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.