Kapa library amplification kit

Exome sequencing was done on two platforms, HiSeq 1500 and NextSeq 550 (Illumina, San Diego, CA, USA).
For the NextSeq 550 platform, exome libraries were prepared with the TruSeq DNA Library Preparation Kit (Illumina, San Diego, CA, USA) and the xGen Exome Research Panel (IDT, Integrated DNA Technologies, Coralville, IA, USA) according to the IDT-Illumina TruSeq DNA Exome protocol (Illumina, San Diego, CA, USA). Sequencing was done using NextSeq 550 (Illumina, San Diego, CA, USA) with paired-end sequencing (150 bp) (11 (link), 12 (link)).
For the HiSeq 1500 platform, exome libraries were prepared using the Kapa Library Amplification Kit (Roche, Basel, Switzerland) and NimbleGen SeqCap EZ Exome v3.0 (Roche, Basel, Switzerland). Sequencing was performed on HiSeq 1500 (Illumina, San Diego, CA, USA) with paired-end sequencing (250 bp).

Template DNA underwent end-repair and A-tailing followed by ligation of adapters (5 μL at 15 μM concentration added to each reaction) using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, Ipswich, MA) following the manufacturer’s instructions. For comparison studies shown in S3c Fig, duplicate ccfDNA libraries were prepared using the Kapa Hyper Prep Kit (Roche, Indianapolis, IN). Following the ligation reaction, gBlock DNA and lambda DNA samples underwent SPRI bead cleanup (Agencourt AMPure XP, Beckman Coulter, Indianapolis, IN) using a 2X SPRI ratio and elution volume of 25 μL IDTE. WBC DNA and ccfDNA underwent SPRI bead cleanup using a 1X SPRI ratio and elution volume of 20 μL IDTE followed by 10 cycles of PCR amplification for ccfDNA using the KAPA Library Amplification Kit (Roche, Indianapolis, IN) and following the manufacturer’s instructions. Samples with singleton and duplex adapters were amplified using 5 μL of 20 μM P5/P7 primers and 20 μM indexing primers, respectively. Singleton adapters, duplex adapters, and the associated primers were obtained from IDT.

All subsequent methods associated with tumor DNA and ccfDNA were performed in separate replicate procedures. 100 ng of WBC DNA and 100 ng of tumor DNA were used as library input. WBC DNA and tumor DNA were sheared using a focused ultrasonicator (S220, Covaris, Woburn, MA) with a targeted size of 175 bp. Two mL plasma equivalents of ccfDNA from pancreatic tumor patients was targeted for library input. Because of a low initial plasma volume and insufficient quantity of ccfDNA, less was used for the second replicate in one patient (Table S1). In some of the post-operative samples, the yield was high and an upper limit of 150 ng for library input was applied (Table S1). To provide a sufficiently complex sample from the four healthy controls, 20 ng of ccfDNA was used as library input. The Kapa HyperPrep Kit (Roche, Indianapolis, IN) and Kapa Library Amplification Kit (Roche) were used for end-repair, A-tailing, adapter ligation, and PCR amplification. Adapters consisted of a single index and single 8-mer UMI (Integrated DNA Technologies [IDT], Coralville, IA). Libraries underwent panel capture enrichment using a custom-designed capture probe set (118 genes, 123 kb; IDT) [14] followed by paired-end sequencing (125 × 2 bp) on a HiSeq 2500 (Illumina, San Diego, CA). The number of paired fastq reads for ccfDNA are detailed in Table S1.