Peripheral blood samples were collected from the ASD and TD group for extracting DNA. Exome capture was performed using the Agilent SureSelect Human All Exon V6 r2 kit followed by Illumina paired-end sequencing.

The raw reads were quality-controlled using FastQC v0.11.2 and then mapped to the human reference genome (hg19) using bwa v0.7.17.74 PCR duplicates were removed using Picard tools v2.18.12. SNVs were identified using FreeBayes v1.2.075 and filtered using bcftools v1.9. SNVs with base quality < 30, coverage < 30, with less than 3 reads mapped to the alternative alleles or located within 5bp nearby a gap were removed.

SNVs presented in the TD groups were removed. Then, SNVs were annotated by the annovar software to extract allele frequencies from the public database Allele frequency of these SNVs in the normal population was extracted from the 1000 Genome project,76 the Exome Aggregation Consortium (ExAC) database,77 and the Chines MillionomeI database (CMDB),78 a large-scale Chinese genomics database including comprehensive variation and their allele frequency information from 141,431 unrelated healthy Chinese individuals. SNVs with MAF < 5% in these public databases and detected in more than two ASD samples were considered as candidates. GO enrichment analysis of genes with SNVs was conducted in R package.

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