Using cell-free dna fragment size to determine copy number variations

A system for evaluation of copy number of a nucleic acid sequence of interest in a test sample, the system comprising:

• a sequencer for receiving nucleic acid fragments from the test sample and providing nucleic acid sequence information of the test sample;
• a processor;
• and
• one or more computer-readable storage media having stored thereon instructions for execution on said processor to evaluate copy number in the test sample using the method comprising:
• (a) receiving sequence reads obtained by sequencing the cell-free nucleic acid fragments in the test sample;
• (b) aligning the sequence reads of the cell-free nucleic acid fragments to a reference genome comprising the sequence of interest, thereby providing test sequence tags, wherein the reference genome is divided into a plurality of bins;
• (c) determining sizes of at least some of the cell-free nucleic acid fragments in the test sample;
• (d) weighting the test sequence tags based on the sizes of cell-free nucleic acid fragments from which the tags are obtained;
• (e) calculating coverages for the bins based on the weighted tags of (d);
• and
• (f) identifying a copy number variation in the sequence of interest from the calculated coverages.

A method for determining a copy number variation of a nucleic acid sequence of interest in a test sample comprising cell-free nucleic acid fragments originating from two or more genomes, the method comprising:

• (a) receiving sequence reads obtained by sequencing the cell-free nucleic acid fragments in the test sample;
• (b) aligning the sequence reads of the cell-free nucleic acid fragments to a reference genome comprising the sequence of interest, thereby providing test sequence tags, wherein the reference genome is divided into a plurality of bins;
• (c) determining sizes of the cell-free nucleic acid fragments existing in the test sample;
• (d) determining, in bins of the reference genome, including bins in the sequence of interest, values of a fragment size parameter biased toward fragment sizes characteristic of one of the genomes;
• (e) providing a global profile for the bins of the sequence of interest, wherein the global profile comprises expected values of the parameter in bins of the sequence of interest, and wherein the expected values of the parameter are obtained from a training set of unaffected training samples comprising nucleic acids sequenced and aligned in substantially the same manner as the nucleic acid fragments of the test sample, the expected values of the parameter exhibiting variation from bin to bin;
• (f) adjusting the values of the parameter of the test sequence tags using the expected values of the parameter in the bins of at least the sequence of interest, thereby obtaining global-profile-corrected values of the parameter for the sequence of interest;
• and
• (g) evaluating a copy number of the sequence of interest in the test sample based on the global-profile-corrected values of the parameter, wherein the global-profile-corrected values of the parameter improve a signal level and/or reduce a noise level for determining the copy number of the sequence of interest.

A method for determining a copy number variation of a nucleic acid sequence of interest in a test sample comprising cell-free nucleic acid fragments originating from two or more genomes, the method comprising:

• (a) receiving sequence reads obtained by sequencing the cell-free nucleic acid fragments in the test sample;
• (b) aligning the sequence reads of the cell-free nucleic acid fragments to a reference genome comprising the sequence of interest, thereby providing test sequence tags, wherein the reference genome is divided into a plurality of bins;
• (c) determining sizes of the cell-free nucleic acid fragments existing in the test sample;
• (d) determining, in bins of the reference genome, including bins in the sequence of interest, a fragment size parameter biased toward fragment sizes characteristic of one of the genomes;
• (e) adjusting the values of the fragment size parameter of the test sequence tags using a relation between GC content levels and the values of the parameter in the test sample, thereby obtaining GC-corrected values of the fragment size parameter for the sequence of interest;
• and
• (f) evaluating a copy number of the sequence of interest in the test sample based on the GC-corrected values of the fragment size parameter, wherein the GC-corrected values of the fragment size parameter improve a signal level and/or reduce a noise level for determining the copy number of the sequence of interest.