Targeted sequencing of 170 cancer-related gene panels was performed using formalin-fixed, paraffin-embedded tissue (FFPE) samples as previously described [13 (link)]. All FFPE materials had a short cold ischemic time not exceeding 2 hours, fixation time ranging from 8 to 72 hours, and were aged between 0 and 9 years.
In brief, approximately 3 µg of genomic DNA was extracted from FFPE tumor tissues, and the sequencing library was prepared using an Agilent SureSelect Target Enrichment Kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s guidelines. High-throughput sequencing was performed using the HiSeq 2500 system (Illumina, San Diego, CA, USA) (Macrogen Inc., Seoul, Korea). After quality control of the FASTQ files, sequencing reads were aligned to the reference genome (GRCh37/hg19) using Burrows-Wheeler Aligner-MEM (BWA-MEM) [14 ]. Single nucleotide variants and small insertions and deletions (INDELs) were detected using the MuTect2 algorithm [15 (link)]. SnpEff and SnpSift v4.3i [16 (link)] with dbNSFP v2.9.3 [17 (link)] were used for variant annotation with various databases including the OncoKB [18 ] and ClinVar archives [19 (link)].
In brief, approximately 3 µg of genomic DNA was extracted from FFPE tumor tissues, and the sequencing library was prepared using an Agilent SureSelect Target Enrichment Kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s guidelines. High-throughput sequencing was performed using the HiSeq 2500 system (Illumina, San Diego, CA, USA) (Macrogen Inc., Seoul, Korea). After quality control of the FASTQ files, sequencing reads were aligned to the reference genome (GRCh37/hg19) using Burrows-Wheeler Aligner-MEM (BWA-MEM) [14 ]. Single nucleotide variants and small insertions and deletions (INDELs) were detected using the MuTect2 algorithm [15 (link)]. SnpEff and SnpSift v4.3i [16 (link)] with dbNSFP v2.9.3 [17 (link)] were used for variant annotation with various databases including the OncoKB [18 ] and ClinVar archives [19 (link)].