Massively parallel sequencing

A custom gene panel (TruSeq® Custom Amplicon Assay, Illumina) was developed, encompassing our candidate gene - SDHAF3 ({"type":"entrez-nucleotide","attrs":{"text":"NM_020186","term_id":"1707761956","term_text":"NM_020186"}}NM_020186); as well as eight known pheochromocytoma/paraganglioma suseptibility genes (MAX [{"type":"entrez-nucleotide","attrs":{"text":"NM_002382","term_id":"1653960896","term_text":"NM_002382"}}NM_002382], SDHB [{"type":"entrez-nucleotide","attrs":{"text":"NM_003000","term_id":"1732746310","term_text":"NM_003000"}}NM_003000], SDHC [{"type":"entrez-nucleotide","attrs":{"text":"NM_003001","term_id":"78711818","term_text":"NM_003001"}}NM_003001], SDHD [{"type":"entrez-nucleotide","attrs":{"text":"NM_003002","term_id":"1519245278","term_text":"NM_003002"}}NM_003002], SDHAF2 [{"type":"entrez-nucleotide","attrs":{"text":"NM_017841","term_id":"300795354","term_text":"NM_017841"}}NM_017841], RET [{"type":"entrez-nucleotide","attrs":{"text":"NM_020975","term_id":"1519313481","term_text":"NM_020975"}}NM_020975], TMEM127 [{"type":"entrez-nucleotide","attrs":{"text":"NM_017849","term_id":"305682550","term_text":"NM_017849"}}NM_017849] and VHL [{"type":"entrez-nucleotide","attrs":{"text":"NM_000551","term_id":"319655736","term_text":"NM_000551"}}NM_000551]). The panel included the protein-coding exons and flanking intronic regions of each of the genes and was created using DesignStudio (Illumina). DNA libraries were prepared (using 250 ng of DNA from each sample) and sequenced on a MiSeq platform (using 2 × 150 bp paired end reads) according to the manufacturer’s instructions (Illumina). FASTQ files (containing reads and their base call quality scores) were generated for each sample; and alignment of reads (banded Smith-Waterman algorithm) and variant calling (GATK [19]) was processed by MiSeq Reporter (version 2.0, Illumina). Annotation of functional consequences to variant calls was performed using ANNOVAR (version 2013Jul [20]), which incorporates various in silico tools, including (but not limited to) PolyPhen-2, SIFT, MutationTaster. Visualisation of reads was performed using IGV (v2.1).