Systems and methods to detect rare mutations and copy number variation

A method for detecting a rare mutation in a cell-free or substantially cell-free sample obtained from a subject, comprising:

• a) sequencing extracellular polynucleotides from a bodily sample from the subject, wherein each of the extracellular polynucleotides generates a plurality of sequence reads;
• b) filtering out reads that fail to meet a set accuracy, quality score, or mapping score threshold;
• c) mapping sequence reads derived from the sequencing onto a reference sequence;
• d) determining unique sequence reads corresponding to the extracellular polynucleotides from among the sequence reads;
• e) identifying a subset of mapped unique sequence reads that include a variant as compared to the reference sequence at each mappable base position;
• f) for each mappable base position, calculating a ratio of (a) a number of mapped unique sequence reads that include a variant as compared to the reference sequence, to (b) a number of total unique sequence reads for each mappable base position;
• and
• g) processing the ratio with a similarly derived number from a reference sample.