JP2021182940A5 - - Google Patents

Claims (53)

(A) With one or more adapters;
(B) One or more capture probe modules with one or more capture probe modules, each of which includes a tail sequence and a capture probe sequence.
A plurality of compositions for targeting gene analysis of DNA fragments containing
Multiple compositions in which the capture probe sequence is less than about 60 nucleotides in length. The plurality of compositions according to claim 1, wherein the DNA fragment is circulating acellular (cfDNA). The plurality of compositions according to claim 1 or 2, wherein each adapter comprises a PCR primer binding site. The plurality of compositions according to any one of claims 1 to 3, wherein each adapter comprises one or more unique read cords. The plurality of compositions according to any one of claims 1 to 4, wherein each adapter comprises one or more sample codes for sample multiplexing. The plurality of compositions according to any one of claims 1 to 5, wherein each adapter comprises one or more anchor sequences. The plurality of compositions according to any one of claims 4 to 6, wherein each read code is configured to identify a unique sequencing lead. The sample code is configured to identify the sample from which the tagged DNA library was derived, the tagged DNA library containing a DNA fragment ligated to an adapter module at each terminal, said one or more. The plurality of compositions according to any one of claims 5 to 7, wherein each of the plurality of adapters comprises one adapter module. The plurality of compositions according to any one of claims 3 to 8, wherein the PCR primer binding site is configured for single primer amplification of a DNA library. The invention according to any one of claims 3 to 9, wherein the PCR primer binding site is about 12 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, or about 40 nucleotides. Multiple compositions of. The plurality of compositions according to any one of claims 4 to 10, wherein the read code is about 2 nucleotides, about 3 nucleotides, or about 5 nucleotides, which is at least 2 nucleotides different from all other read codes. The plurality of compositions according to any one of claims 5 to 11, wherein the sample code is about 2 nucleotides, about 3 nucleotides, or about 5 nucleotides, which is at least 2 nucleotides different from all other sample codes. The plurality of compositions according to any one of claims 6 to 12, wherein the anchor sequence is selected from four nucleotide sequences. The plurality of compositions according to any one of claims 6 to 13, wherein the anchor sequence is about 8 nucleotides, about 12 nucleotides, or about 16 nucleotides. The plurality of compositions according to any one of claims 6 to 14, wherein the 2 nucleotides at the 3'end of each anchor sequence are AA, CC, TT or GG. The plurality of compositions according to any one of claims 1 to 15, wherein the one or more adapters include a plurality of adapter types. 3. The composition according to any one of claims 1 to 16, wherein each adapter comprises a ligation strand and a complementary strand, the ligation strand comprises an adapter module, and the complementary strand is 3 of the ligation strand. 'Multiple compositions that hybridize to the ends to form a partial double strand with the ligation strand. 17. The composition of claim 17, wherein the double-stranded region of the adapter has a blunt end. 17. The double-stranded region of the adapter comprises an overhang at the 3'end of the ligation strand, wherein the overhang is complementary to the 5'-overhang of the DNA fragment ligating therein. Multiple compositions of. The plurality of compositions according to any one of claims 17 to 19, wherein the one or more adapters are used.
(A) A step of ligating a plurality of adapters with a DNA fragment to generate a plurality of complexes containing the DNA fragment ligated with the ligation strand of the adapter.
(B) With the step of contacting the plurality of complexes with one or more enzymes to form a tagged library containing a plurality of adapters-tagged DNA fragments.
