DNA isolation: cancer genomic DNA was extracted from formalin-fixed paraffin-embedded tissue using the MagCore® Genomic DNA FFPE One-Step Kit (RBC Bioscience, Taiwan, China). The quality was quantified using DeNovix DS-11 Spectrophotometer (DeNovix, Wilmington, DE, USA) and QuantiFluo® ONE dsDNA System (Promega, WI, USA).
The assay generated a library of 207 gene-specific amplicons and targeted ~2800 clinically relevant mutations.
Sequencing: the products were analyzed by next-generation sequencing (NGS) using the Illumina platform, MiSeq Dx.
Data analysis: an analysis of the NGS data was performed using the GALAXY platform (usegalaxy.org). Sequencing reads (FASTQ files) were aligned to the human reference genome hg19 using the Bowtie2 tool. Variant calling was performed using the Varscan2 tool. Parameters used for the analysis were minimum allele frequency—0.05, minimum quality—20, and minimum coverage ×80. All variants were annotated with ANNOVAR (Available online:https://wannovar.wglab.org , accessed on 2 May 2022). The results were visualized using the R Bioconductor package Maftools (Available online: http://bioconductor.org/ , accessed on 2 May 2022) [19 (link)].
The assay generated a library of 207 gene-specific amplicons and targeted ~2800 clinically relevant mutations.
Sequencing: the products were analyzed by next-generation sequencing (NGS) using the Illumina platform, MiSeq Dx.
Data analysis: an analysis of the NGS data was performed using the GALAXY platform (usegalaxy.org). Sequencing reads (FASTQ files) were aligned to the human reference genome hg19 using the Bowtie2 tool. Variant calling was performed using the Varscan2 tool. Parameters used for the analysis were minimum allele frequency—0.05, minimum quality—20, and minimum coverage ×80. All variants were annotated with ANNOVAR (Available online:
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