Truseq exome enrichment guide

Ethics approval was obtained from the local ethic committee, as well as the informed concern from patients from families A and B. The study was conducted in accordance with the Declaration of Helsinki.
Whole exome sequencing (WES) of the proband’s DNA was performed in line with the protocol from Illumina’s TruSeq Exome Enrichment Guide. A SureSelect Human All Exon 50 Mb Kit (from Agilent Technologies, Santa Clara, CA, USA). Hybridized fragments were bound to streptavidin beads, while non-hybridized fragments were washed out. The library was then verified on an Agilent 2100 Bioanalyzer and sequenced on a HiSeq 2000 instrument (Illumina) from Intelliseq LLC (Kraków, Poland).

Whole exome sequencing (WES) of the proband’s DNA was performed in line with the protocol from Illumina’s TruSeq Exome Enrichment Guide. A SureSelect Human All Exon 50 Mb Kit (from Agilent Technologies). Hybridized fragments were bound to streptavidin beads, while non-hybridized fragments were washed out. The library was then verified on an Agilent 2100 Bioanalyzer, and sequenced on a HiSeq 2000 instrument (Illumina) from Intelliseq LLC (Kraków, Poland).

Human exome capture was performed following the protocol from Illumina’s TruSeq Exome Enrichment Guide (Illumina, San Diego, CA, USA). Illumina’s TruSeq 62 Mb Exome Enrichment kit was used as exome enrichment probe sets, and 5 μg of genomic DNA in 80 μL of Buffer EB (Qiagen) was fragmented in a Biorupter UCD-200(Diagenode, Belgium) to sizes of 100–500 bp. DNA concentration was estimated by OD₂₆₀ measurement and quantitative real-time polymerase chain reaction (PCR) analysis. Captured DNA libraries were sequenced with the Illumina HiSeq 2000, yielding 200 (2 × 100) base pairs from the final library fragments using V2 reagent. Base calling was performed with CASAVA 1.8 software (Illumina). The reads were aligned with the human genome reference sequence (UCSC hg19) using the Burrows-Wheeler Alignment (BWA) tool, version 0.5.9rc1. Variants (SNPs and indels) were called with vcftools of SAMTools software version 0.1.16 [22 (link)]. High VarQuality SNPs were annotated with Perlscript into functional categories such as missense, nonsense, splice sites, coding, non-coding and untranslated regions (UTRs). Amino acid substitution where an amino acid substitution affects protein function was annotated with SIFT (Sorting Intolerant From Tolerant) and PolyPhen-2 (Polymorphism Phenotyping v2).