Methods for detecting nucleic acid variants

Patent metadata

Field	Value
Publication number	US-12437839-B2
Application number	US-202418617448-A
Country	US
Kind code	B2
Filing date	Mar 26, 2024
Priority date	May 3, 2019
Publication date	Oct 7, 2025
Grant date	Oct 7, 2025

Abstract

Methods for detecting a short genetic variant in a test sample are described herein. In some exemplary methods, the short genetic variant is called using one or match scores, which are determined using one or more sequencing data sets obtained from a test nucleic acid molecule, wherein the test sequencing data sets are determined by sequencing the test nucleic acid molecule using non-terminating nucleotides provided in separate nucleotide flows according to a flow-cycle order. Also described herein are methods of sequencing a test nucleic acid molecule using two or more different flow-cycle orders and/or extended flow cycle orders having five or more nucleotide flows per flow cycle.

First claim

What is claimed is:

1. A method for detecting a disease based on single nucleotide variants identified from sequencing, comprising:
selecting a set of single nucleotide variants (SNV) loci from a disease-associated SNV locus panel, wherein the disease-associated SNV locus panel had been or is generated by sequencing a nucleic acid sample derived from diseased tissue from a subject, and wherein the SNV loci in the selected set of SNV loci are associated with a diseased sequencing data set that differs from a reference sequencing data set associated with a reference sequence across at least one flow cycle when the diseased sequencing data set and the reference sequencing data set are obtained by sequencing using non-terminating nucleotides provided in separate flow positions according to a flow-cycle order;
sequencing a cell-free nucleic acid sample from the subject using non-terminating nucleotides provided in separate flow positions according to a flow-cycle order to obtain a cell-free nucleic acid data set, wherein the mean sequencing depth of the cell-free nucleic acid sequencing data set (D) is less than 10;
determining a fraction value (F) by processing a total number of SNV reads detected at the set of SNV loci in the cell-free nucleic acid sequencing data set (N total ), a number of loci selected in the set of SNV loci (N var ), a mean sequencing depth of the cell-free nucleic acid sequencing data set (D), and a sequencing false positive error rate (E), wherein the fraction value is determined as:

F
=

N
total

N
var

⁢
D

-
E

;

and
calling a presence, absence, progression, or regression of the disease in the subject based on the fraction value or a degree of change in the fraction value from a prior fraction value determined for the subject.

2. The method of claim 1 , further comprising generating the disease-associated SNV locus panel by (i) sequencing the nucleic acid sample derived from the diseased tissue using non-terminating nucleotides provided in separate flow positions according to the flow-cycle order to generate the diseased sequencing data set and determine a first set of SNV loci and (ii) filtering the first set of SNV loci to remove germline variants and non-disease related somatic variants.

3. The method of claim 2 , wherein the germline variants or the non-disease related somatic variants, or both, are determined by sequencing a second nucleic acid sample derived from non-diseased tissue from the subject.

4. The method of claim 2 , further comprising one or more of:
filtering the first set of SNV loci to remove SNV loci supported by only one sequencing read;
filtering the first set of SNV loci to remove SNV loci not supported by complementary sequencing reads; and
filtering the first set of SNV loci to remove SNV loci present in a general population of individuals at an allele frequency greater than a predetermined threshold.

5. The method of claim 2 , wherein generating the disease-associated SNV locus panel further comprises filtering the first set of SNV loci to include only those SNV loci that correspond to differences between the diseased sequencing data set and a reference sequencing data set across at least one flow cycle, wherein the diseased sequencing data set and the reference sequencing data set are generated by sequencing the nucleic acid sample derived from the diseased tissue and a reference sample, respectively, using non-terminating nucleotides provided in separate flow positions according to the flow-cycle order.

6. The method of claim 1 , wherein the cell-free nucleic acid sample from the subject is a fluidic sample.

7. The method of claim 1 , wherein the disease is cancer.

8. The method of claim 1 , further comprising re-sequencing the cell-free nucleic acid sample according to a different flow-cycle order, wherein the different flow-cycle order results in another cell-free nucleic acid sequencing data set with a different sequencing false positive error rate at a subset of the set of SNV loci.

9. The method of claim 1 , wherein the sequencing is untargeted sequencing.

10. The method of claim 9 , wherein the cell-free nucleic acid sequencing data set is obtained from untargeted whole genome sequencing.

11. The method of claim 1 , wherein the disease-associated SNV locus panel comprises single nucleotide polymorphism (SNP) loci, indel loci, or both.

12. The method of claim 1 , wherein the set of SNV loci comprises about 300 or more loci.

13. The method of claim 1 , wherein the set of SNV loci are selected based on a false positive rate of each individual loci.

14. The method of claim 1 , wherein the cell-free nucleic acid sequencing data set is obtained using surface-based sequencing, and wherein the cell-free nucleic acid sample is not amplified.

15. The method of claim 1 , wherein the cell-free nucleic acid sequencing data set is obtained without the use of unique molecular identifiers (UMIs) or without the use of sample identification barcodes or both.

16. The method of claim 1 , wherein the sequencing false positive error rate is measured using a panel of control loci.

17. The method of claim 1 , further comprising generating a report that indicates the presence, absence, or level of disease in the subject.

Assignees

• Ultima Genomics Inc

Inventors

• Almogy Gilad
• Pratt Mark
• Barad Omer
• Faigler Simchon
• Oberstrass Florian

Classifications

• G06F17/00 Primary

  Digital computing or data processing equipment or methods, specially adapted for specific functions (information retrieval, database structures or file system structures therefor G06F16/00) · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title