the scope of this disclosure. For example, while method steps or functions are given in a given order, other embodiments may implement the functions in a different order or may implement these functions substantially simultaneously. The teachings of this disclosure provided herein can be applied in a suitable manner to other procedures or methods. The various embodiments described herein may be combined to provide further embodiments. Where appropriate, aspects of this disclosure may be modified to provide further embodiments of this disclosure by utilizing the combinations, functions, and concepts referenced and applied above. The above and other changes may be made to this disclosure in light of the detailed description. All such modifications fall within the scope of the invention as defined in the appended claims.

[0159] Specific elements in any of the foregoing embodiments may be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with particular embodiments of this disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, but not all embodiments must exhibit such advantages to fall within the scope of this disclosure.

[0160] The techniques described herein are further illustrated by the following embodiments, but should in no way be construed as being further limited to the techniques described herein.

[0161] Some implementations of the technology described herein can be defined according to any of the following numbered paragraphs:

[0162] 1. A method for determining a nucleotide sequence adjacent to a known target nucleotide sequence, the method comprising:

[0163] (a) Under hybridization conditions, a target nucleic acid molecule containing the known target nucleotide sequence is contacted with an initiation target-specific primer;

[0164] (b) Perform a template-dependent extension reaction, which is initiated by a hybridization initiation target-specific primer and uses the target nucleic acid molecule as a template;

[0165] (c) Under hybridization conditions, the product of step (b) is contacted with a set of tailed random primers;

[0166] (d) Perform a template-dependent extension reaction, which is initiated by a hybridization tailed random primer and uses a portion of the target nucleic acid molecule downstream of the hybridization site as a template;

[0167] (e) Amplify a portion of the target nucleic acid molecule and the tailed random primer sequence using a first tail primer and a first target-specific primer;

[0168] (f) Amplify a portion of the amplicon generated in step (e) using a second tail primer and a second target-specific primer;

[0169] (g) Sequencing the amplified portion from step (f) using the first and second sequencing primers;

[0170] The tailed random primer group includes single-stranded oligonucleotide molecules, which have the same 5' nucleic acid sequence as the first sequencing primer and a 3' nucleic acid sequence containing about 6 to about 12 random nucleotides;

[0171] Wherein, the first target-specific primer contains a nucleic acid sequence that can be specifically annealed to the known target nucleotide sequence of the target nucleic acid at an annealing temperature;

[0172] The second target-specific primer comprises a 3' portion and a 5' portion, wherein the 3' portion comprises a nucleic acid sequence capable of specifically annealing to a portion of the known target nucleotide sequence contained in the amplicon generated by step (e), and the 5' portion comprises the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer;

[0173] Wherein, the first tail primer contains the same nucleic acid sequence as the tailed random primer; and

[0174] The second tail primer contains a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer.

[0175] 2. A method for determining a nucleotide sequence adjacent to a known target nucleotide sequence, the method comprising:

[0176] (a) Under hybridization conditions, a target nucleic acid molecule containing the known target nucleotide sequence is contacted with a set of tailed random primers;

[0177] (b) Perform a template-dependent extension reaction, which is initiated by a hybridization tailed random primer and uses a portion of the target nucleic acid molecule downstream of the hybridization site as a template;

[0178] (c) Under hybridization conditions, the product of step (b) is contacted with the initiation target-specific primer;

[0179] (d) Perform a template-dependent extension reaction, which is initiated by a hybridization initiation target-specific primer and uses the target nucleic acid molecule as a template;

[0180] (e) Amplify a portion of the target nucleic acid molecule and the tailed random primer sequence using a first tail primer and a first target-specific primer;

[0181] (f) Amplify a portion of the amplicon generated in step (e) using a second tail primer and a second target-specific primer;

[0182] (g) Sequencing the amplified portion from step (f) using the first and second sequencing primers;

[0183] The tailed random primer group includes single-stranded oligonucleotide molecules, which have the same 5' nucleic acid sequence as the first sequencing primer and a 3' nucleic acid sequence containing about 6 to about 12 random nucleotides;

[0184] Wherein, the first target-specific primer contains a nucleic acid sequence that can be specifically annealed to the known target nucleotide sequence of the target nucleic acid at an annealing temperature;

[0185] The second target-specific primer comprises a 3' portion and a 5' portion, wherein the 3' portion comprises a nucleic acid sequence capable of specifically annealing to a portion of the known target nucleotide sequence contained in the amplicon generated by step (c), and the 5' portion comprises the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer;

[0186] Wherein, the first tail primer contains the same nucleic acid sequence as the tailed random primer; and

[0187] The second tail primer contains a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer.

[0188] 3. The method as described in any of paragraphs 1-2, the method further comprising the step of contacting the sample and product with RNase after extension of the starting target-specific primer.

[0189] 4. The method described in any of paragraphs 1-3, wherein the tailed random primer can form a hairpin loop structure.

[0190] 5. The method described in any of paragraphs 1-4, wherein the starting target-specific primer is the same as the first target-specific primer.

[0191] 6. The method as described in any of paragraphs 1-5, wherein, between the 5' nucleic acid sequence identical to the first sequencing primer and the 3' nucleic acid sequence containing 6-12 random nucleotides, the tailed random primer further comprises a barcode portion containing 6-12 random nucleotides.

[0192] 7. A method for determining a nucleotide sequence adjacent to a known target nucleotide sequence, the method comprising:

[0193] (a) Under hybridization conditions, a target nucleic acid molecule containing the known target nucleotide sequence is contacted with a set of tailed random primers;

[0194] (b) Perform a template-dependent extension reaction, which is initiated by a hybridization tailed random primer and uses a portion of the target nucleic acid molecule downstream of the hybridization site as a template;

[0195] (c) Amplify a portion of the target nucleic acid molecule and the tailed random primer sequence using the first tail primer and the first target-specific primer;

[0196] (d) Amplify a portion of the amplicon generated in step (c) using the second tail primer and the second target-specific primer;

[0197] (e) Sequencing the amplified portion from step (d) using the first and second sequencing primers;

[0198] The tailed random primer group includes single-stranded oligonucleotide molecules, which have the same 5' nucleic acid sequence as the first sequencing primer, a middle barcode portion containing 6-12 random nucleotides, and a 3' nucleic acid sequence containing about 6-12 random nucleotides.

[0199] Wherein, the first target-specific primer contains a nucleic acid sequence that can be specifically annealed to the known target nucleotide sequence of the target nucleic acid at an annealing temperature;

[0200] The second target-specific primer comprises a 3' portion and a 5' portion, wherein the 3' portion comprises a nucleic acid sequence capable of specifically annealing to a portion of the known target nucleotide sequence contained in the amplicon generated by step (c), and the 5' portion comprises the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer;

[0201] Wherein, the first tail primer contains the same nucleic acid sequence as the tailed random primer; and

[0202] The second tail primer contains a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer.

[0203] 8. The method as described in paragraph 7, wherein each tailed random primer further comprises a spacer region nucleic acid sequence between the 5' nucleic acid sequence identical to the first sequencing primer and the 3' nucleic acid sequence containing about 6 to about 12 random nucleotides.

[0204] 9. The method as described in paragraph 7 or 8, wherein, after the extension step, the unhybridized primers are removed from the reaction.

[0205] 10. The method as described in any of paragraphs 7-9, wherein the second tail primer is nested relative to the first tail primer by at least 3 nucleotides.

