Libraries for sWGS were prepared from 100ng DNA using modified TruSeq
Nano DNA LT Sample Prep Kit (Illumina) protocol41 (link). Quality and quantity of the libraries were assessed with
DNA-7500 kit on 2100 Bioanalyzer (Agilent Technologies) and with Kapa Library
Quantification kit (Kapa Biosystems) according to the manufacturer's
protocols. Sixteen to twenty barcoded libraries were pooled together in
equimolar amounts and each pool was sequenced on HiSeq4000 in SE-50bp mode.
Prior to sequencing we estimated the required sequencing depth by
adapting calculations made in previous work that explored the relationship
between sequencing depth (reads per sample) and copy number calling
accuracy42 (link). Based on these analyses,
we devised a power calculator for sWGS copy number analysis (seeURL 1 , described in 43 (link)). We estimated that with an average ploidy of 3 and
purity of 0.65, a sequencing depth of at least 2.7 million reads is required to
detect single, clonal copy-number changes (minimum 60kb) at 90% power and alpha
0.05. After analysis we determined that BritROC 3-star samples had an average
purity of 0.66, ploidy of 2.7, and were sequenced to an average depth of 8.6
million reads. This allowed us to detect single copy-number changes with 90%
power, and alpha 0.05 down to subclonal frequencies of 55%.
Nano DNA LT Sample Prep Kit (Illumina) protocol41 (link). Quality and quantity of the libraries were assessed with
DNA-7500 kit on 2100 Bioanalyzer (Agilent Technologies) and with Kapa Library
Quantification kit (Kapa Biosystems) according to the manufacturer's
protocols. Sixteen to twenty barcoded libraries were pooled together in
equimolar amounts and each pool was sequenced on HiSeq4000 in SE-50bp mode.
Prior to sequencing we estimated the required sequencing depth by
adapting calculations made in previous work that explored the relationship
between sequencing depth (reads per sample) and copy number calling
accuracy42 (link). Based on these analyses,
we devised a power calculator for sWGS copy number analysis (see
purity of 0.65, a sequencing depth of at least 2.7 million reads is required to
detect single, clonal copy-number changes (minimum 60kb) at 90% power and alpha
0.05. After analysis we determined that BritROC 3-star samples had an average
purity of 0.66, ploidy of 2.7, and were sequenced to an average depth of 8.6
million reads. This allowed us to detect single copy-number changes with 90%
power, and alpha 0.05 down to subclonal frequencies of 55%.
