Blood samples were collected in EDTA containing tubes. Genomic DNA was isolated with QIAamp DNA Mini QIAcube kit (QIAGEN, Germany) according to the manufacturer’s instructions. DNA concentrations were measured with the QubitTM Fluorometric Quantitation system (Thermo Fisher Scientific) using Qubit HS DNA Assay kit (Thermo Scientific, US). DNA libraries were obtained using the BRCA Hereditary Cancer MASTR Plus, Multiplicom (Agilent, United States) kit. Variant screening on 26 risk carrying genes for hereditary cancers like breast, ovarian and colorectal cancer (ABRAXAS1, ATM, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MEN1, MLH1, MRE11, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, and XRCC2) has been performed by this kit which contained five multiplex PCR primer pools. 10 ng of DNA per primer pool was used for multiplex PCR amplification, followed by barcode ligation and purification with Agentcourt AMPureXP reagent (Beckman Coulter, Beverly, MA, United States). Quantity and quality of prepared libraries were assessed by QubitTM Fluorometric Quantitation system (Thermo Fisher Scientific). For library preparation 4 ng DNA was used. After libraries were prepared, sequence analysis was performed with Illumina MiSeq instrument using MiSeq Reagent v3 kit (Illumina, US). All sequencing data were submitted to Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra/PRJNA895859 ).
Bioinformatics analysis was performed using the software Sophia Genetics DDM (Sophia Genetics v4.2). GRCh37/hg19 was used as the reference genome. During variant calling, a minimum sequence coverage depth and variant fraction parameters were set to 30x and 20%, respectively. Variants were classified according to the ACMG Guidelines (Richards et al., 2015 (link)) using databases of ClinVar (Landrum et al., 2014 (link)), BRCAExchange, OMIM®, dbSNP (v.155), gnomAD (v2.1.1), in silico pathogenicity classifiers of MutationTaster (Schwarz et al., 2010 (link)), SIFT (Ng and Henikoff, 2003 (link)), PolyPhen-2 (Adzhubei et al., 2013 ), REVEL (Ioannidis et al., 2016 (link)). All variants with minor allele frequency (MAF2) of less than 1% in gnomAD database were considered.
Bioinformatics analysis was performed using the software Sophia Genetics DDM (Sophia Genetics v4.2). GRCh37/hg19 was used as the reference genome. During variant calling, a minimum sequence coverage depth and variant fraction parameters were set to 30x and 20%, respectively. Variants were classified according to the ACMG Guidelines (Richards et al., 2015 (link)) using databases of ClinVar (Landrum et al., 2014 (link)), BRCAExchange, OMIM®, dbSNP (v.155), gnomAD (v2.1.1), in silico pathogenicity classifiers of MutationTaster (Schwarz et al., 2010 (link)), SIFT (Ng and Henikoff, 2003 (link)), PolyPhen-2 (Adzhubei et al., 2013 ), REVEL (Ioannidis et al., 2016 (link)). All variants with minor allele frequency (MAF2) of less than 1% in gnomAD database were considered.
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