[0206] 11. The method as described in any of paragraphs 7-10, wherein the first target-specific primer further comprises a 5' tag sequence portion comprising a high-GC-content nucleic acid sequence that is substantially not complementary to or substantially identical to any other portion of any primer.

[0207] 12. The method described in any of paragraphs 7-11, wherein the second tail primer is the same as the full-length first sequencing primer.

[0208] 13. The method as described in any of paragraphs 7-12, wherein the portion of the target-specific primer specifically annealed to the known target will be specifically annealed in PCR buffer at a temperature of approximately 65°C.

[0209] 14. The method as described in any of paragraphs 7-13, wherein the sample comprises genomic DNA.

[0210] 15. The method as described in any of paragraphs 7-14, wherein the sample comprises RNA, and the method further comprises a first step of subjecting the sample to a reverse transcriptase protocol.

[0211] 16. The method as described in any of paragraphs 7-15, wherein the nucleic acid present in the sample has not been cleaved or digested.

[0212] 17. The method as described in any of paragraphs 7-16, wherein the sample comprises single-stranded gDNA or single-stranded cDNA.

[0213] 18. The method as described in any of paragraphs 7-17, wherein the reverse transcriptase protocol includes the use of random hexamers.

[0214] 19. The method as described in any of paragraphs 7-18, wherein the gene rearrangement includes the known target sequence.

[0215] 20. The method as described in paragraph 19, wherein the gene rearrangement is present in a nucleic acid selected from the group consisting of genomic DNA, RNA and cDNA.

[0216] 21. The method as described in any of paragraphs 19-20, wherein the gene rearrangement includes oncogenes.

[0217] 22. The method as described in paragraph 21, wherein the gene rearrangement includes a fusion oncogene.

[0218] 23. The method described in any of paragraphs 7-22, wherein the nucleic acid product is sequenced by next-generation sequencing.

[0219] 24. The method as described in paragraph 23, wherein the next-generation sequencing method comprises methods selected from the group consisting of: Ion Torrent, Illumina, SOLiD, 454, massively parallel signature sequencing, solid-phase reversible dye-terminated sequencing, and DNA nanosphere sequencing.

[0220] 25. The method described in any of paragraphs 7-24, wherein the first and second sequencing primers are compatible with the selected next-generation sequencing method.

[0221] 26. The method as described in any of paragraphs 7-25, wherein the method comprises contacting the sample or a separate portion of the sample with a plurality of sets of first target-specific primers and second target-specific primers.

[0222] 27. The method as described in any of paragraphs 7-26, wherein the method comprises contacting a single reaction mixture containing the sample with a plurality of sets of first target-specific primers and second target-specific primers.

[0223] 28. The method as described in any of paragraphs 7-27, wherein multiple sets of first target-specific primers and second target-specific primers are specifically annealed to a known target nucleotide sequence contained in a single gene.

[0224] 29. The method as described in any of paragraphs 7-28, wherein at least two sets of first target-specific primers and second target-specific primers are specifically annealed to different portions of a known target nucleotide sequence.

[0225] 30. The method as described in any of paragraphs 7-29, wherein at least two sets of first target-specific primers and second target-specific primers are specifically annealed to different portions of a single gene containing a known target nucleotide sequence.

[0226] 31. The method as described in any of paragraphs 7-30, wherein at least two sets of first target-specific primers and second target-specific primers are specifically annealed to different exons of a gene containing a known target nucleotide sequence.

[0227] 32. The method described in any of paragraphs 7-31, wherein the plurality of first target-specific primers contain the same 5' tag sequence portion.

[0228] 33. The method described in any of paragraphs 7-32, wherein each tailed random primer in the tailed random primer group further comprises the same sample barcode portion.

[0229] 34. The method as described in paragraph 33, wherein multiple samples are each contacted with a separate tailed random primer group having a sample barcode portion; wherein each tailed random primer group has a different sample barcode portion; and wherein the samples are merged after step (b).

[0230] 35. The method described in any of paragraphs 7-34, wherein each amplification step includes a PCR amplification protocol cycle set of 5 to 20 cycles in length.

[0231] 36. The method as described in any of paragraphs 7-35, wherein the target-specific primers and the tail primers are designed such that they are specifically annealed to their complementary sequences at an annealing temperature of about 61°C-72°C.

[0232] 37. The method as described in any of paragraphs 7-36, wherein the target-specific primers and the tail primers are designed such that they are specifically annealed to their complementary sequences at an annealing temperature of about 65°C.

[0233] 38. The method as described in any of paragraphs 7-37, wherein the target nucleic acid molecule is derived from a sample, optionally a biological sample obtained from a subject.

[0234] 39. The method as described in paragraph 38, wherein the sample is obtained from a subject requiring treatment for a disease associated with a genetic alteration.

[0235] 40. The method as described in paragraph 39, wherein the disease is cancer.

[0236] 41. The method as described in paragraph 38, wherein the sample comprises a population of tumor cells.

[0237] 42. The method as described in paragraph 38, wherein the sample is a tumor biopsy.

[0238] 43. The method as described in paragraph 40, wherein the cancer is lung cancer.

[0239] 44. The method described in any of paragraphs 7-43, wherein the disease-related gene contains the known target sequence.

[0240] 45. The method as described in paragraph 38, wherein the gene rearrangement product in the sample contains the known target sequence.

[0241] 46. ​​The method as described in paragraph 45, wherein the gene rearrangement product is an oncogene.

[0242] 47. A method for preparing nucleic acids for analysis, the method comprising:

[0243] (a) Under conditions that promote template-specific hybridization and extension of the target-specific primer, a nucleic acid template containing the first strand of the target nucleic acid is contacted with a target-specific primer containing a complementary target-specific hybridization sequence; and

[0244] (b) Under conditions that promote template-specific hybridization and extension of at least one of a plurality of different primers, a nucleic acid template containing a second strand complementary to the first strand of the target nucleic acid is contacted with a plurality of different primers that share a common sequence, the common sequence being the 5' end of a different hybridization sequence;

[0245] The process generates an extension product, which simultaneously contains a sequence with target-specific primer characteristics and a sequence with characteristics of at least one of multiple different primers.

[0246] 48. The method as described in paragraph 47, wherein the target nucleic acid is ribonucleic acid.

[0247] 49. The method as described in paragraph 47, wherein the target nucleic acid is deoxyribonucleic acid.

[0248] 50. The method described in any of paragraphs 47-49, wherein steps (a) and (b) are performed sequentially.

[0249] 51. The method as described in any of paragraphs 47-50, wherein the nucleic acid template in step (a) comprises an extension product generated from the extension and hybridization of at least one of the plurality of different primers in step (b).

[0250] 52. The method as described in any of paragraphs 47-50, wherein the nucleic acid template in step (b) comprises the extension product generated by the extension and hybridization of the target-specific primer in step (a).

[0251] 53. The method as described in paragraph 48, wherein the target nucleic acid is a messenger RNA encoding a chromosomal segment containing gene rearrangements.

[0252] 54. The method as described in paragraph 49, wherein the target nucleic acid is a chromosomal segment containing a portion of a gene rearrangement.

[0253] 55. The method as described in paragraph 48, wherein the gene rearrangement is an inversion, deletion, or translocation.

[0254] 56. The method as described in any of paragraphs 47-55, the method further comprising amplifying the extended product.

[0255] 57. The method as described in any of paragraphs 47-55, the method further comprising contacting the extended product or the amplified extended product with the fixed oligonucleotide under conditions where hybridization occurs between the extended product and the fixed oligonucleotide.

