Non-invasive in vitro methods for detecting somatic mutations

Abstract

The present invention relates to an in vitro method for detecting somatic mutations in humans, comprising the following steps: (i) providing a human sample, preferably a blood sample from a subject, more preferably a plasma sample, or a serum sample, or a buffy coat sample; (ii) preparing a first nucleic acid sequencing library from nucleic acids present in the sample; (iii) hybridizing one or more TAC oligonucleotides from a first TAC oligonucleotide pool (TAC oligonucleotide-1) into the first nucleic acid library, thereby isolating a first subset of library nucleic acid molecules; (iv) sequencing the first subset of library nucleic acid molecules, comparing the determined sequence with a human reference sequence, thereby creating a first group of putatively useful sequence variants (PISV1); and (v) preparing The process includes (i) to (iv) hybridizing one or more TAC oligonucleotides from a second, smaller TAC oligonucleotide pool containing TAC oligonucleotides (TAC oligonucleotide-2) specific to the first group of nucleic acid molecules of the above beneficial sequence variants into a first library of nucleic acid molecules, thereby isolating a second subset of library nucleic acid molecules; (vi) sequencing the second subset of library nucleic acid molecules and comparing the determined sequences with human reference sequences to create a second group of putative beneficial sequence variants (PISV2); and (vii) analyzing the putative beneficial sequence variants (PISV2) to detect somatic mutations in a sample from the subject. Steps (i) to (iv) are collectively referred to as Step A. Steps (v) to (vii) are collectively referred to as Step B.

Claims

1. The following steps: (i) A step of providing a human sample, preferably a blood sample from a subject, more preferably a plasma sample, serum sample, or buffy coat sample, (ii) A step of preparing a first nucleic acid sequencing library from nucleic acids present in the sample, (iii) A step of hybridizing one or more TAC oligonucleotides from a first TAC oligonucleotide pool (TAC oligonucleotide 1) into the first nucleic acid library, thereby isolating a first subset of the library nucleic acid molecules, (iv) A step of sequencing a first subset of the library nucleic acid molecules, comparing the determined sequences with human reference sequences, thereby creating a first group of presumptively useful sequence variants (PISV1), (v) A step of hybridizing one or more TAC oligonucleotides from a second TAC oligonucleotide pool, which includes TAC oligonucleotides (TAC oligonucleotide 2) that are specific to the first group of nucleic acid molecules of the presumed beneficial sequence variants, to the first library of nucleic acid molecules, thereby isolating a second subset of the library nucleic acid molecules; (vi) A step of sequencing a second subset of the library nucleic acid molecules, comparing the determined sequences with human reference sequences, thereby creating a second group of doctrine-beneficial sequence variants (PISV2), (vii) The step of analyzing the presumably beneficial sequence variant (PISV2) and thereby detecting somatic mutations in the sample from the subject. An in vitro method for detecting somatic mutations in humans, including [specific example].

2. The method according to claim 1, wherein the second TAC oligonucleotide pool (TAC oligonucleotide-2) is a subset of the first TAC oligonucleotide pool.

3. The method according to claim 1 or 2, wherein each sequence within the first group of estimated beneficial sequence variants (PISV1) is ranked according to its informational value based on likelihood statistics.

4. Each sequence within the first group of estimated beneficial sequence variants (PISV1) is ranked according to its information value based on likelihood statistics, which is an ensemble learning method, (i) The number of cfDNA nucleic acid molecules in which variant alleles exist, (ii) Average mapping quality of sequenced cfDNA nucleic acid molecules relative to the reference genome sequence, (iii) The ratio of high-quality sequenced cfDNA nucleic acid molecules supporting the variant to low-quality sequenced cfDNA nucleic acid molecules supporting the variant, (iv) The ratio of a sequenced cfDNA nucleic acid molecule supporting a phasing single nucleotide polymorphism to the single nucleotide polymorphism when a nearby single nucleotide polymorphism exists, wherein the single nucleotide variant and the single nucleotide polymorphism are separated by a maximum of 110 bp. (v) Frequency of single nucleotide variants in disease-specific databases The method according to any one of claims 1 to 3, including

5. The method according to any one of claims 1 to 4, wherein the TAC oligonucleotides of the first TAC oligonucleotide pool (TAC oligonucleotide 1) and the second TAC oligonucleotide pool (TAC oligonucleotide 2) are 150 to 260 base pairs long, designed to bind to a target region, and have a 5' end and a 3' end.

6. The method according to any one of claims 1 to 5, wherein the GC content of the pool of TAC oligonucleotides, determined by calculating the GC content of each member in the pool of TAC oligonucleotides, is 19% to 80%.

7. The method according to any one of claims 1 to 6, wherein the pool of TAC oligonucleotides comprises a plurality of TAC oligonucleotide families, each of which is directed to a different target region, each of which comprises a plurality of member sequences, and each member sequence binds to the same target region but has different start and / or end positions with respect to the reference coordinate system of the target region, and furthermore, the start and / or end positions of each member sequence within the TAC oligonucleotide family are shifted by 5 to 10 base pairs with respect to the reference coordinate system of the target region.

8. The method according to any one of claims 1 to 7, wherein the second TAC oligonucleotide pool (TAC oligonucleotide 2) includes a specified fraction of the first TAC oligonucleotide pool (TAC oligonucleotide 1) and constitutes 0.1%, 0.25%, 0.5%, 0.75%, or 1.0% of the first TAC oligonucleotide pool (TAC oligonucleotide 1), but most preferably constitutes about 0.5% of the first TAC oligonucleotide pool (TAC oligonucleotide 1).

9. The method according to any one of claims 1 to 8, wherein the pool of TAC oligonucleotides 2 is designed to capture regions spanning the locations of the top 20, 30, 40, 50, 60, 70, 80, 90, or 96 somatic single nucleotide variant candidates selected in step A.

10. The method according to any one of claims 1 to 9, wherein step vi) of claim 1 comprises amplifying and sequencing a second subset of the library nucleic acid molecules to obtain at least 200,000 cfDNA nucleic acid sequences per target region.

11. The method according to any one of claims 1 to 10, wherein, in order to determine the proportion of sequences resulting from clonal hematopoiesis, the buffy coat fraction of the blood is analyzed separately from the plasma sample.

12. The method according to claim 1, wherein the human sample is a blood sample, or a serum sample, or a buffy coat sample, or a urine sample, or a sputum sample, or an ascites sample, or a cerebrospinal fluid sample, or a pleural exudate sample, or a saliva sample, or a bronchoalveolar lavage fluid, or a sample of aspirated fluid from various parts of the body.

13. The method according to claim 1, wherein the human sample is a tissue sample or a fecal sample.

14. The method according to claim 1, used for detecting donor-derived cell-free DNA.

15. The method according to claim 1, wherein the alignment in step B is performed using a patient-specific consensus sequence generated in step A.

16. The method according to any one of claims 1 to 15, wherein the single nucleotide variant and the single nucleotide polymorphism are separated by a maximum of 100 bp, 120 bp, 130 bp, 140 bp, or 150 bp.

17. The method according to any one of claims 1 to 16, wherein the TAC oligonucleotide comprises biotin modification.

18. A kit for carrying out the method described in claim 1, i) The TAC oligonucleotide, ii) One or more components for isolating cell-free DNA from a biological sample, iii) One or more components for preparing the sequencing library, iv) One or more components for amplifying and / or sequencing the enriched library, and optionally, v) Software for performing statistical analysis A kit including a container.

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