Haloplex custom target enrichment kit

An Agilent Haloplex custom target enrichment kit that captured all exons in 89 genes related to melanoma and tumors from mucous membranes [5 (link), 16 (link)–19 (link)] was designed using the Agilent SureDesign software (Agilent Technologies, Santa Clara, CA). The genes on the panel are listed in Additional file 2: Table S2. Target-enriched libraries were constructed from genomic DNA (3 μg), following an instruction from HaloPlex Target Enrichment for Illumina Kit (Agilent Technologies) [13 (link)]. Only library fragments within 175 to 625 bp were considered for the final quantification, normalization, and pooling. The final multiplexed Haloplex custom target library pool was sequenced on the Illumina HiSeq 2500 (Illumina, San Diego, CA) on rapid mode using paired-end 100 bp reads. Overall, the panel showed high coverage rates (median 349x) for all the target regions.

The study was performed according to the Declaration of Helsinki, and approval was obtained from the Ethical Review Boards of Almazov National Medical Research Centre, approval number 2014/95. Written informed consent was obtained from the study subject, including a consent for publication of the clinical case. Target next generation sequencing was performed on Illumina MiSeq using Haloplex custom target enrichment kit (Agilent; Waldbronn, Germany) with a panel of 108 genes associated with cardiac disorders as earlier described (Kostareva et al., 2016 (link)). All disease-related genetic variants were subsequently validated by Sanger sequencing and classified according to American College of Medical Genetics guidelines (Richards et al., 2015 (link)).