Multiple compositions configured to ligate a DNA fragment to generate a tagged DNA library using a method comprising. 20. The composition of claim 20, wherein the complementary strand is configured to migrate from the partial duplex of step (b). The plurality of compositions according to claim 20 or 21, comprising the one or more enzymes of step (b), wherein the one or more enzymes optionally include a DNA polymerase. Multiple compositions. The plurality of compositions according to any one of claims 20 to 22, comprising a DNA ligase for ligating the plurality of adapters with a DNA fragment in step (a), wherein the DNA ligase is required. Is a T4 DNA ligase, a plurality of compositions. The plurality of compositions according to any one of claims 1 to 23, wherein the one or more adapters each comprises one or more sequencing primer binding sites. The plurality of compositions according to any one of claims 1 to 24, wherein the one or more adapters each comprises one or more sequences for DNA sequencing. The plurality of compositions according to any one of claims 1 to 25, wherein the capture probe sequence is about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, or about 45 nucleotides. Claims 1-26, wherein the capture probe sequence is about 25 to about 45 nucleotides, about 30 to about 45 nucleotides, about 35 to about 45 nucleotides, about 35 to about 40 nucleotides, or about 40 to about 45 nucleotides. A plurality of compositions according to any one. The plurality of compositions according to any one of claims 1-27, wherein the capture probe sequence is substantially complementary to the target region in the tagged DNA library. The plurality of compositions according to any one of claims 1-28, wherein the capture probe sequence has at least 70% complementarity with a target region in a tagged DNA library. The plurality of compositions according to any one of claims 1 to 29, wherein the capture probe sequence can hybridize to a target region in a tagged DNA library. 13. Multiple compositions. The plurality of compositions according to any one of claims 1 to 31, wherein the tail sequence comprises a PCR primer binding site. The plurality of compositions according to any one of claims 1 to 32, wherein the tail sequence comprises a sequencing primer binding site. The plurality of compositions according to any one of claims 1 to 33, which comprises a plurality of capture probe modules. 34. Multiple compositions. The plurality of compositions according to claim 34 or 35, wherein each capture probe of the plurality of capture probe modules is configured to hybridize to its target sequence within about 200 bp of any other capture probe. .. One of claims 34 to 36, wherein each of the capture probe modules of the plurality of capture probe modules hybridizes to the tagged DNA library fragment to form a plurality of DNA library fragment-capture probe module complex. Multiple compositions according to the section. 37. Composition. The plurality of compositions according to any one of claims 31 to 38, wherein the partner oligo comprises a biotin molecule. Any one of claims 37-39 comprising one or more enzymes for enzymatically treating a DNA library fragment-capture probe module complex to produce an enzymatically treated complex. A plurality of compositions according to. The 5'-3'DNA polymerase is configured such that the one or more enzymes use the DNA library fragment as a template to extend each capture probe of the DNA library fragment-capture probe module complex. 40. The composition according to claim 40. The plurality of compositions according to claim 40 or 41, comprising one or more reagents for amplifying the enzymatically treated complex to produce an amplified hybrid nucleic acid molecule. , A plurality of compositions comprising a sequence in which each amplified hybrid nucleic acid molecule can hybridize to a capture probe module and a complement to the tail sequence of said capture probe module. The plurality of compositions according to claim 42, comprising a component for performing quantitative gene analysis on the amplified hybrid nucleic acid molecule, wherein the quantitative gene analysis produces a plurality of sequencing reads. A plurality of compositions comprising DNA sequencing of the amplified hybrid nucleic acid molecule. 13. The composition of claim 43, wherein the quantitative genetic analysis is used to detect one or more genetic lesions associated with a genetic disorder. 44. The composition of claim 44, wherein the one or more genetic lesions comprise a nucleotide transition or transversion, a nucleotide insertion or deletion, a genomic rearrangement, or a change in the number of copies. 44. The composition of claim 44 or claim 45, wherein the genetic disorder is cancer. The DNA of the tagged DNA library is from a biological sample of interest selected from the group consisting of amniotic fluid, blood, plasma, serum, semen, lymph, cerebrospinal fluid, ophthalmic fluid, urine, saliva, feces, mucus and sweat. The plurality of compositions according to any one of claims 8 to 46, which are isolated. The plurality of compositions according to any one of claims 8 to 47, comprising a reagent for producing the tagged DNA library. The composition of claim 48 comprising one or more reagents for performing end repair, wherein the DNA fragment of the tagged DNA library is end repair prior to ligation to an adapter. There are multiple compositions. 49. The composition of claim 49, wherein the one or more reagents for performing end repair comprises one or more enzymes that produce blunt ends, 5'-overhangs or 3'-overhangs. thing. The plurality of compositions according to any one of claims 1 and 3 to 50, wherein the DNA fragment is genomic DNA, recombinant DNA, synthetic DNA or cDNA. The plurality of compositions according to any one of claims 1 to 51, wherein the average size of the DNA fragment is between about 150 nucleotides and about 400 nucleotides. The plurality of compositions according to any one of claims 1 to 52, wherein the DNA fragment has an average size of about 150 nucleotides.