Nextseq reagent kit v3

Selected tumor DNA sample for NGS were prepared following the manufacturer’s protocol using TruSight Tumor 170 (Illumina). Samples were sequenced on Illumina NextSeq 550 Sequencing System with NextSeq Reagent kit v3 (Illumina) following the manufacturer’s protocol and recommendations for quality control. Read alignment to the hg19 reference genome and variant calling was performed using TruSight Tumor 170 v2 Local App software (Illumina). Variant annotation was performed using Variant Studio 3.0 software and Alamut Visual v2.14 software. Tumor mutational burden was defined as the number of somatic variants in coding regions, base substitution, and small indels (insertions and deletions less than 20 nucleotides) per megabase (mut/Mb) of gene panel [31 (link),32 (link)].

RNA sequencing was performed using the TruSight RNA Pan-Cancer panel (Illumina, San Diego, CA) which contains 1385 cancer genes and enables fusion calling and variant detection within the panel. Twenty nanograms of RNA was processed according to the manufacturer’s protocol and was sequenced on a Next Seq 550 system (Illumina, San Diego, CA) using the NextSeq^® Reagent Kit v3 (150 cycles) with a PE NextSeq^® Flow Cell. Data analyses were performed using the Illumina BaseSpace apps TopHat Alignment (version 1.0.0, read mapping on hg19 reference genome by TopHat21, fusion calling by TopHat-Fusion2) and RNA-seq Alignment (version 1.1.0, read mapping on hg19 reference genome by STAR3, fusion calling by Manta4 using standard settings) (BaseSpace.illumina.com/apps">https://BaseSpace.illumina.com/apps). Fusion transcripts with a low number of split-reads were excluded as likely false positives. The raw data of the sequence variants were converted to variant call format (vcf) files and analysed in Variant Studio software v.4.0.