2.5 Library construction and sequencing
Library construction was performed according to the standard procedures of Illumina (Illumina, Inc., USA), which included terminal repair, adaptor connection, and PCR enrichment briefly. The Neonatal Gene Capture Kit- NeoEXOME™ (MyGenostics GenCap® Enrichment technologies, China) was designed based on the targeted genes in our project, and used for hybridization capture of relevant target regions. The final library was constructed after hybridization capture, PCR, and then determined by Qubit® 3.0 Fluorometer and Agilent 2100 Bioanalyzer system (Agilent Technologies, USA). After QC (quality control), the enriched library was sequenced by using HiSeq X Ten System (Illumina, Inc., USA) for 150-bp double-terminal sequencing22.
After sequencing, the reads were compared to the UCSC HG19 reference genome, and variants were identified using a bioinformatics process set by MyGenostics (based on GATK (The Genome Analysis Toolkit)). Variation was annotated using ANNOVAR databases. Mutations with high population frequency were filtered reference to dbSNP 1381000 Genome Project, ESP6500SI, and ExAC (the Exome Aggregation Consortium) browser.