DNA and RNA sequencing
tumor, primary tumors
TUMOR, PRIMARY TUMORS
DNA and RNA material was isolated from FFPE sections of primary tumors or FF tissue sections of biopsies of metastatic lesions containing at least 30% tumor cells. DNA and RNA isolation was done simultaneously using the Qiagen AllPrep DNA/RNA FF kit for FF tissue and the Qiagen AllPrep DNA/RNA FFPE kit for FFPE blocks, according to manufacturer’s instructions. Germline DNA was isolated from peripheral blood using the QIAsymphony DSP DNA midi kit. The total amount of DNA was quantified on a Nanodrop 2000 (Thermo Fisher). The amount of double-stranded DNA (dsDNA) in the genomic DNA samples was quantified using an Invitrogen Qubit dsDNA high-sensitivity assay kit. A maximum of 2,000 ng of dsDNA was fragmented by Covaris shearing. Samples were purified using 2× Agencourt AMPure XP PCR purification beads according to the manufacturer’s instructions. The sheared DNA samples were quantified and qualified on a BioAnalyzer system using the Agilent Technologies DNA7500 assay kit. With an input of a maximum of 1 μg of sheared DNA, library preparation for Illumina sequencing was performed using a KAPA HTP prep kit for FF DNA (KAPA Biosystems, KK8234) or a KAPA Hyper prep kit (KAPA Biosystems, KK8504) for FFPE DNA. Libraries were amplified with four (FF) or six (FFPE) PCR cycles and cleaned with 1× AMPure XP beads. Concentrations were measured with DNA7500 chips on a BioAnalyzer system. Six pools of six to seven samples were created using 500 ng of each indexed sample of FF DNA. Two pools of six to seven samples were created using 65 ng of each indexed sample of FFPE DNA. Two microliters of IDT TS-mix universal blockers and 5 μl of Invitrogen human Cot-1 DNA were added to each pool. Each pool was dried with a concentrator (Eppendorf). To each dried pool, 8.5 μl of hybridization buffer, 3.4 μl of hybridization component A (SeqCap hybridization and wash kit, Roche) and 1.1 μl of nuclease-free water were added to rehydrate the pool. Each pool was incubated at room temperature for 10 min, followed by an incubation at 96 °C for 10 min. Samples were hybridized with the IDT xGen exome research panel v1.0. The pool was captured and washed following the IDT protocol and amplified using 10 PCR cycles. The amplified pool was purified using AMPure XP beads (Beckman Coulter). The purified pools were quantified on an Agilent Bioanalyzer 7500 system, and one sequence pool was made by equimolar pooling. The sequence pool was diluted to a final concentration of 10 nM and subjected to sequencing on an Illumina NovaSeq 6000 machine with an SP2 300 cycle kit for a paired-end, 150-base pair run for FF samples and with an SP 200 cycle kit for a paired-end, 100-base pair run for FFPE samples, according to manufacturer’s instructions.Quality and quantity of the total RNA were assessed using the 2100 Bioanalyzer and a Nano chip (Agilent). The percentage of RNA fragments with >200-nucleotide fragment distribution values (DV200) were determined using the region analysis method according to the manufacturer’s instructions (Illumina, technical-note-470-2014-001). Strand-specific libraries were generated using the TruSeq RNA exome library prep kit (Illumina) according to the manufacturer’s instructions (Illumina, 1000000039582v01). Briefly, total RNA was fragmented (only for FF material), random primed and reverse transcribed using SuperScript II reverse transcriptase (Invitrogen, 18064-014) with the addition of actinomycin D. Second-strand synthesis was performed using polymerase I and RNaseH with the replacement of dTTP for dUTP. The generated cDNA fragments were 3′-end adenylated and ligated to Illumina paired-end sequencing adapters and subsequently amplified by 15 cycles of PCR. The libraries were validated on a 2100 Bioanalyzer using a 7500 chip (Agilent) followed by a 1–4 plex library pooling containing up to 200 ng of each sample. The pooled libraries were enriched for target regions using the probe Coding Exome Oligos set (CEX, 45MB) according to the manufacturer’s instructions (Illumina, 1000000039582v01). Briefly, cDNA libraries and biotin-labeled capture probes were combined and hybridized using a denaturation step of 95 °C for 10 min and an incubation step from 94 °C to 58 °C with a ramp of 18 cycles, a 1-min incubation and 2 °C per cycle. The hybridized target regions were captured using streptavidin magnetic beads and subjected to two stringency washes, an elution step and a second round of enrichment followed by a cleanup using AMPure XP beads (Beckman, A63881) and PCR amplification of 10 cycles. The target enriched pools were analyzed on a 2100 Bioanalyzer using a 7500 chip (Agilent), diluted and subsequently pooled equimolar into a multiplex sequencing pool. The libraries were sequenced with 54 paired-end reads on a NovaSeq 6000 using an SP reagent kit v1.5 (100 cycles; Illumina).