Non-invasive assessment of chromosome alterations using change in subsequence mappability

1. A method for determining the presence or absence of one or more chromosome alterations in a test sample of nucleic acids, which method comprises:

(a) obtaining sequence reads from high-throughput sequencing of the test sample of nucleic acids and a reference sample of nucleic acids;

(b) generating a plurality of sequence read subsequences for each obtained sequence read for the test sample and a plurality of sequence read subsequences from each read for the reference sample,

(c) aligning each of the sequence read subsequences to a reference genome to determine the mappability of the sequence read subsequence using a computer processor,

(d) identifying a subset of the sequence reads for which there is a change in the mappability of one or more of the subsequences of each identified sequence read for the test sample and for the reference sample;

(e) comparing (i) the number of each of the sequence reads in the subset identified in (d) from the test sample, to (ii) the number of each of the sequence reads in the subset identified in (d) from the reference sample, thereby generating a comparison; and

(f) determining the presence or absence of one or more chromosome alterations for the sample according to the comparison in (e).

2. The method of claim 1 , wherein the sequence reads have been mapped to a reference genome or a portion thereof, and wherein the method comprises, prior to (b), identifying a subset of sequence reads for which all bases do not align with the reference genome or portion thereof, and performing (b), (c), (d), (e), and (f) for the subset of sequence reads.

3. The method of claim 1 , wherein the sequence reads are single-end sequence reads, the sequence reads are discordant reads, and the change in mappability is determined for the discordant reads.

4. The method of claim 1 , wherein the sequence reads are paired-end sequence reads and the sequence reads are discordant read pairs.

5. The method of claim 1 , wherein the method comprises identifying discordant read pairs thereby providing discordant read mates, and wherein the change in mappability in (d) is determined for the discordant read mates.

6. The method of claim 1 , comprising identifying a candidate breakpoint for each sequence read in the subset prior to the comparing in (e), wherein the candidate breakpoint for each sequence read is identified according to the change in the mappability.

7. The method of claim 6 , wherein the comparing in (e) comprises comparing (i) the number of each of the sequence reads in the subset identified in (d) from the sample associated with the candidate breakpoint, to (ii) the number of each of the sequence reads in the subset identified in (d) from the reference sample associated with the candidate breakpoint.

8. The method of claim 1 , wherein the identifying in (d) comprises generating a fitted relationship between the mappability and the length of each of the sequence read subsequences of each of the reads, and wherein the change in mappability of the sequence reads subsequence is determined from a slope in the relationship.

9. The method of claim 1 , wherein the comparison in (e) is determined according to a z-score between the number of sequence reads in (e)(i) and the number of sequence reads in (e)(ii).

10. The method of claim 1 , wherein determining the presence of the one or more chromosome alterations in (f) comprises identifying a substantially greater number of sequence reads from the test sample compared to the reference sample in the comparison of (e).

11. The method of claim 1 , wherein a breakpoint is identified according to the comparison in (e).

12. The method of claim 1 , wherein a first breakpoint and a second breakpoint are identified according to the comparison in (e), and wherein the presence of a chromosome alteration is identified in (f) according to the first and the second breakpoints.

13. The method of claim 1 , comprising filtering the sequence reads.

14. The method of claim 13 , wherein the filtering is chosen from one or more of (i) removing low quality reads, (ii) removing concordant reads, (iii) removing PCR duplicated reads, (iv) removing reads mapped to mitochondrial DNA, (v) removing reads mapped to repetitive elements, (vi) removing unmappable reads, (vi) removing reads comprising stepwise multiple alignments, and (vii) removing reads mapped to a centromere.

15. The method of claim 13 , wherein the filtering comprises removing one or more singleton events.

16. The method of claim 13 , wherein the filtering comprises removing the subset of reads identified in (d) in instances where the number of each of the sequence reads in the subset from the sample are substantially similar to the number of each of the sequence reads in the subset from the reference.

17. The method of claim 5 , comprising filtering reads, wherein the filtering comprises removing one or both of the discordant read mates provided from each identified read pair.

18. The method of claim 1 , wherein the sequence reads are of circulating, cellfree nucleic acids.

19. The method of claim 18 , wherein the circulating, cell-free nucleic acids are from serum or plasma.

20. The method of claim 1 , wherein the one or more chromosome alterations comprise one or more of a chromosome translocation, a balanced chromosome translocation, a chromosome deletion, a chromosome inversion, and a heterologous insertion.