Sureselect human all exon 50 mb kit

Exonic sequences from DNA samples of the patient were enriched with the SureSelect Human All Exon 50 Mb Kit (Agilent Technologies, Santa Clara, CA, USA). Sequences (100-bp paired-end) were generated on a HiSeq2000 (Illumina, San Diego, CA, USA). Read alignment and variant calling were performed with DNAnexus (Palo Alto, CA, USA) using default parameters with the human genome assembly hg19 (GRCh37) as reference. Sequencing of the coding region of RBM7 has been performed by intronic primers (Supplementary Materials). Parental consent was given for genetic studies. The study was performed with the approval of the ethical committees of Hadassah Medical Center and the Israeli Ministry of Health.

Exome sequencing was performed on one affected child from all families using the SureSelect Human All Exon 50Mb Kit (Agilent Technologies UK, Cat. No G3370A). Sequencing was performed with the SOLiD4 System (Applied Biosystems) with 50 bp fragment reads (in families B and C) or the Illumina Analyzer IIx with 76 bp paired end reads (in families A and D). Raw sequencing reads were mapped to the GRCh37 reference human genome and changes compared to this reference sequence identified. Analyses focused on non-synonymous coding, nonsense, splice site variants, and indels involving exons. Potentially pathogenic mutations were identified based upon being unknown variants or those where the rare allele frequency was <1%, how well the site was conserved throughout evolution, and re-examination of the sequence reads containing potential mutations using the Integrated Genome Viewer. Analysis of the 84 genes in the candidate interval in family A revealed only one rare potentially pathogenic variant in PLAA (Table S2). This c.68G>T mutation in PLAA was found to segregate correctly by Sanger sequencing in families A, B, and C.

Whole blood genomic DNA was fragmented to 150–200 bp by Adaptive Focused Acoustics (Covaris), end-paired, adenylated and ligated to adapters. Exonic sequences were enriched using Agilent SureSelect Target Enrichment (Agilent SureSelect Human All Exon 50 Mb kit). The captured fragments were purified and sequenced on a GAIIx platform using 75 bp paired-end reads. Bioinformatic analysis was performed using an in-house algorithm based on published tools. Sequence was aligned to the human reference genome (UCSC hg19), using NovoAlign (www.novocraft.com). The aligned sequence files were reformatted using SAMtools and duplicate sequence reads were removed using Picard. Single base variants were identified using Varscan (v2.2) and indels were identified using Dindel (v1.01). The raw lists of variants were filtered to include variants within the Sequence Capture target regions (±500 bp). On target variants were annotated using wAnnovar and common variants with a minor allele frequency > 0.02 that were present in the 1000 Genomes (February 2012 data release), the NHLBI-5400 Exome Sequencing Project and 191 unrelated in-house exomes were excluded. Rare, protein altering, homozygous and compound heterozygous variants that fitted the recessive disease model were identified.

Whole-exome sequencing was performed as 54 bp paired-end runs on a Genome Analyzer IIx system (Illumina) after in-solution enrichment of exonic sequences (SureSelect Human All Exon 50 Mb kit, Agilent). Reads are mapped to the reference genome, duplicate fragments are marked using Picard and GATK is then used to recalibrate base qualities. We used the human genome assembly hg19 (GRCh37) as reference.