Ez exome 2

Bar-coded DNA libraries were prepared and whole exome capture was performed (EZ Exome 2.0, Roche, Basel, Switzerland) by the Yale Center for Genome Analysis. Illumina HiSeq 2000 and 2500 instruments were used for sequencing samples pooled 6 per lane, with 75 bp paired-end reads. Resulting reads were aligned to the human reference sequence (hg18 or hg19) with Efficient Large-scale Alignment of Nucleotide Databases software (ELAND, Illumina, San Diego, CA) and processed via a SAMtools-based Perl script to trim sequence to targeted intervals and remove PCR duplicates. Single nucleotide variants (SNVs) and deletions and insertions (indels) were identified using SAMtools software. Variants were annotated for functional impact using a Perl script, and filtered in Excel to exclude frequent variants present in dbSNP (Build 140), 1000 Genomes, the NHLBI exome database (release ESP6500SI-V2) and in 2577 control exomes, and to examine coding mutations (missense, nonsense, and splice site SNVs and indels) with SAMtools quality scores ≥50 and coverage ≥8. Aligned reads were examined with the Broad Institute Integrative Genomics Viewer (IGV) to exclude SNVs resulting from alignment error.

Each patient and available first generation relatives underwent phlebotomy for chromosomal microarray analysis of peripheral blood lymphocytes (Claritas Genomics, Cambridge MA). Whole exome sequencing was provided by Yale University Centers for Mendelian Genomics on a Illumina HiSeq 2000 instrument with blood samples pooled 6 per lane. Libraries (TruSeq DNA v2 Sample Preparation kit; Illumina, San Diego, CA) and whole exome capture (EZ Exome 2.0, Roche) were performed according to standard protocols. FASTQs were aligned by Codified Genomics (proprietary algorithm, Houston, TX).
Medical records of the patients and their families were collected by The Manton Center for Orphan Disease Research, Gene Discovery Core under informed consent governed by the Institutional Review Board of Boston Children's Hospital. A standard assessment was performed, which documented physical features and recorded medical, developmental, psychiatric, and family illness history, supplemented by medical records (Table I).