[0256] 58. The method as described in any of the preceding paragraphs, wherein the target nucleic acid comprises a target portion having a known sequence and a flanking portion having an unknown sequence.

[0257] 59. The method as described in paragraph 58, wherein the different hybridization sequences are complementary to the flanking portion.

[0258] 60. The method as described in any of paragraphs 58 or 59, wherein the target-specific hybridization sequence is complementary to the target portion.

[0259] 61. The method as described in any of paragraphs 47-60, wherein the target-specific primer further comprises: the 5' end of the target-specific hybridization sequence; at least one of a barcode sequence, a adapter sequence, and an index sequence.

[0260] 62. The method as described in any of paragraphs 47-60, wherein the common sequence comprises at least one of a barcode sequence, a connector sequence, and an index sequence.

[0261] 63. The method as described in any of paragraphs 1-62, wherein the adapter sequence is a cleavable adapter sequence for immobilizing oligonucleotides in a flow cell.

[0262] Some implementations of the technology described herein can be defined according to any of the following numbered paragraphs:

[0263] 1. A method for preparing nucleic acids for analysis, the method comprising:

[0264] (a) Under conditions that promote template-specific hybridization and target-specific primer extension, a nucleic acid template containing the first strand of the target nucleic acid is contacted with a target-specific primer containing a complementary target-specific hybridization sequence; and

[0265] (b) Under conditions that promote template-specific hybridization and extension of at least one of a plurality of different primers, a nucleic acid template containing a second strand complementary to the first strand of the target nucleic acid is contacted with a plurality of different primers that share a common sequence, the common sequence being the 5' end of a different hybridization sequence;

[0266] The process generates an extension product, which simultaneously contains a sequence with target-specific primer characteristics and a sequence with characteristics of at least one of multiple different primers.

[0267] 2. The method as described in paragraph 1, wherein the target nucleic acid is ribonucleic acid.

[0268] 3. The method as described in paragraph 1, wherein the target nucleic acid is deoxyribonucleic acid.

[0269] 4. The method described in any of paragraphs 1-3, wherein steps (a) and (b) are performed sequentially.

[0270] 5. The method as described in any of paragraphs 1-4, wherein the nucleic acid template in step (a) comprises an extension product generated from the extension and hybridization of at least one of the plurality of different primers in step (b).

[0271] 6. The method as described in any of paragraphs 1-4, wherein the nucleic acid template in step (b) comprises the extension product generated by the extension and hybridization of the target-specific primer in step (a).

[0272] 7. The method as described in paragraph 2, wherein the target nucleic acid is a messenger RNA encoding a chromosomal segment containing gene rearrangements.

[0273] 8. The method as described in paragraph 3, wherein the target nucleic acid is a chromosomal segment containing a portion of a gene rearrangement.

[0274] 9. The method as described in paragraph 8, wherein the gene rearrangement is an inversion, deletion, or translocation.

[0275] 10. The method as described in any of paragraphs 1-9, wherein the method further comprises amplifying the extended product.

[0276] 11. The method as described in any of paragraphs 1-9, the method further comprising contacting the extension product or the amplified extension product with the fixed oligonucleotide under conditions where hybridization occurs between the extension product and the fixed oligonucleotide.

[0277] 12. The method as described in any of the preceding paragraphs, wherein the target nucleic acid comprises a target portion having a known sequence and a flanking portion having an unknown sequence.

[0278] 13. The method as described in paragraph 12, wherein the different hybridization sequences are complementary to the flanking portion.

[0279] 14. The method as described in paragraph 12 or 13, wherein the target-specific hybridization sequence is complementary to the target portion.

[0280] 15. The method as described in any of paragraphs 1-14, wherein the target-specific primer further comprises: the 5' end of the target-specific hybridization sequence; at least one of a barcode sequence, a adapter sequence, and an index sequence.

[0281] 16. The method as described in any of paragraphs 1-14, wherein the common sequence comprises at least one of a barcode sequence, a connector sequence, and an index sequence.

[0282] 17. The method as described in paragraph 15 or 16, wherein the adapter sequence is a cleavable adapter sequence for immobilizing oligonucleotides in a flow cell.

[0283] 18. A method for determining a nucleotide sequence adjacent to a known target nucleotide sequence, the method comprising:

[0284] (a) Under hybridization conditions, a target nucleic acid molecule containing the known target nucleotide sequence is contacted with an initiation target-specific primer;

[0285] (b) Perform a template-dependent extension reaction, which is initiated by a hybridization initiation target-specific primer and uses the target nucleic acid molecule as a template;

[0286] (c) Under hybridization conditions, the product of step (b) is contacted with a set of tailed random primers;

[0287] (d) Perform a template-dependent extension reaction, which is initiated by a hybridization tailed random primer and uses a portion of the target nucleic acid molecule downstream of the hybridization site as a template;

[0288] (e) Amplify a portion of the target nucleic acid molecule and the tailed random primer sequence using a first tail primer and a first target-specific primer;

[0289] (f) Amplify a portion of the amplicon generated in step (e) using a second tail primer and a second target-specific primer;

[0290] (g) Sequencing the amplified portion from step (f) using the first sequencing primer and the second sequencing primer;

[0291] The tailed random primer group includes single-stranded oligonucleotide molecules, which have the same 5' nucleic acid sequence as the first sequencing primer and a 3' nucleic acid sequence containing about 6 to about 12 random nucleotides;

[0292] Wherein, the first target-specific primer contains a nucleic acid sequence that can be specifically annealed to the known target nucleotide sequence of the target nucleic acid at an annealing temperature;

[0293] The second target-specific primer comprises a 3' portion and a 5' portion, wherein the 3' portion comprises a nucleic acid sequence capable of specifically annealing to a portion of the known target nucleotide sequence contained in the amplicon generated by step (e), and the 5' portion comprises the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer;

[0294] Wherein, the first tail primer contains the same nucleic acid sequence as the tailed random primer; and

[0295] The second tail primer contains a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer.

[0296] 19. A method for determining a nucleotide sequence adjacent to a known target nucleotide sequence, the method comprising:

[0297] (a) Under hybridization conditions, a target nucleic acid molecule containing the known target nucleotide sequence is contacted with a set of tailed random primers;

[0298] (b) Perform a template-dependent extension reaction, which is initiated by a hybridization tailed random primer and uses a portion of the target nucleic acid molecule downstream of the hybridization site as a template;

[0299] (c) Under hybridization conditions, contact the product of step (b) with the initiation target-specific primer;

[0300] (d) Perform a template-dependent extension reaction, which is initiated by a hybridization initiation target-specific primer and uses the target nucleic acid molecule as a template;

[0301] (e) Amplify a portion of the target nucleic acid molecule and the tailed random primer sequence using a first tail primer and a first target-specific primer;

[0302] (f) Amplify a portion of the amplicon generated in step (e) using a second tail primer and a second target-specific primer;

[0303] (g) Sequencing the amplified portion from step (f) using the first sequencing primer and the second sequencing primer;

[0304] The tailed random primer group includes single-stranded oligonucleotide molecules, which have the same 5' nucleic acid sequence as the first sequencing primer and a 3' nucleic acid sequence containing about 6 to about 12 random nucleotides;

[0305] Wherein, the first target-specific primer contains a nucleic acid sequence that can be specifically annealed to the known target nucleotide sequence of the target nucleic acid at an annealing temperature;

[0306] The second target-specific primer comprises a 3' portion and a 5' portion, wherein the 3' portion comprises a nucleic acid sequence capable of specifically annealing to a portion of the known target nucleotide sequence contained in the amplicon generated by step (c), and the 5' portion comprises the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer;

[0307] Wherein, the first tail primer contains the same nucleic acid sequence as the tailed random primer; and

[0308] The second tail primer contains a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer.

