1% HCC78 in C1 cfDNA
"Indexed Illumina NGS libraries were prepared from plasma DNA and shorn cell line genomic DNA. Plasma DNA was used for library construction without additional fragmentation. Cell line genomic DNA was sheared prior to library construction with a Covaris S2 instrument using the recommended settings for 200 bp fragments. The NGS libraries were constructed using the KAPA Library Preparation Kit (Kapa Biosystems) employing a DNA Polymerase possessing strong 3'-5' exonuclease (or proofreading) activity and displaying the lowest published error rate (i.e. highest fidelity) of all commercially available B-family DNA polymerases. The manufacturer?s protocol was modified to incorporate with-bead enzymatic and cleanup steps using Agencourt AMPure XP beads (Beckman-Coulter). Ligation was performed for 16 h at 16 ?C using 100-fold molar excess of indexed Illumina TruSeq adapters. Single-step size selection was performed by adding 40 ?L (0.8X) of PEG buffer to enrich for ligated DNA fragments. The ligated fragments were then amplified using 500 nM Illumina backbone oligonucleotides and 4?9 PCR cycles, depending on input DNA mass. Library purity and concentration was assessed by spectrophotometer (NanoDrop 2000) and qPCR (KAPA Biosystems), respectively. Fragment length was determined on a 2100 Bioanalyzer using the DNA 1000 Kit (Agilent). Hybrid selection was performed with a custom SeqCap EZ Choice Library (Roche NimbleGen). NimbleGen SeqCap EZ Choice was used according to the manufacturer?s protocol with modifications. Between 9 and 12 indexed Illumina libraries were included in a single capture hybridization. Following hybrid selection, the captured DNA fragments were amplified with 12 to 14 cycles of PCR using 1X KAPA HiFi Hot Start Ready Mix and 2 ?M Illumina backbone oligonucleotides in 4 to 6 separate 50 ?L reactions. The reactions were then pooled and processed with the QIAquick PCR Purification Kit (Qiagen). Multiplexed libraries were sequenced using 100 bp paired-end runs on an Illumina HiSeq 2000."