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 cell-free 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:
• (a) receive sequence reads obtained by sequencing the cell-free nucleic acid fragments in the test sample;
• (b) align the sequence reads of the cell-free nucleic acid fragments or aligning fragments containing the sequence reads to bins of 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) determine fragment sizes of at least some of the cell-free nucleic acid fragments present in the test sample;
• (d) for cell-free nucleic acid fragments determined as being in a first size domain, calculate first coverages of the sequence tags for the bins of the reference genome by, for each bin:
• (i) determining a number of sequence tags aligning to the bin, and
• (ii) normalizing the number of sequence tags aligning to the bin by accounting for bin-to-bin variations due to factors other than copy number variation;
• (e) for cell-free nucleic acid fragments determined as being in a second size domain, calculate second coverages of the sequence tags for the bins of the reference genome by, for each bin:
• (i) determining a number of sequence tags aligning to the bin, and
• (ii) normalizing the number of sequence tags aligning to the bin by accounting for bin-to-bin variations due to factors other than copy number variation;
• and
• (f) determine a copy number variation in the sequence of interest using a likelihood ratio calculated from the first coverages and the second coverages.

A method for determining a copy number variation (CNV) 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 or aligning fragments containing the sequence reads to bins of 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 fragment sizes of the cell-free nucleic acid fragments existing in the test sample;
• (d) calculating coverages of the sequence tags for the bins of the reference genome using sequence tags for the cell-free nucleic acid fragments having sizes in a first size domain;
• (e) calculating coverages of the sequence tags for the bins of the reference genome using sequence tags for the cell-free nucleic acid fragments having sizes in a second size domain, wherein the second size domain is different from the first size domain;
• (f) calculating size characteristics for the bins of the reference genome using the fragment sizes determined in (c);
• and
• (g) determining a copy number variation in the sequence of interest using the coverages calculated in (d) and (e) and the size characteristics calculated in (f).