[0309] 20. The method as described in any of paragraphs 18-19, the method further comprising the step of contacting the sample and product with RNase after extension of the starting target-specific primer.

[0310] 21. The method described in any of paragraphs 1-3, wherein the tailed random primer can form a hairpin loop structure.

[0311] 22. The method described in any of paragraphs 1-4, wherein the starting target-specific primer is the same as the first target-specific primer.

[0312] 23. The method as described in any of paragraphs 1-5, wherein, between the 5' nucleic acid sequence identical to the first sequencing primer and the 3' nucleic acid sequence containing 6-12 random nucleotides, the tailed random primer further comprises a barcode portion containing 6-12 random nucleotides. 7. A method for determining a nucleotide sequence adjacent to a known target nucleotide sequence, the method comprising:

[0313] (a) Under hybridization conditions, a target nucleic acid molecule containing the known target nucleotide sequence is contacted with a set of tailed random primers;

[0314] (b) Perform a template-dependent extension reaction, which is initiated by a hybridization tailed random primer and uses a portion of the target nucleic acid molecule downstream of the hybridization site as a template;

[0315] (c) Amplify a portion of the target nucleic acid molecule and the tailed random primer sequence using the first tail primer and the first target-specific primer;

[0316] (d) Amplify a portion of the amplicon generated in step (c) using the second tail primer and the second target-specific primer;

[0317] (e) Sequencing the amplified portion from step (d) using the first sequencing primer and the second sequencing primer;

[0318] The tailed random primer group includes single-stranded oligonucleotide molecules, which have the same 5' nucleic acid sequence as the first sequencing primer, a barcode portion containing 6-12 random nucleotides in the middle, and a 3' nucleic acid sequence containing about 6-12 random nucleotides.

[0319] Wherein, the first target-specific primer contains a nucleic acid sequence that can be specifically annealed to the known target nucleotide sequence of the target nucleic acid at an annealing temperature;

[0320] The second target-specific primer comprises a 3' portion and a 5' portion, wherein the 3' portion comprises a nucleic acid sequence capable of specifically annealing to a portion of the known target nucleotide sequence contained in the amplicon generated by step (c), and the 5' portion comprises the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer;

[0321] Wherein, the first tail primer contains the same nucleic acid sequence as the tailed random primer; and

[0322] The second tail primer contains a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer.

[0323] 24. The method as described in paragraph 23, wherein each tailed random primer further comprises a spacer region nucleic acid sequence between the 5' nucleic acid sequence identical to the first sequencing primer and the 3' nucleic acid sequence containing about 6 to about 12 random nucleotides.

[0324] 25. The method as described in paragraph 23 or 24, wherein, after the extension step, the unhybridized primers are removed from the reaction.

[0325] 26. The method as described in any of paragraphs 23-25, wherein the second tail primer is nested relative to the first tail primer by at least 3 nucleotides.

[0326] 27. The method as described in any of paragraphs 23-26, wherein the first target-specific primer further comprises a 5' tag sequence portion comprising a high-GC-content nucleic acid sequence that is substantially not complementary to or substantially identical to any other portion of any primer.

[0327] 28. The method as described in any of paragraphs 23-27, wherein the second tail primer is the same as the full-length first sequencing primer.

[0328] 29. The method as described in any of paragraphs 23-28, wherein the portion of the target-specific primer specifically annealed to the known target is specifically annealed in PCR buffer at a temperature of approximately 65°C.

[0329] 30. The method as described in any of paragraphs 23-29, wherein the sample comprises genomic DNA.

[0330] 31. The method as described in any of paragraphs 23-30, wherein the sample comprises RNA, and the method further comprises a first step of subjecting the sample to a reverse transcriptase protocol.

[0331] 32. The method as described in any of paragraphs 23-31, wherein the nucleic acid present in the sample has not been cleaved or digested, or wherein the sample contains single-stranded gDNA or single-stranded cDNA.

[0332] 33. The method as described in any of paragraphs 23-32, wherein the reverse transcriptase protocol includes the use of random hexamers.

[0333] 34. The method described in any of paragraphs 23-33, wherein the gene rearrangement includes the known target sequence.

[0334] 35. The method as described in paragraph 34, wherein the gene rearrangement is present in a nucleic acid selected from the group consisting of genomic DNA, RNA and cDNA.

[0335] 36. The method described in any of paragraphs 34-35, wherein the gene rearrangement includes oncogenes.

[0336] 37. The method as described in paragraph 36, wherein the gene rearrangement includes a fusion oncogene.

[0337] 38. The method described in any of paragraphs 23-37, wherein the nucleic acid product is sequenced by next-generation sequencing.

[0338] 39. The method as described in paragraph 38, wherein the next-generation sequencing method comprises methods selected from the group consisting of: Ion Torrent, Illumina, SOLiD, 454, massively parallel signature sequencing, solid-phase reversible dye-terminated sequencing, and DNA nanosphere sequencing.

[0339] 40. The method described in any of paragraphs 23-39, wherein the first and second sequencing primers are compatible with the selected next-generation sequencing method.

[0340] 41. The method as described in any of paragraphs 23-40, wherein the method comprises contacting the sample or a separate portion of the sample with a plurality of sets of first target-specific primers and second target-specific primers.

[0341] 42. The method as described in any of paragraphs 23-41, wherein the method comprises contacting a single reaction mixture containing the sample with a plurality of sets of first target-specific primers and second target-specific primers.

[0342] 43. The method as described in any of paragraphs 23-42, wherein multiple sets of first target-specific primers and second target-specific primers are specifically annealed to a known target nucleotide sequence contained in a single gene.

[0343] 44. The method as described in any of paragraphs 23-43, wherein at least two sets of first target-specific primers and second target-specific primers are specifically annealed to different portions of a known target nucleotide sequence.

[0344] 45. The method as described in any of paragraphs 23-44, wherein at least two sets of first target-specific primers and second target-specific primers are specifically annealed to different portions of a single gene containing a known target nucleotide sequence.

[0345] 46. ​​The method as described in any of paragraphs 23-45, wherein at least two sets of first target-specific primers and second target-specific primers are specifically annealed to different exons of a gene containing a known target nucleotide sequence.

[0346] 47. The method described in any of paragraphs 23-46, wherein the plurality of first target-specific primers contain the same 5' tag sequence portion.

[0347] 48. The method described in any of paragraphs 23-47, wherein each tailed random primer in the tailed random primer group further comprises the same sample barcode portion.

[0348] 49. The method as described in paragraph 48, wherein multiple samples are each contacted with a separate set of tailed random primers having a sample barcode portion; wherein each set of tailed random primers has a different sample barcode portion; and wherein, after step (b), the samples are combined.

[0349] 50. The method described in any of paragraphs 23-49, wherein each amplification step includes a PCR amplification protocol cycle set of 5 to 20 cycles in length.

[0350] 51. The method as described in any of paragraphs 23-50, wherein the target-specific primers and the tail primers are designed such that they are specifically annealed to their complementary sequences at an annealing temperature of about 61°C-72°C.

