Detecting and classifying copy number variation
The invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest. The method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability. The method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions. CNV that can be determined according to the method include trisomies and monosomies of any one or more of chromosomes 1-22, X and Y, other chromosomal polysomies, and deletions and/or duplications of segments of any one or more of the chromosomes, which can be detected by sequencing only once the nucleic acids of a test sample.
1. A method for classifying a copy number variation in a fetal genome, the method comprising:
(a) receiving sequence reads from fetal and maternal nucleic acids in a maternal test sample obtained from a mother carrying a fetus, wherein the sequence reads are provided in an electronic format;
(b) aligning the sequence reads to one or more bins from a reference sequence using a computing apparatus and thereby providing sequence tags corresponding to the sequence reads, wherein each bin contains a subsequence within the reference sequence;
(c) computationally identifying a number of those sequence tags that are from one or more bins by using the computing apparatus and determining that a first bin of interest in the fetal genome harbors a copy number variation;
(d) calculating a first fetal fraction value by a first method that does not use information from sequence tags from the first bin of interest;
(e) calculating a second fetal fraction value by a second method that uses information from the sequence tags from the first bin of interest, wherein the second method includes a ploidy assumption that the fetal genome has a complete aneuploidy within the subsequence of the first bin of interest; and
(f) comparing the first fetal fraction value and the second fetal fraction value and using the comparison to classify the copy number variation of the first bin of interest in the fetal genome as harboring a complete aneuploidy or another type of copy number variation.
2. The method of claim 1 , wherein the comparing the first fetal fraction value and the second fetal fraction value comprises determining whether the two fetal fraction values are approximately equal.
3. The method of claim 2 , further comprising determining that the two fetal fraction values are approximately equal, and thereby determining that the ploidy assumption of the second method is true.
4. The method of claim 2 , wherein comparing the first fetal fraction value and the second fetal fraction value indicates that the two fetal fraction values are not approximately equal, and further comprising analyzing the tag information for the first bin of interest to determine whether (i) the first bin of interest harbors a partial aneuploidy, or (ii) the fetus is a mosaic.
5. The method of claim 4 , wherein analyzing the tag information for the first bin of interest comprises:
dividing the sequence for the first bin of interest into a plurality of sub-bins;
determining whether any of said sub-bins contains significantly more or significantly less nucleic acid than one or more other sub-bins; and
if any of said plurality of sub-bins contain significantly more or significantly less nucleic acid than one or more other sub-bins, determining that the first bin of interest harbors a partial aneuploidy.
6. The method of claim 5 , further comprising determining that a sub-bin of the first bin of interest containing significantly more or significantly less nucleic acid than one or more other sub-bins harbors the partial aneuploidy.
7. The method of claim 4 , wherein analyzing the tag information for the first bin of interest comprises:
dividing the sequence for the first bin of interest into a plurality of sub-bins;
determining whether any of said sub-bins contains significantly more or significantly less nucleic acid than one or more other sub-bins; and
if none of said sub-bins contain significantly more or significantly less nucleic acid than one or more other sub-bins, determining that the fetus is a mosaic.
8. The method of claim 1 , wherein if the first fetal fraction value is not approximately equal to the second fetal fraction value,
(i) determining whether the copy number variation results from a partial aneuploidy or a mosaic; and
(ii) if the copy number variation results from a partial aneuploidy, determining a locus of the partial aneuploidy on the first bin of interest.
9. The method of claim 8 , wherein determining the locus of the partial aneuploidy on the first bin of interest comprises categorizing the sequence tags for the first bin of interest into sub-bins of blocks of nucleic acids in the first bin of interest; and counting sequence tags in each sub-bin.
10. The method of claim 1 , further comprising sequencing cell free DNA from the maternal test sample to provide the sequence reads.
11. The method of claim 10 , further comprising obtaining the maternal test sample from a pregnant organism.
12. The method of claim 1 , wherein the aligning in (b) comprises using the computing apparatus to align at least about 1 million reads.
13. A computer program product comprising a non-transitory computer readable medium on which is provided program instructions for classifying a copy number variation in a fetal genome, the program instructions comprising:
(a) code for receiving sequence reads from fetal and maternal nucleic acids in a maternal test sample obtained from a mother carrying a fetus, wherein the sequence reads are provided in an electronic format;
(b) code for aligning the sequence reads to one or more bins from a reference sequence using a computing apparatus and thereby providing sequence tags corresponding to the sequence reads, wherein each bin contains a subsequence within the reference sequence;
(c) code for computationally identifying a number of those sequence tags that are from one or more bins by using the computing apparatus and determining that a first bin of interest in the fetal genome harbors a copy number variation;
(d) code for calculating a first fetal fraction value by a first method that does not use information from sequence tags from the first bin of interest;
(e) code for calculating a second fetal fraction value by a second method that uses information from the sequence tags from the first bin of interest, wherein the second method includes a ploidy assumption that the fetal genome has a complete aneuploidy within the subsequence of the first bin of interest; and
(f) code for comparing the first fetal fraction value and the second fetal fraction value and using the comparison to classify the copy number variation of the first bin of interest in the fetal genome as harboring a complete aneuploidy or another type of copy number variation.
