Methods

Laboratory section

A fragment library was collected from total DNA with KAPA HyperPlus kit (Roche, Switzerland) in accordance with the manufacturer's instructions. DNA was fragmented with the Fragmentase in a range of 150-220 bp. The concentration of the libraries was measured after amplification by means of Qubit (ThermoFisher Scientific, USA) in accordance with the manufacturer's instructions. The size of ready-made libraries and possible presence of primer/adapter dimers were determined by Agilent High Sensitivity DNA Kit (Agilent, USA), the optimal length of fragments with adapters was 290-330 bp. Prepared libraries were mixed, and then double hybridized with SeqCap EZ Choice panel probes in accordance with the manufacturer's Protocol (panel size 2,424,836 p. o.). Hybridization was performed at 47 °C for 16 hours. Hybrid complexes were enriched with SeqCap Capture beads, washed from non-specific fragments and amplified with KAPA HiFi HS MasterMix (Roche, Switzerland) for 5 cycles. And after that, the hybridization procedure was repeated as described above. The final amplification of the enriched libraries took 16 cycles.

Bioinformation section

Sequencing data are analyzed in accordance with recommendations of the GATK Best Practices (Broad Institute) to search germline mutations and cDNA mutations. Somatic mutations detected with Mutect2 are evaluated by means of the FilterMutectCalls, FilterAlignmentArtifacts, and FilterByOrientationBias, tools helping to filter false-positive variants.

Each block of calculation is performed in an isolated environment with optimal resource allocation and maximal parallelization of processes. Post-processing of variants is performed using individual quality filters, that significantly improve the sensitivity and specificity of indicators to detect mutations.

The computing pipeline automatically adjusts to the size of input files and distributes the load across the required number of containers, which makes possible to process data of any size - from target panels to exomes and genomes - quickly and efficiently.