[0351] 52. The method as described in any of paragraphs 23-51, wherein the target-specific primers and the tail primers are designed such that they are specifically annealed to their complementary sequences at an annealing temperature of about 65°C.

[0352] 53. The method as described in any of paragraphs 23-52, wherein the target nucleic acid molecule is derived from a sample, optionally a biological sample obtained from a subject.

[0353] 54. The method as described in paragraph 53, wherein the sample is obtained from a subject requiring treatment for a disease associated with a genetic alteration.

[0354] 55. The method as described in paragraph 54, wherein the disease is cancer.

[0355] 56. The method as described in paragraph 53, wherein the sample comprises a population of tumor cells.

[0356] 57. The method as described in paragraph 53, wherein the sample is a tumor biopsy.

[0357] 58. The method as described in paragraph 55, wherein the cancer is lung cancer.

[0358] 59. The method described in any of paragraphs 23-58, wherein the disease-related gene contains the known target sequence.

[0359] 60. The method as described in paragraph 53, wherein the gene rearrangement product in the sample contains the known target sequence.

[0360] 61. The method as described in paragraph 60, wherein the gene rearrangement product is an oncogene.

[0361] Example

[0362] Example 1: A method for amplifying 3' fusion events using reverse transcriptase, tailed random oligonucleotides, and gene-specific oligonucleotides.

[0363] First-chain synthesis

[0364] As a first step in amplifying the 3' fusion event for sequence analysis, RNA is obtained from a sample isolated from the subject. The following reaction assembly is performed on ice to synthesize the first cDNA strand:

[0365] ● 12 μl of purified RNA and H2O

[0366] ●2 μl of 1.2 μg / μl random primer #1 (9mer)

[0367] ●2μl dNTP

[0368] The reaction was transferred to a thermal cycler and incubated at 65°C for 5 minutes. Then, the reaction was centrifuged and incubated on ice for at least one minute.

[0369] For the above reaction, the preparation on ice is as follows:

[0370] ●2μl 10X M-MuLV reverse transcriptase buffer

[0371] ●1μl 40U / μl RNase inhibitor

[0372] ●1μl 200U / μl M-MuLV enzyme

[0373] The reaction mixture was briefly centrifuged to collect the reaction material at the bottom of the tube, and then placed back on ice. The reaction was then incubated at 42°C for 60 minutes, followed by incubation at 4°C.

[0374] ExoI processing

[0375] Add the following to the above reaction.

[0376] ●1 μl 20 U / μl exonuclease I

[0377] Mix the reaction mixture and briefly centrifuge to collect the reaction material at the bottom of the tube, then incubate at 37°C for 10 minutes. Next, add 1.28 μl of 1N NaOH, mix by up-and-down pipetting, and then centrifuge to collect the material. Incubate the reaction at 80°C for 10 minutes, then add 4 μl of 10 mM Tris (pH 8.3) and mix by up-and-down pipetting. Transfer 20 μl of the exonuclease-treated DNA solution to a new 200 μl PCR tube on ice.

[0378] Second-strand cDNA synthesis

[0379] Prepare the following reactions:

[0380] ●From the above 20μl DNA solution

[0381] ● 11 μl of nuclease-free H2O

[0382] ●4μl 10X PCR Buffer II

[0383] ●4μl 3μM gene-specific primer #1

[0384] ●1μl 0.5mM dNTP

[0385] Mix the reaction mixture by blowing and aspiration, then briefly centrifuge to collect the material and place it on ice. Incubate the reaction at 95°C for 3 minutes, then at 22°C for 10 seconds, followed by incubation at 4°C until proceeding to the next step. Incubate the reaction on ice for at least one minute.

[0386] Add the following to the reaction:

[0387] ●1μl 400U / μl Manta 1.0 DNA polymerase (high concentration)

[0388] The reaction was incubated at 25°C for 10 seconds, then at 70°C for 10 minutes, and kept at 4°C until the next step was carried out.

[0389] DNA purified with AMPure beads #1

[0390] Add the following to the above reaction.

[0391] ●88.4μl AMPure beads

[0392] Mix the suspension thoroughly and incubate at room temperature for 5 minutes. Collect the beads using a magnet for 2–4 minutes until the solution becomes clear. Discard the supernatant and wash the beads twice with 200 μl of 70% ethanol on the magnet. After the second wash, allow the beads to dry at room temperature for 5 minutes. Finally, elute the DNA by removing the tube from the magnet and resuspending the beads in 12 μl of 10 mM Tris-HCl (pH 8.3) elution buffer included in the AMPure kit. Place the RNA-bead solution on the magnet for 2 minutes. Then, transfer the DNA solution to a new PCR tube, ensuring that the beads are not transferred to a new tube.

[0393] It should be understood that, in some embodiments, the ratio of beads to the reaction mixture can affect the size of the returned fragments. In some embodiments, for the purpose of fusion detection, all or substantially all fragments are longer than 60 nt (e.g., 30 nt on each side of the fusion breakpoint or node), thereby enabling easy identification of the genes.

[0394] Amplification #1

[0395] Prepare the following reactions:

[0396] ● 10 μl of purified DNA from purification #1 above

[0397] ●4μl 5X Phoenix Hot Start Buffer

[0398] ●2μl 2mM dNTP

[0399] ●2μl 10μM P5_barcode primer

[0400] ●2μl 3μM gene-specific primer #1

[0401] ● 0.5-2 unit polymerases (e.g., Pheonix Hot Start Taq, VeraSeq)

[0402] The reaction was incubated as follows:

[0403] ●Step 1: Incubate at 95℃ for 3 minutes

[0404] ●Step 2: Incubate at 95℃ for 30 seconds

[0405] ●Step 3: Incubate at 65℃ for 5 minutes, then return to Step 2 for a total of 14 cycles.

[0406] ●Step 4: Incubate at 72℃ for 2 minutes

[0407] ●Step 5: 4℃, until the plan is continued.

[0408] DNA was purified using AMPure beads #2.

[0409] Add the following to the above reaction.

[0410] ●36.4μl AMPure beads

[0411] Mix the suspension thoroughly and incubate at room temperature for 5 minutes. Collect the beads using a magnet for 2–4 minutes until the solution becomes clear. Discard the supernatant and wash the beads twice with 200 μl of 70% ethanol on the magnet. After the second wash, allow the beads to dry at room temperature for 5 minutes. Finally, elute the DNA by removing the tube from the magnet and resuspending the beads in 9 μl of 10 mM Tris-HCl (pH 8.3) elution buffer included in the AMPure kit. Place the RNA-bead solution on the magnet for 2 minutes. Then, transfer the DNA solution to a new PCR tube, ensuring that the magnetic beads are not transferred to the new tube.

[0412] Amplification #2

[0413] Prepare the following reactions:

[0414] ●8.5 μl of purified DNA from purification #2 above

[0415] ●4μl 5X Phoenix Hot Start Buffer

[0416] ●2μl 2mM dNTP

[0417] ●2μl 10μM P5_29bp primer

[0418] ●2μl 10μM P7 barcode primer

[0419] ●2μl 3μM gene-specific primer #2

[0420] ●0.2μl 5U / μl Phoenix Hot Start Taq polymerase

[0421] ●2μl 10μM P7 barcode primer

[0422] The reaction was incubated as follows:

[0423] ●Step 1: Incubate at 95℃ for 3 minutes

[0424] ●Step 2: Incubate at 95℃ for 30 seconds

[0425] ●Step 3: Incubate at 65℃ for 5 minutes, then return to Step 2 for a total of 14 cycles.