14. A system for classifying a copy number variation in a fetal genome, the system comprising:
(a) an interface for receiving at least about 10,000 sequence reads from fetal and maternal nucleic acids in a maternal test sample obtained from a mother carrying a fetus, wherein the sequence reads are provided in an electronic format;
(b) memory for storing, at least temporarily, a plurality of said sequence reads;
(c) a processor designed or configured with p(i) aligning the sequence reads to one or more reference sequences and thereby providing sequence tags corresponding to the sequence reads;
(ii) identifying a number of those sequence tags that are from one or more bins of interest and determining that a first bin of interest in the fetal genome harbors a copy number variation, wherein each bin contains a subsequence within the reference sequence;
(iii) calculating a first fetal fraction value by a first method that does not use information from sequence tags from the first bin of interest;
(iv) calculating a second fetal fraction value by a second method that uses information from the sequence tags from the first bin of interest, wherein the second method includes a ploidy assumption that the fetal genome has a complete aneuploidy within the subsequence of the first bin of interest; and
(v) comparing the first fetal fraction value and the second fetal fraction value and using the comparison to classify the copy number variation of the first bin of interest in the fetal genome as harboring a complete aneuploidy or another type of copy number variation.
15. The computer program product of claim 13 , wherein the comparing the first fetal fraction value and the second fetal fraction value comprises determining whether the two fetal fraction values are approximately equal.
16. The computer program product of claim 15 , the program instructions further comprising instructions for determining that the two fetal fraction values are approximately equal, and thereby determining that the ploidy assumption of the second method is true.
17. The computer program product of claim 15 , the program instructions further comprising instructions for determining that the two fetal fraction values are not approximately equal, and analyzing the tag information for the first bin of interest to determine whether (i) the first bin of interest harbors a partial aneuploidy, or (ii) the fetus is a mosaic.
18. The computer program product of claim 17 , wherein analyzing the tag information for the first bin of interest comprises:
dividing the sequence for the first bin of interest into a plurality of sub-bins;
determining whether any of said sub-bins contains significantly more or significantly less nucleic acid than one or more other sub-bins; and
if any of said plurality of sub-bins contain significantly more or significantly less nucleic acid than one or more other sub-bins, determining that the first bin of interest harbors a partial aneuploidy.
19. The computer program product of claim 17 , wherein analyzing the tag information for the first bin of interest comprises:
dividing the sequence for the first bin of interest into a plurality of sub-bins;
determining whether any of said sub-bins contains significantly more or significantly less nucleic acid than one or more other sub-bins; and
if none of said sub-bins contain significantly more or significantly less nucleic acid than one or more other sub-bins, determining that the fetus is a mosaic.
20. The system of claim 14 , wherein the comparing the first fetal fraction value and the second fetal fraction value comprises determining whether the two fetal fraction values are approximately equal.
21. The system of claim 20 , the program instructions further comprising instructions for determining that the two fetal fraction values are approximately equal, and thereby determining that the ploidy assumption of the second method is true.
22. The system of claim 20 , the program instructions further comprising instructions for determining that the two fetal fraction values are not approximately equal, and analyzing the tag information for the first bin of interest to determine whether (i) the first bin of interest harbors a partial aneuploidy, or (ii) the fetus is a mosaic.
23. The system of claim 22 , wherein analyzing the tag information for the first bin of interest comprises:
dividing the sequence for the first bin of interest into a plurality of sub-bins;
determining whether any of said sub-bins contains significantly more or significantly less nucleic acid than one or more other sub-bins; and
if any of said plurality of sub-bins contain significantly more or significantly less nucleic acid than one or more other sub-bins, determining that the first bin of interest harbors a partial aneuploidy.
24. The system of claim 22 , wherein analyzing the tag information for the first bin of interest comprises:
dividing the sequence for the first bin of interest into a plurality of sub-bins;
determining whether any of said sub-bins contains significantly more or significantly less nucleic acid than one or more other sub-bins; and
if none of said sub-bins contain significantly more or significantly less nucleic acid than one or more other sub-bins, determining that the fetus is a mosaic.