Non-invasive in vitro diagnostic methods

Abstract

The present invention comprises 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) the nucleic acid molecules of the first group of putatively useful sequence variants. The present invention relates to an in vitro method for diagnosing, prognosing, controlling and predicting therapeutic responses to specific human diseases, comprising the steps of (i) to (iv) hybridizing one or more TAC oligonucleotides from a second, smaller TAC oligonucleotide pool containing a TAC oligonucleotide specific to (TAC oligonucleotide-2) to 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, comparing the determined sequences with human reference sequences, thereby creating a second group of putatively beneficial sequence variants (PISV2); and (vii) analyzing the putatively beneficial sequence variants (PISV2), thereby diagnosing, prognosing, and controlling drug responses to specific human disease conditions. Steps (i) to (iv) are collectively referred to as Step A. Steps (v) to (vii) are collectively referred to as Step B.

Claims

1. below: (i) A step of using 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 process of analyzing the presumably beneficial sequence variant (PISV2) and thereby diagnosing, prognosing, and controlling the response to drugs for a specific human disease state. In vitro methods for the diagnosis, prognosis, treatment response control, and response prediction of specific human diseases, including

2. The method according to claim 1, wherein the disease is selected from the group including proliferative disorders such as cancer, autoimmune diseases, neurological disorders, hematopoietic disorders, and neurodevelopmental disorders.

3. The method according to claim 1 or 2, wherein the disease is cancer, and the cancer is selected from the group including carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, melanoma, and brain tumors and spinal cord tumors.

4. The method according to any one of claims 1 to 3, wherein the disease is minimal residual disease (MRD).

5. The method according to any one of claims 1 to 4, wherein the second TAC oligonucleotide pool (TAC oligonucleotide 2) is a subset of the first TAC oligonucleotide pool.

6. The method according to any one of claims 1 to 5, wherein each sequence within the first group of estimated beneficial sequence variants (PISV1) is ranked according to its informational value based on likelihood statistics.

7. The method according to claim 6, wherein the likelihood statistic for a single nucleotide variant represents the probability that the single nucleotide variant is a true somatic single nucleotide variant, and the likelihood statistic is calculated using a regression model, the regression model comprising one or more of the following steps. (i) Average mapping quality of aligned cfDNA nucleic acid molecules, (ii) The ratio of high-quality aligned cfDNA nucleic acid molecules supporting variant nucleotides to low-quality aligned cfDNA nucleic acid molecules supporting variant nucleotides. (iii) The average distance from the endpoints of one or more aligned cfDNA nucleic acid molecules to the position of the single nucleotide variant, (iv) The Levenshtein distance of the sequence of one or more aligned cfDNA nucleic acid molecules from the reference genome sequence, (v) Frequency of the position of one or more single nucleotide variants in a cohort of normal reference samples, (vi) When a nearby single nucleotide polymorphism is present, the ratio of a sequenced cfDNA nucleic acid molecule supporting a phased single nucleotide variant to the single nucleotide polymorphism, such that the single nucleotide variant and the single nucleotide polymorphism are separated by a maximum of 110 bp. (vii) Frequency of single nucleotide variants in disease-specific databases, (viiii) The predicted functional impact of the single-nucleotide variant.

8. The method according to any one of claims 1 to 7, wherein the buffy coat of the sample is treated according to the process described in step B, and thereafter, in order to determine the presence of cell-free tumor-derived DNA nucleic acid molecules, somatic single nucleotide variant candidates detected in both step A and the buffy coat are removed from the classification model.

9. The method according to any one of claims 1 to 8, 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' and a 3'.

10. The method according to any one of claims 1 to 9, wherein the GC content of the pool of TAC oligonucleotides is 19% to 80%, determined by calculating the GC content of each member in the pool of TAC oligonucleotides.

11. The method according to any one of claims 1 to 10, wherein the pool of TAC oligonucleotides comprises a plurality of TAC oligonucleotide families, each directed toward a different target region, each TAC oligonucleotide family comprises a plurality of member sequences, 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 further, the start and / or end positions of the member sequences within the TAC oligonucleotide family are shifted by 5 to 10 base pairs with respect to the reference coordinate system of the target region.

12. The method according to any one of claims 1 to 11, wherein the second TAC oligonucleotide pool (TAC oligonucleotide-2) includes a defined fraction of the first TAC oligonucleotide pool (TAC oligonucleotide-1) and constitutes 0.1%, 0.25%, 0.5%, 0.75%, 1.0%, or 5% 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).

13. The method according to any one of claims 1 to 12, 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.

14. The method according to any one of claims 1 to 13, 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.

15. The method according to any one of claims 1 to 14, 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 and the serum sample.

16. 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, or a tissue sample, or a fecal sample.

17. The method according to claim 1, for use in detecting donor-derived cell-free DNA.

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

19. The method according to any one of claims 1 to 18, 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.

20. The method according to any one of claims 1 to 19, wherein the TAC oligonucleotide includes biotin modification.

21. 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 enriching the sequencing library, v) One or more components for amplifying and / or sequencing the enriched library, vi) Software for performing statistical analysis A kit including a container.

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