[0426] ●Step 4: Incubate at 72℃ for 2 minutes

[0427] ●Step 5: 4℃, until the plan is continued.

[0428] DNA purified with AMPure beads #3

[0429] Add the following to the above reaction.

[0430] ●37.3μl AMPure beads

[0431] Mix the suspension thoroughly and incubate at room temperature for 5 minutes. Collect the beads using a magnet for 2–4 minutes until the solution becomes clear. Discard the supernatant and wash the beads twice with 200 μl of 70% ethanol on the magnet. After the second wash, allow the beads to dry at room temperature for 5 minutes. Finally, elute the DNA by removing the tube from the magnet and resuspending the beads in 20 μl of 10 mM Tris-HCl (pH 8.3) elution buffer included in the AMPure kit. Place the RNA-bead solution on the magnet for 2 minutes. Then, transfer the DNA solution to a new PCR tube, ensuring that the magnetic beads are not transferred to the new tube.

[0432] Quantitative analysis of library concentration

[0433] The concentrations of the libraries prepared using the above protocol were quantified using the Illumina Kapa Biosystems qPCR kit. The barcoded libraries were pooled at equimolar concentrations. The libraries were then loaded onto the Illumina MiSeq at XpM using the MiSeq v2 300 cycling kit, following the manufacturer's instructions. The samples were sequenced using 2x150 bp reads (index reads with 7 bases encoded).

[0434] Example 2: A method for amplifying 5' fusion events using reverse transcriptase, gene-specific oligonucleotides, and tailed random oligonucleotides.

[0435] First-chain synthesis

[0436] As a first step in amplifying the 5' fusion event for sequence analysis, RNA is obtained from a sample isolated from the subject. The following reaction assembly is performed on ice to synthesize the first cDNA strand:

[0437] ● 12 μl of purified RNA and H2O

[0438] ●2μl gene-specific primer #1

[0439] ●2μl dNTP

[0440] The reaction was transferred to a thermal cycler and incubated at 65°C for 5 minutes. Then, the reaction was centrifuged and incubated on ice for at least one minute.

[0441] For the above reaction, the preparation on ice is as follows:

[0442] ●2μl 10X M-MuLV reverse transcriptase buffer

[0443] ●1μl 40U / μl RNase inhibitor

[0444] ●1μl 200U / μl M-MuLV enzyme

[0445] The reaction mixture was briefly centrifuged to collect the reaction material at the bottom of the tube, and then placed back on ice. The reaction was then incubated at 42°C for 60 minutes, followed by incubation at 4°C.

[0446] ExoI processing

[0447] Add the following to the above reaction.

[0448] ●1 μl 20 U / μl exonuclease I

[0449] Mix the reaction mixture and briefly centrifuge to collect the reaction material at the bottom of the tube, then incubate at 37°C for 10 minutes. Next, add 1.28 μl of 1N NaOH, mix by pipetting, and then centrifuge to collect the material. Incubate the reaction at 80°C for 10 minutes, then add 4 μl of 10 mM Tris (pH 8.3) and mix by pipetting. Transfer 20 μl of the exonuclease-treated DNA solution to a new 200 μl PCR tube on ice.

[0450] Second-strand cDNA synthesis

[0451] Prepare the following reactions:

[0452] ●From the above 20μl DNA solution

[0453] ● 11 μl of nuclease-free H2O

[0454] ●4μl 10X PCR Buffer II

[0455] ●4 μl of 1.2 μg / μl random primers (9mer)

[0456] ●1μl 0.5mM dNTP

[0457] Mix the reaction mixture by blowing and aspiration, then briefly centrifuge to collect the material and place it on ice. Incubate the reaction at 95°C for 3 minutes, then at 22°C for 10 seconds, followed by incubation at 4°C until proceeding to the next step. Incubate the reaction on ice for at least one minute.

[0458] Add the following to the reaction:

[0459] ●1μl 400U / μl Manta 1.0 DNA polymerase (high concentration)

[0460] The reaction was incubated at 25°C for 10 seconds, then at 70°C for 10 minutes, and kept at 4°C until the next step was carried out.

[0461] DNA purified with AMPure beads #1

[0462] Add the following to the above reaction.

[0463] ●88.4μl AMPure beads

[0464] Mix the suspension thoroughly and incubate at room temperature for 5 minutes. Collect the beads using a magnet for 2–4 minutes until the solution becomes clear. Discard the supernatant and wash the beads twice with 200 μl of 70% ethanol on the magnet. After the second wash, allow the beads to dry at room temperature for 5 minutes. Finally, elute the DNA by removing the tube from the magnet and resuspending the beads in 12 μl of 10 mM Tris-HCl (pH 8.3) elution buffer included in the AMPure kit. Place the RNA-bead solution on the magnet for 2 minutes. Then, transfer the DNA solution to a new PCR tube, ensuring that the magnetic beads are not transferred to the new tube.

[0465] Amplification #1

[0466] Prepare the following reactions:

[0467] ● 10 μl of purified DNA from purification #1 above

[0468] ●4μl 5X Phoenix Hot Start Buffer

[0469] ●2μl 2mM dNTP

[0470] ●2μl 10μM P5_barcode primer

[0471] ●2μl 3μM gene-specific primer #1

[0472] ● 0.5-2 unit polymerases (e.g., Pheonix Hot Start Taq, VeraSeq)

[0473] The reaction was incubated as follows:

[0474] ●Step 1: Incubate at 95℃ for 3 minutes

[0475] ●Step 2: Incubate at 95℃ for 30 seconds

[0476] ●Step 3: Incubate at 65℃ for 5 minutes, then return to Step 2 for a total of 14 cycles.

[0477] ●Step 4: Incubate at 72℃ for 2 minutes

[0478] ●Step 5: 4℃, until the plan is continued.

[0479] DNA was purified using AMPure beads #2.

[0480] Add the following to the above reaction.

[0481] ●36.4μl AMPure beads

[0482] Mix the suspension thoroughly and incubate at room temperature for 5 minutes. Collect the beads using a magnet for 2–4 minutes until the solution becomes clear. Discard the supernatant and wash the beads twice with 200 μl of 70% ethanol on the magnet. After the second wash, allow the beads to dry at room temperature for 5 minutes. Finally, elute the DNA by removing the tube from the magnet and resuspending the beads in 9 μl of 10 mM Tris-HCl (pH 8.3) elution buffer included in the AMPure kit. Place the RNA-bead solution on the magnet for 2 minutes. Then, transfer the DNA solution to a new PCR tube, ensuring that the magnetic beads are not transferred to the new tube.

[0483] Amplification #2

[0484] Prepare the following reactions:

[0485] ●8.5 μl of purified DNA from purification #2 above

[0486] ●4μl 5X Phoenix Hot Start Buffer

[0487] ●2μl 2mM dNTP

[0488] ●2μl 10μM P5_29bp primer

[0489] ●2μl 10μM P7 barcode primer

[0490] ●2μl 3μM gene-specific primer #2

[0491] ●0.2μl 5U / μl Phoenix Hot Start Taq polymerase

[0492] ●2μl 10μM P7 barcode primer

[0493] The reaction was incubated as follows:

[0494] ●Step 1: Incubate at 95℃ for 3 minutes

[0495] ●Step 2: Incubate at 95℃ for 30 seconds

[0496] ●Step 3: Incubate at 65℃ for 5 minutes, then return to Step 2 for a total of 14 cycles.

