Systems and methods for identifying and quantifying gene copy number variations

1. A method of identifying and quantifying copy number variations in a gene of interest from a genomic deoxyribonucleic acid (DNA) sample, the method comprising;

fragmenting a genomic DNA sample having an unknown copy number for regions of a target gene to produce a plurality of polynucleotide fragments;

isolating a plurality of target polynucleotide fragments from the plurality of polynucleotide fragments by using a plurality of breakpoint probes to isolate target polynucleotide fragments comprising one or more breakpoint regions, each of the plurality of target polynucleotide fragments including at least a portion of the target gene, wherein the plurality of breakpoint probes target a chromosome region where one or more breakpoints in structural variation are selected from the group consisting of: duplication, deletion, insertion, translocation, interchange, fusion, and inversion that have been observed in one or more other samples;

sequencing the plurality of target polynucleotide fragments to obtain a plurality of fragment sequences; and

via a computing device: aligning fragment sequences of the plurality of fragment sequences to a reference sequence;

calculating read depths for base positions of the plurality of target polynucleotide fragments relative to each base position of the reference sequence;

calculating copy number likelihoods for each base position of the reference sequence based on the read depths to make a call on at least one copy number variation in the target gene;

performing a breakpoint analysis on a set of fragment sequences of the plurality of fragment sequences to:

identify at least one sequence variation located between selected breakpoint regions of the target gene, wherein the selected breakpoint regions comprise sequence regions that include a breakpoint between adjacent base pairs that result in the at least one sequence variation, and wherein the breakpoint comprises a point in a sequencing read located between a region of the target gene that matches the reference sequence and a region of the target gene that differs from the reference sequence; and

calculate modified copy number likelihoods for base positions of the reference sequence based on the at least one sequence variation, the modified copy number likelihoods each including a modification to a respective copy number likelihood indicating an increase or decrease in evidence for a copy number variation in the target gene at the corresponding base position of the reference sequence; and

correcting, based on the modified copy number likelihoods for the base positions of the reference sequence, the call on the at least one copy number variation in the target gene when the modified copy number likelihoods breach a threshold probability.

2. The method of claim 1 , further comprising:

partitioning each of the plurality of fragment sequences to either the target gene or a homolog of the target gene;

wherein aligning the fragment sequences of the plurality of fragment sequences to the reference sequence comprises aligning fragment sequences partitioned to the target gene to the reference sequence, which is a target gene reference sequence.

3. The method of claim 2 , further comprising aligning additional fragment sequences partitioned to the homolog of the target gene to a homolog gene reference sequence.

4. The method of claim 1 , wherein the set of fragment sequences on which the breakpoint analysis is performed comprises fragment sequences that are at least partially located between the selected breakpoint regions of the target gene.

5. The method of claim 1 , wherein the set of fragment sequences on which the breakpoint analysis is performed comprises fragment sequences that include the selected breakpoint regions of the target gene.

6. The method of claim 5 , wherein the fragment sequences that include the selected breakpoint regions of the target gene include a sequence variation on one side of at least one of the selected breakpoint regions.

7. The method of claim 1 , wherein the plurality of breakpoint probes comprise capture probes that hybridize to a selected breakpoint region observed in at least one other sample.

8. The method of claim 7 , wherein the plurality of capture probes are hybrid capture probes.

9. The method of claim 7 , wherein the set of fragment sequences on which the breakpoint analysis is performed comprises fragment sequences having portions that hybridize to the breakpoint probes.

10. The method of claim 9 , wherein the breakpoint probes hybridize to sequence regions located closest to the selected breakpoint regions with respect to the reference sequence.

11. The method of claim 9 , wherein the breakpoint probes hybridize to sequence regions located between the selected breakpoint regions with respect to the reference sequence.

12. The method of claim 1 , wherein performing the breakpoint analysis on the set of fragment sequences of the plurality of fragment sequences further comprises determining a likelihood that each of a plurality of types of sequence variation are present between the selected breakpoint regions of the target gene.

13. The method of claim 12 , wherein the plurality of types of sequence variation respectively correspond to a plurality of types of copy number variation and no copy number variation.

14. The method of claim 13 , wherein the plurality of types of copy number variation respectively correspond to at least one deletion and at least one duplication of one or more of the regions of the target gene.

15. The method of claim 12 , wherein determining whether the target gene includes at least one copy number variation comprises determining, above a threshold probability, that the at least one sequence variation is present between the selected breakpoint regions of the target gene based on the respective likelihoods calculated for the plurality of types of sequence variation.

16. The method of claim 12 , wherein the plurality of types of sequence variation include a deletion, an insertion, an inversion, a translocation, an interchange, and a fusion.

17. The method of claim 1 , wherein the breakpoint analysis is performed when the copy number likelihoods calculated based on read depths for base positions located between the selected breakpoint regions are below a specified threshold.

18. The method of claim 1 , wherein the reference sequence comprises a sequence from a reference genome.

19. The method of claim 1 , wherein calculating the modified copy number likelihoods for the base positions of the reference sequence comprises calculating normalized read depths for the base positions of the plurality of target polynucleotide fragments relative to each base position of the reference sequence.

20. A system for identifying and quantifying copy number variations in a gene of interest from a genomic DNA sample, the system comprising:

(1) a next generation sequencing device that:

(i) fragments a genomic DNA sample having an unknown copy number for regions of a target gene to produce a plurality of polynucleotide fragments;

(ii) isolates a plurality of target polynucleotide from the plurality of polynucleotide fragments by using a plurality of breakpoint probes to isolate target polynucleotide fragments comprising one or more breakpoint regions, each of the plurality of target polynucleotide fragments including at least a portion of the target gene, wherein the plurality of breakpoint probes target a chromosome region where one or more breakpoints in structural variation are selected from the group consisting of: duplication, deletion, insertion, translocation, interchange, fusion, and inversion that have been observed in one or more other samples; and

(iii) sequences the plurality of target polynucleotide fragments to obtain a plurality of fragment sequences;

(2) an alignment module, stored in memory, that aligns fragment sequences of the plurality of fragment sequences to a reference sequence;

(3) a read depth module, stored in memory, that:

(i) calculates read depths for base positions of the plurality of target polynucleotide fragments relative to each base position of the reference sequence; and

(ii) calculates copy number likelihoods for each base position of the reference sequence based on the read depths to make a call on at least one copy number variation in the target gene;

(4) a breakpoint module, stored in memory, that performs a breakpoint analysis on a set of fragment sequences of the plurality of fragment sequences to:

(i) identify at least one sequence variation located between selected breakpoint regions of the target gene, wherein the selected breakpoint regions comprise sequence regions that include a breakpoint between adjacent base pairs that result in the at least one sequence variation, and wherein the breakpoint comprises a point in a sequencing read located between a region of the target gene that matches the reference sequence and a region of the target gene that differs from the reference sequence; and

(ii) calculate modified copy number likelihoods for base positions of the reference sequence based on the at least one sequence variation, the modified copy number likelihoods each including a modification to a respective copy number likelihood indicating an increase or decrease in evidence for a copy number variation in the target gene at the corresponding base position of the reference sequence;

(5) a copy number module, stored in memory, that corrects, based on the modified copy number likelihoods for the base positions of the reference sequence, the call on the at least one copy number variation in the target gene when the modified copy number likelihoods breach a threshold probability; and

(6) at least one physical processor that executes the alignment module, the read depth module, the breakpoint module, and the copy number module.