Next-Generation sequencing
The amount of amplifiable DNA (ng) was calculated according to the Archer PreSeq DNA Calculator Assay Protocol (Archer DX, Boulder, CO, USA). After fragmentation of the genomic DNA, libraries were created by the Archer VariantPlex Solid Tumor Kit (Archer DX, Boulder, CO, USA). The KAPA Universal Library Quantification Kit (Kapa Biosystems, Roche, Basel, Switzerland) was used for the final quantification of the libraries.
The MiSeq System (MiSeq Reagent kit v3 600 cycles, Illumina, San Diego, CA, USA) was used for sequencing. The libraries (final concentration of 4 nM, pooled by equal molarity) were denatured by adding 0.2 nM NaOH and diluted to 40 pM with hybridization buffer from Illumina (San Diego, CA, USA). The final loading concentration was 8 pM libraries and 1% PhiX. Sequencing was conducted according to the MiSeq instruction manual. Captured libraries were sequenced in a multiplexed fashion with paired end run to obtain 2x150 bp reads with at least 250X depth of coverage. The trimmed fastq files were generated using MiSeq reporter (Illumina, San Diego, CA, USA).
Raw sequence data were analysed with Archer analysis software (version 6.2.; Archer DX, Boulder, CO, USA) for the presence of single-nucleotide variants (SNVs) as well as insertions and deletions (indels). For the alignment, the human reference genome GRCh37 (equivalent UCSC version hg19) was built. Molecular barcode (MBC) adapters were used to count unique molecules and characterized sequencer noise, revealing mutations below standard NGS-based detection thresholds. The sequence quality for each sample was assessed and the cutoff was set to 5% variant allele fraction. Large insertion/deletion (>50 bp) and complex structural changes could not be captured by the method. The results were described using the latest version of Human Genome Variation Society nomenclature for either the nucleotide or protein level. Individual gene variants were cross-checked in the COSMIC (Catalogue of Somatic Mutations in Cancer) and ClinVar databases for clinical relevance.We used gnomAD v.2.1.1 population database to compare the significance of each gene alterations which is included in our Archer NGS analysis system.