[0497] ●Step 4: Incubate at 72℃ for 2 minutes

[0498] ●Step 5: 4℃, until the plan is continued.

[0499] DNA purified with AMPure beads #3

[0500] Add the following to the above reaction.

[0501] ●37.3μl AMPure beads

[0502] Mix the suspension thoroughly and incubate at room temperature for 5 minutes. Collect the beads using a magnet for 2–4 minutes until the solution becomes clear. Discard the supernatant and wash the beads twice with 200 μl of 70% ethanol on the magnet. After the second wash, allow the beads to dry at room temperature for 5 minutes. Finally, elute the DNA by removing the tube from the magnet and resuspending the beads in 20 μl of 10 mM Tris-HCl (pH 8.3) elution buffer included in the AMPure kit. Place the RNA-bead solution on the magnet for 2 minutes. Then, transfer the DNA solution to a new PCR tube, ensuring that the magnetic beads are not transferred to the new tube.

[0503] Quantitative analysis of library concentration

[0504] The concentrations of the libraries prepared using the above protocol were quantified using the Illumina Kapa Biosystems qPCR kit. The barcoded libraries were pooled at equimolar concentrations. The libraries were then loaded onto the Illumina MiSeq at XpM using the MiSeq v2 300 cycling kit, following the manufacturer's instructions. The samples were sequenced using 2x150 bp reads (index reads with 7 bases encoded).

Claims

1. A method for preparing nucleic acids for analysis, the method comprising: (a) Under conditions that promote template-specific hybridization and target-specific primer extension, a first nucleic acid template containing the first strand of the target nucleic acid is contacted with a complementary target-specific primer containing the target-specific hybridization sequence; (b) Under conditions that promote template-specific hybridization and extension of at least one of a plurality of different hairpin primers, a second nucleic acid template containing a sequence of a second strand complementary to the first strand of the target nucleic acid is contacted with a plurality of different hairpin primers, each hairpin primer containing two complementary common sequences forming a stem-loop hairpin structure by base pairing, the stem-loop hairpin structure being the 5' end of the different hybridization sequences, wherein the stem-loop hairpin structure contains at least one index sequence, barcode sequence or adapter sequence flanked by the two complementary common sequences; The process involves generating an extension product, which simultaneously comprises a sequence characteristic of the target-specific primer and a sequence characteristic of at least one of the plurality of different hairpin primers; and (c) Amplify the extended product.

2. The method as described in claim 1, wherein, The target nucleic acid is ribonucleic acid.

3. The method as described in claim 1, wherein, The target nucleic acid is deoxyribonucleic acid (DNA).

4. The method according to any one of claims 1-3, wherein, Perform steps (a) and (b) sequentially.

5. The method according to any one of claims 1-3, wherein, The nucleic acid template in step (a) comprises an extension product generated from the extension and hybridization of at least one of the plurality of different hairpin primers in step (b).

6. The method according to any one of claims 1-3, wherein, The nucleic acid template in step (b) contains an extension product generated from the extension and hybridization of the target-specific primers in step (a).

7. The method of claim 2, wherein, The target nucleic acid is a messenger RNA encoding a chromosomal segment containing gene rearrangements.

8. The method of claim 3, wherein, The target nucleic acid is a segment of chromosome containing a portion of a gene rearrangement.

9. The method of claim 8, wherein, The gene rearrangement is an inversion, deletion, or translocation.

10. The method of any one of claims 1-3, further comprising contacting the extended product or the amplified extended product with the fixed oligonucleotide under conditions where hybridization occurs between the extended product and the fixed oligonucleotide.

11. The method according to any one of claims 1-3, wherein, The target nucleic acid comprises a target portion having a known sequence and a flanking portion having an unknown sequence.

12. The method of claim 11, wherein, Different hybridization sequences are complementary to the flanking portion.

13. The method of claim 11, wherein, The target-specific hybridization sequence is complementary to the target region.

14. The method according to any one of claims 1-3, wherein, The target-specific primer further comprises: the 5' end of the target-specific hybridization sequence; and at least one barcode sequence, adapter sequence, or index sequence.

15. The method of claim 1, wherein, The adapter sequence is a cleavable adapter sequence used to immobilize oligonucleotides in a flow cell.

16. A method for determining a nucleotide sequence adjacent to a known target nucleotide sequence, the method comprising: (a) Under hybridization conditions, a target nucleic acid molecule containing the known target nucleotide sequence is contacted with an initiating target-specific primer; (b) Perform a template-dependent extension reaction, which is initiated by a hybridization initiation target-specific primer and uses the target nucleic acid molecule as a template; (c) Under hybridization conditions, the product of step (b) is contacted with a set of tailed random primers, each tailed random primer containing two complementary common sequences that form a stem-loop hairpin structure by base pairing, wherein the stem-loop hairpin structure is the 5' end of different hybridization sequences, wherein the stem-loop hairpin structure contains at least one index sequence, barcode sequence or adapter sequence flanked by the two complementary common sequences. (d) Perform a template-dependent extension reaction, which is initiated by a hybridization tailed random primer and uses a portion of the target nucleic acid molecule downstream of the hybridization site as a template; (e) Amplify a portion of the target nucleic acid molecule and the tailed random primer sequence using the first tail primer and the first target-specific primer; (f) Amplify a portion of the amplicon generated in step (e) using the second tail primer and the second target-specific primer; (g) Sequencing the amplified portion from step (f) using the first and second sequencing primers; The tailed random primer group includes oligonucleotide molecules, which have the same 5' nucleic acid sequence as the first sequencing primer and a 3' nucleic acid sequence containing 6-12 random nucleotides; Wherein, the first target-specific primer contains a nucleic acid sequence that can be specifically annealed to the known target nucleotide sequence of the target nucleic acid at an annealing temperature; The second target-specific primer comprises a 3' portion and a 5' portion, wherein the 3' portion comprises a nucleic acid sequence capable of specifically annealing to a portion of the known target nucleotide sequence contained in the amplicon generated by step (e), and the 5' portion comprises the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer; Wherein, the first tail primer contains the same nucleic acid sequence as the tailed random primer; and The second tail primer contains a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer.

17. A method for determining a nucleotide sequence adjacent to a known target nucleotide sequence, the method comprising: (a) Under hybridization conditions, a target nucleic acid molecule containing the known target nucleotide sequence is contacted with a set of tailed random primers, each tailed random primer containing two complementary common sequences that form a stem-loop hairpin structure by base pairing, the stem-loop hairpin structure being the 5' end of different hybridization sequences, wherein the stem-loop hairpin structure contains at least one index sequence, barcode sequence or adapter sequence flanked by the two complementary common sequences; (b) Perform a template-dependent extension reaction, which is initiated by a hybridization tailed random primer and uses a portion of the target nucleic acid molecule downstream of the hybridization site as a template; (c) Under hybridization conditions, the product of step (b) is contacted with the initiation target-specific primer; (d) Perform a template-dependent extension reaction, which is initiated by a hybridization initiation target-specific primer and uses the target nucleic acid molecule as a template; (e) Amplify a portion of the target nucleic acid molecule and the tailed random primer sequence using the first tail primer and the first target-specific primer; (f) Amplify a portion of the amplicon generated in step (e) using the second tail primer and the second target-specific primer; (g) Sequencing the amplified portion from step (f) using the first and second sequencing primers; The tailed random primer group includes oligonucleotide molecules, which have the same 5' nucleic acid sequence as the first sequencing primer and a 3' nucleic acid sequence containing 6-12 random nucleotides; Wherein, the first target-specific primer contains a nucleic acid sequence that can be specifically annealed to the known target nucleotide sequence of the target nucleic acid at an annealing temperature; The second target-specific primer comprises a 3' portion and a 5' portion, wherein the 3' portion comprises a nucleic acid sequence capable of specifically annealing to a portion of the known target nucleotide sequence contained in the amplicon generated by step (c), and the 5' portion comprises the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer; Wherein, the first tail primer contains the same nucleic acid sequence as the tailed random primer; and The second tail primer contains a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer.

