Sequencing libraries were prepared with a custom Ion AmpliSeq Breast Cancer Panel targeting 14 known breast cancer driver and focal mutations (table S3 ) using the Ion AmpliSeq Library Preparation protocol with 3 ng of cfDNA, according to manufacturer's instructions. After barcoding, libraries were quantified using qPCR, diluted to 100 pM, and pooled. Libraries were templated with the Ion OneTouch2 system (Life Technologies), and sequenced on a PI chip using the Ion PI OT2 200 Kit (Life Technologies), 520 flows, and an average amplicon length of 97 bases to a mean depth of x9183. The sequencing resulted in 1042543-5763164 reads per sample.
Ion torrent Variant caller v4.0-r73742 with no Hotspot region and configuration “Germ Line Low Stringency” was used for calling variants. Read counts for all positions were computed using pileup (samtools v1.1(21 (link))) and this data was analysed for possible variants using custom perl and R scripts. Variants at > 3% reported by both analysis methods and not reported in 1000 Genomes Project database (www.1000genomes.org ) were identified as possible somatic mutations. The data was cross referenced against the Cosmic database v70 (cancer.sanger.ac.uk) to identify possible hotspot mutations Variants not appearing in the 1000 genomes database were taken forward for development of digital PCR assays.
Ion torrent Variant caller v4.0-r73742 with no Hotspot region and configuration “Germ Line Low Stringency” was used for calling variants. Read counts for all positions were computed using pileup (samtools v1.1(21 (link))) and this data was analysed for possible variants using custom perl and R scripts. Variants at > 3% reported by both analysis methods and not reported in 1000 Genomes Project database (
