Sureselect v6 kit

Whole-exome of the DNA samples was captured and enriched strictly deferring to the protocol of commercially available Agilent SureSelect v6 Kit on Agilent Liquid Chip Capture System. Specifically, prepped DNA fragments were hybridized with biotinylated RNA library “baits”, and then mixed with streptavidin coated magnetic beads. Beads-conjunct fractions were selectively captured by magnet and put to washing and digestion of RNA “baits” subsequently. The fractions obtained were then amplified by PCR for the following sequencing. Captured libraries were firstly testified for its quality including insert size and effective concentration by Agilent 2100 and Q-PCR respectively. Qualified libraries were sequenced on illumina Novaseq 6000 sequencing platform with reads length of 150bp. Raw image data were converted into raw sequence reads through Base Calling analysis.

We extracted DNA and RNA from formalin-fixed, paraffin-embedded tissues. After hematoxylin and eosin (H&E)-stained slide review and tumor tissue selection, we manually microdissected the corresponding tissue into 20 unstained, 5-μm-thick tissue sections (10 slides for DNA sequence and 10 for RNA). We purified DNA from the sample using a QIAmp FFPE DNA kit (Qiagen, Germany). For whole-exome sequencing, a minimum of 100 ng of DNA was used for the sequence library. DNA quality was determined by Caliper BioAnalyzer 2100 Instrument (Agilent Technologies, Santa Clara, CA, USA) and was confirmed by real-time PCR before sequencing. DNA samples were submitted to WuXi NextCode for next-generation sequencing using the low-input Agilent SureSelect V6 Kit. Paired-end sequencing (2 × 150 bp reads) was performed on successful DNA libraries using an Illumina HiSeq X-Ten after whole-exome capture. For RNA sequencing, we purified RNA with RNeasy FFPE kit from FFPE slides. RNA quality was determined with DV₂₀₀ value (DV₂₀₀ > 30%) by Caliper BioAnalyzer 2100 Instrument. RNA samples were also submitted to WuXi NextCode for next-generation sequencing with TruSeq RNA Exome. Paired-end sequencing (2 × 150 bp reads) was performed on successful RNA libraries using the Illumina HiSeq X-Ten as mentioned above.

For whole‐exome sequencing profiling, DNA was extracted from tumor biopsies and peripheral blood mononuclear cells (PBMC) using AllPrep DNA/RNA and protein kit (QIAGEN) following the manufacturer’s instructions. DNA was quantified with Qubit 4 fluorimeter, and the quality was verified by gel electrophoresis. The library preparation was performed by Biodiversa (http://www.biodiversa.it/) using the Agilent SureSelect V6 kit. Paired‐end sequencing (2× 150 bp) was carried out on Illumina HiSeq 4000.