Sureselectxt human all exon v5

Paired samples were obtained from the University of Maryland Brain and Tissue Bank as detailed in S1 Table. Individuals were diagnosed with ASD (n = 12) or were controls; criteria for diagnosing ASD included the Autism Diagnostic Interview-Revised (ADI-R), Childhood Autism Rating Scale (CARS), and Autism Diagnostic Observation Schedule (ADOS) as detailed S1 Table. DNA was extracted from tissue dissections according to protocols in the QIAGEN Genomic DNA Handbook. Exonic regions were selectively captured using Agilent SureSelectXT Human All Exon V5. Sequencing was performed at the Center for Inherited Disease Research at Johns Hopkins generating 100 bp sequence reads on an Illumina HiSeq. CIDRSeqSuite version 3.0.1 was used for processing of the raw data files. BCL files were converted to qseq format using Illumina’s BCL converter. qseq files were then demultiplexed and converted to FASTQ files using a custom demultiplexer. Paired-end alignment was performed using BWA aln to the 1000 genomes hg19/GRCh37 reference genome [34 (link)]. SAM files were sorted, converted to BAM, and duplicates were marked with Picard. GATK was used for local realignment and base quality score recalibration [35 (link),36 (link)]. Quality metrics for these data are provided in S2 Table.

Genomic DNA (1 µg) from each sample was sheared using the Covaris S220 (Covaris, Woburn, MA, USA), and a library was constructed using SureSelect XT Human All Exon V5 and a SureSelect XT Reagent Kit, HSQ (Agilent Technologies). The kit captures 335,756 exons of 21,058 genes, covering ~ 71 Mb of the human genome. After enriched exome libraries were multiplexed, the libraries were sequenced on a HiSeq 2500 platform (Illumina, San Diego, CA, USA). Briefly, a paired-end DNA sequencing library was prepared by gDNA shearing, end-repair, A-tailing, paired-end adaptor ligation, and amplification. After hybridization of the library with bait sequences for 16 h, the captured library was purified and amplified with an index barcode tag, and the library quality and quantity were measured. Sequencing was performed in the 100-bp paired-end mode of the TruSeq Rapid PE Cluster Kit and TruSeq Rapid SBS Kit (Illumina).

Whole‐exome sequencing was performed on DNA extracted from formalin‐fixed paraffin‐embedded (FFPE) tumor samples (Maxwell^® 16 FFPEPlus LEV DNA Purification Kit, Promega, Madison, WI, USA) in addition to freshly obtained peripheral blood or normal tissue. WES Libraries were prepared according to manufacturer’s protocol (SureSelect XT Human All Exon v5, Agilent, Santa Clara, CA, USA). Finally, libraries were sequenced in a HiSeq2500 (Illumina, San Diego, CA, USA), 2X100 Paired‐end. Reads were aligned against the hg19 reference genome.

Genomic DNA from three fresh LGACC specimens was extracted using a DNA kit (QIAamp; Qiagen, Chatsworth, CA, USA) and DNA from 11 FFPE sections was purified with an isolation kit (RecoverAll Total Nucleic Acid Isolation Kit; Thermo Fisher Scientific, Rockland, DE, USA) following the manufacturer's instructions. The quantity and quality of DNA was evaluated by a spectrophotometer (NanoDrop 8000; Thermo Fisher Scientific) and automated electrophoresis tool (Bioanalyzer 2000; Agilent, Palo Alto, CA, USA), respectively. Whole exome sequencing was conducted in the Sequencing Core facility at the John P. Hussman Institute for Human Genomics in the University of Miami. Briefly, DNA samples were sheared using a sonicator (E210; Covaris, Woburn, MA, USA) and the whole exome was captured using a commercial kit (SureSelect XT Human All Exon V5; Agilent). To sequence the enriched 50-Mb exomes, a three-plex strategy per lane was conducted on a sequencer (HiSeq 2000; Illumina, San Diego, CA, USA) using 125-bp paired-end reads, which yielded an average of approximately ×100 coverage depth at targeted regions.