# US 2019/0172550 A1 · Methods and systems for analyzing nucleic acid sequences

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- Number: US 2019/0172550 A1
- Document ID: US20190172550A1
- Status: Published application
- Kind: A1
- Application number: 16/206,976
- Filed: 2018-11-30
- Published: 2019-06-06
- Earliest claimed: 2017-11-30
- Assignees: Grail, LLC
- Applicants: GRAIL, Inc.
- Inventors: Srinka GHOSH
- Web page: https://patentgrep.com/patent/US20190172550A1

## CPC

- G16B 20/10: Ploidy or copy number detection
- G16B 20/20: Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
- G16B 30/20: Sequence assembly
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

Methods of identifying changes in genomic DNA copy number are disclosed. This disclosure provides methods for detecting chromosomal aberrations in a subject using Hidden Markov modeling. In some cases, methods provided herein use de novo sequence assembly to detect chromosomal aberrations in a subject. The methods can be used to detect copy number changes in cancerous tissue compared to normal tissue. The methods can be used to diagnose cancer and other diseases associated with chromosomal anomalies.

## Claims

### Claim 1

A method of analyzing nucleic acid molecules from a biological sample of a subject of an organism, the method comprising:
(a) obtaining, in a computer system, sequence reads of the nucleic acid molecules from the biological sample of the subject;
(b) identifying, in the computer system, a first genomic region in a reference genome of the organism with a mappability score below a threshold and a second genomic region in the reference genome of the subject with a mappability score above the threshold, wherein the second genomic region is adjacent to one end of the first genomic region;
(c) performing, in the computer system, local de novo assembly of a first subset of the sequence reads that align to the first genomic region and a second subset of the sequence reads that align to the second genomic region, thereby generating contigs, wherein at least one of the contigs extends over a portion of the first genomic region and a portion of the second genomic region; and
(d) recovering, in the computer system, sequence data corresponding to the contigs within the first genomic region, wherein the recovered genomic sequence data comprise at least a portion of the first subset of sequence read.

### Claim 2 (depends on 1)

The method of claim 1, further comprising identifying a third genomic region in the reference genome having a mappability score above the threshold, wherein the third genomic region is located on the other end of the first genomic region, wherein the contigs are generated by local de novo assembly of the first subset, the second subset, and the third subset of the sequence reads, and wherein at least one of the contigs extends over a portion of the first genomic region and a portion of the third genomic region.

### Claim 3 (depends on 1)

The method of claim 1, wherein the reference genome comprises a plurality of genomic regions, and the method further comprises:
(e) repeating steps (b)-(d) for each of the genomic regions having a mappability score below the threshold, thereby recovering portions of the sequence data from all of the genomic regions having mappability scores below the threshold.

### Claim 4 (depends on 3)

The method of claim 3, further comprising:
combining the recovered sequence data from (e) with sequence data from genomic regions that have mappability scores above the threshold.

### Claim 5 (depends on 4)

The method of claim 4, further comprising:
normalizing the combined sequence data using a covariate of GC content.

### Claim 6

(canceled)

### Claim 7 (depends on 1)

The method of claim 1, wherein the local de novo assembly is performed using an algorithm selected from the group consisting of a greedy algorithm assembler, graph method assembler, string graph assembler, De Bruijn graph assembler, Spades, Ray, ABySS, ALLPATHS-LG, and Trinity.

### Claim 8 (depends on 1)

The method of claim 1, wherein the local de novo assembly comprises creation of a scaffold comprising the contigs, and wherein the recovered sequence data align to the scaffold.

### Claim 9 (depends on 8)

The method of claim 8, wherein the sequence reads comprise paired-end sequence reads, and the scaffold is created by ordering the contigs based on paired-end information of the paired-end sequence reads.

### Claim 10 (depends on 1)

The method of claim 1, wherein the recovered sequence data comprise sequence reads that align uniquely to the contigs within the first genomic region.

