Basecalling software 1

Exome libraries were prepared using the Agilent SureSelect All Human Exon v5 capture kit^{5 (link)}. Briefly, the qualified genomic DNA samples were randomly fragmented into base pair peak of 150 to 200 bp, and adapters ligated to both ends of the resulting fragments. Adapter-ligated templates were purified by the Agencourt AMPure SPRI beads, and fragments with insert size of about 200 bp were excised. Extracted DNA was amplified by ligation-mediated polymerase chain reaction (LM-PCR), purified, and hybridized to the SureSelect Biotinylated RNA Library (BAITS) for enrichment. Hybridized fragments were bound to the streptavidin beads whereas non-hybridized fragments were washed away after 24 h. Captured LM-PCR products were subjected to Agilent 2100 Bioanalyzer to estimate the magnitude of enrichment. Each captured library was independently loaded on Hiseq 4000 platform for high-throughput sequencing to ensure that each sample meets the desired average fold-coverage. Raw image files were processed by Illumina base calling Software 1.7 with default parameters, and the sequences were generated as 90 bp paired-end reads. All the experimental protocols were approved by the NUS, OSHE.

Raw image files were processed using Illumina base-calling software 1.7 with default parameters, and the sequences for each individual were generated as 90-bp paired-end reads. After filtering, high-quality reads were aligned to the human genome (GRCH37, UCSC hg19) using the Burrows-Wheeler Aligner program [10 (link)]. SAMtools [11 (link)] and SOAPsnp [12 (link)] were used for the identification of small insertions or deletions (InDels) and single nucleotide polymorphisms (SNPs), respectively. SNPs were called using SOAPsnp with options:-r 0.00005-e 0.0001-t-u-L 90-Q L. Next, filters of quality score (≥20), neighbor distance (≥5) and depth (≥4) were applied to the SNPs calling results. Finally, ANNOVAR [13 (link)] was used for the annotation of InDels and SNPs.

The sequencing data (raw data) generated from the Illumina software (Illumina basecalling Software 1.7) was needed to conduct cleaning and mapping. The adapter sequence in the raw data and low quality sequences which had too many unknown bases or low base quality were excluded. Clean data was produced and aligned by BWA (http://biobwa.sourceforge.net/) and formatted the sequence into binary BAM files. The BAM format files were established mate information of the alignment, added read group information and removed duplicate reads caused by PCR. Clean reads were processed by mapped to the reference human genome (GRCh37/hg19) from UCSC database (http://genome.ucsc.edu/) using SOAPalinger (http://soap.genomics.org.cn/index.html). Single Nucleotide Polymorphisms (SNPs) were detected according to SOAPsnp (http://soap.genomics.org.cn/soapsnp.html). Indels were aligned to the reference human genome from UCSC using BWA and further conduct with the Genome Analysis Toolkit (GATK v1.6) for recalling. Variants in the non-coding region and synonymous mutations were removed. SNPs and indels with higher frequency (>0.5%) noted in dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/), 1000 Genomes (ftp://www.1000genome.org), HapMap were also filtered out. Quality Control (QC) was processed in the steps of the clean data, the alignment, and the identified variant.

Exome capture was performed in the proband and his parents by BGI–Shenzhen using NimbleGen SeqCap EZ Human Exome Library v3.0 (Roche NimbleGen, Inc., Madison, WI, USA) according to the manufacturer’s protocols, and sequencing was performed using a HiSeq2000 platform (Illumina, San Diego, CA, USA). Illumina base calling Software 1.7 was used with default parameters to process the raw image files and to sequence the individual products as 90-bp paired-end reads. The sequenced reads were aligned to the human genome reference (UCSC hg19 version, build37.1) using SOAP aligner/SOAP2[27 (link)]. SNP or indels were called using Soapsnp [28 (link)] software and BWA [29 (link)], respectively. The alignment results were identified using GATK [30 (link)] to identify the breakpoints.

The qualified genomic DNA sample was randomly fragmented by Covaris and the size of the library fragments is mainly distributed between 250 bp and 300 bp. Then adapters were ligated to both ends of the resulting fragments. Extracted DNA was then amplified by ligation-mediated PCR (LM-PCR), purified, and hybridized to the NimbleGen 44 M human exome array for enrichment. Each captured library was then loaded on Hiseq2000 platform, and we performed high-throughput sequencing for each captured library. Each sample was sequenced at the mean depth of 100× to achieve high sensitivity and accuracy for mutations detection. Raw image files were processed by Illumina basecalling Software 1.7 for base-calling with default parameters and the sequences of each individual were generated as 90 bp pair-end reads.