18. The method of any one of claims 16-17, the method further comprising the step of contacting the sample and product with RNase after the extension of the starting target-specific primer.

19. The method according to any one of claims 16-17, wherein, The starting target-specific primer is the same as the first target-specific primer.

20. The method according to any one of claims 16-17, wherein, Between the 5' nucleic acid sequence identical to the first sequencing primer and the 3' nucleic acid sequence containing 6-12 random nucleotides, each tailed random primer contains the barcode sequence, wherein the barcode sequence contains 6-12 random nucleotides.

21. A method for determining a nucleotide sequence adjacent to a known target nucleotide sequence, the method comprising: (a) Under hybridization conditions, a target nucleic acid molecule containing the known target nucleotide sequence is contacted with a set of tailed random primers, each tailed random primer containing two complementary common sequences that form a stem-loop hairpin structure by base pairing, the stem-loop hairpin structure being the 5' end of different hybridization sequences, wherein the stem-loop hairpin structure contains at least one index sequence, barcode sequence or adapter sequence flanked by the two complementary common sequences; (b) Perform a template-dependent extension reaction, which is initiated by a hybridization tailed random primer and uses a portion of the target nucleic acid molecule downstream of the hybridization site as a template; (c) Amplify a portion of the target nucleic acid molecule and the tailed random primer sequence using the first tail primer and the first target-specific primer; (d) Amplify a portion of the amplicon generated in step (c) using the second tail primer and the second target-specific primer; (e) Sequencing the amplified portion from step (d) using the first and second sequencing primers; The tailed random primer group includes oligonucleotide molecules, which have the same 5' nucleic acid sequence as the first sequencing primer, a middle barcode portion containing 6-12 random nucleotides, and a 3' nucleic acid sequence containing 6-12 random nucleotides. Wherein, the first target-specific primer contains a nucleic acid sequence that can be specifically annealed to the known target nucleotide sequence of the target nucleic acid at an annealing temperature; The second target-specific primer comprises a 3' portion and a 5' portion, wherein the 3' portion comprises a nucleic acid sequence capable of specifically annealing to a portion of the known target nucleotide sequence contained in the amplicon generated by step (c), and the 5' portion comprises the same nucleic acid sequence as the second sequencing primer, and the second target-specific primer is nested relative to the first target-specific primer; Wherein, the first tail primer contains the same nucleic acid sequence as the tailed random primer; and The second tail primer contains a portion of the same nucleic acid sequence as the first sequencing primer, and the second tail primer is nested relative to the first tail primer.

22. The method of claim 21, wherein, Between the 5' nucleic acid sequence identical to that of the first sequencing primer and the 3' nucleic acid sequence containing 6-12 random nucleotides, each tailed random primer further includes a spacer region nucleic acid sequence.

23. The method of claim 21 or 22, wherein, After the extension step, the unhybridized primers are removed from the reaction.

24. The method of claim 21 or 22, wherein, The second tail primer is nested with at least 3 nucleotides relative to the first tail primer.

25. The method of claim 21 or 22, wherein, The first target-specific primer further includes a 5' tag sequence portion, which contains a high-GC-content nucleic acid sequence that is not complementary to or identical to any other portion of any primer.

26. The method of claim 21 or 22, wherein, The second tail primer is the same as the full-length first sequencing primer.

27. The method of claim 21 or 22, wherein, The portion of the target-specific primer that is specifically annealed to the known target will be specifically annealed in PCR buffer at 65°C.

28. The method of claim 21 or 22, wherein, The sample contains genomic DNA.

29. The method of claim 21 or 22, wherein, The sample contains RNA, and the method further includes a first step of subjecting the sample to a reverse transcriptase protocol.

30. The method of claim 21 or 22, wherein, The nucleic acid present in the sample has not been cleaved or digested, or the sample contains single-stranded gDNA or single-stranded cDNA.

31. The method of claim 21 or 22, wherein, The reverse transcriptase protocol includes the use of random hexamers.

32. The method of claim 21 or 22, wherein, Gene rearrangements include known target sequences.

33. The method of claim 32, wherein, The gene rearrangement exists in nucleic acids selected from the group consisting of genomic DNA, RNA and cDNA.

34. The method of claim 32, wherein, The gene rearrangements include oncogenes.

35. The method of claim 34, wherein, The gene rearrangement includes fusion oncogenes.

36. The method of claim 21 or 22, wherein, The nucleic acid product was sequenced using next-generation sequencing.

37. The method of claim 36, wherein, The next-generation sequencing methods include those selected from the group consisting of: Ion Torrent, Illumina, SOLiD, 454, massively parallel signature sequencing, solid-phase reversible dye-terminated sequencing, and DNA nanosphere sequencing.

38. The method of claim 37, wherein, The first and second sequencing primers are compatible with the selected next-generation sequencing method.

39. The method of claim 21 or 22, wherein, The method includes contacting the sample or a separate portion of the sample with multiple sets of first target-specific primers and second target-specific primers.

40. The method of claim 21 or 22, wherein, The method includes contacting a single reaction mixture containing the sample with multiple sets of first target-specific primers and second target-specific primers.

41. The method of claim 21 or 22, wherein, Multiple sets of first-target-specific primers and second-target-specific primers are specifically annealed to known target nucleotide sequences contained in individual genes.

42. The method of claim 21 or 22, wherein, At least two sets of first target-specific primers and second target-specific primers specifically anneal to different parts of the known target nucleotide sequence.

43. The method of claim 21 or 22, wherein, At least two sets of first target-specific primers and second target-specific primers specifically anneal to different parts of a single gene containing a known target nucleotide sequence.

44. The method of claim 21 or 22, wherein, At least two sets of first target-specific primers and second target-specific primers specifically anneal to different exons of a gene containing a known target nucleotide sequence.

45. The method of claim 21 or 22, wherein, Multiple first-target-specific primers contain the same 5' tag sequence portion.

46. ​​The method of claim 21 or 22, wherein, Each tailed random primer in the tailed random primer group further contains the same sample barcode portion.

47. The method of claim 46, wherein, Multiple samples are each contacted with a separate set of tailed random primers having a sample barcode portion; wherein each set of tailed random primers has a different sample barcode portion; and wherein, after step (b), the samples are combined.

48. The method of claim 21 or 22, wherein, Each amplification step includes a PCR amplification protocol cycle set with a length of 5 to 20 cycles.

49. The method of claim 21 or 22, wherein, The target-specific primers and the tail primers are designed to specifically anneal to their complementary sequences at an annealing temperature of 61°C-72°C.

50. The method of claim 21 or 22, wherein, The target-specific primers and the tail primers are designed such that they are specifically annealed to their complementary sequences at an annealing temperature of 65°C.

Images