### Claim 11

(canceled)

### Claim 12 (depends on 4)

The method of claim 4, further comprising:
segmenting the combined sequence data to identify a chromosomal aberration.

### Claim 13 (depends on 12)

The method of claim 12, wherein the combined sequence data is segmented into a copy number state, the copy number state selected from the group consisting of a copy number loss state, a copy number gain state, and a copy number normal state.

### Claim 14 (depends on 13)

The method of claim 13, wherein the copy number state comprises the copy number normal state, and wherein a variability of the copy number normal state is determined using at least one of a healthy reference sample and a tumor reference sample.

### Claim 15 (depends on 12)

The method of claim 12, wherein the segmenting comprises using hidden Markov model statistical analysis.

### Claim 16

(canceled)

### Claim 17

(canceled)

### Claim 18 (depends on 12)

The method of claim 12, wherein said chromosomal aberration comprises a copy number aberration.

### Claim 19 (depends on 18)

The method of claim 18, wherein the copy number aberration is from a tumor.

### Claim 20 (depends on 12)

The method of claim 12, wherein said chromosomal aberration is identified in the absence of identifying one or more single nucleotide polymorphisms in said nucleic acid molecules.

### Claim 21

(canceled)

### Claim 22

(canceled)

### Claim 23 (depends on 1)

The method of claim 1, wherein the mappability score of the first genomic region and the second genomic region is determined according to the following:
partitioning the first genomic region into a first plurality of partitioned sequences and the second genomic region into a second plurality of partitioned sequences, and determining the mappability score of the first genomic region based, at least in part, on a first percentage of the first plurality of partitioned sequences that uniquely align to the first genomic region, and the mappability score of the second genomic region based, at least in part, on a second percentage of the second plurality of partitioned sequences that uniquely align to the second genomic region.

### Claim 24

(canceled)

### Claim 25 (depends on 1)

The method of claim 1, wherein the nucleic acid molecules comprise cell-free nucleic acid molecules.

### Claim 26

A non-transitory computer-readable medium comprising machine executable code that, upon execution by one or more computer processors, implements a method of analyzing nucleic acid molecules from a biological sample of a subject of an organism, said method comprising:
(a) obtaining sequence reads of the nucleic acid molecules from the biological sample of the subject;
(b) identifying a first genomic region in a reference genome of the organism with a mappability score below a threshold and a second genomic region in the reference genome of the subject with a mappability score above the threshold, wherein the second genomic region is adjacent to one end of the first genomic region;
(c) performing local de novo assembly of a first subset of the sequence reads that align to the first genomic region and a second subset of the sequence reads that align to the second genomic region, thereby generating contigs, wherein at least one of the contigs extends over a portion of the first genomic region and a portion of the second genomic region; and
(d) recovering sequence data corresponding to the contigs within the first genomic region, wherein the recovered genomic sequence data comprise at least a portion of the first subset of sequence read.

### Claim at position 27

-50. (canceled)

### Claim 51

A system for analyzing nucleic acid molecules from a biological sample of a subject of an organism, comprising:
a database that stores a plurality of sequence reads generated upon sequencing said nucleic acid molecules; and
one or more computer processors operatively coupled to said database, wherein said one or more computer processors are individually or collectively programmed to execute general instructions for:
(a) identifying a first genomic region in a reference genome of the organism with a mappability score below a threshold and a second genomic region in the reference genome of the subject with a mappability score above the threshold, wherein the second genomic region is adjacent to one end of the first genomic region;
(b) performing local de novo assembly of a first subset of the sequence reads that align to the first genomic region and a second subset of the sequence reads that align to the second genomic region, thereby generating contigs, wherein at least one of the contigs extends over a portion of the first genomic region and a portion of the second genomic region; and
(c) recovering sequence data corresponding to the contigs within the first genomic region, wherein the recovered genomic sequence data comprise at least a portion of the first subset of sequence read.

### Claim at position 29

-74. (canceled)