Computational protocol: Targeted/exome sequencing identified mutations in ten Chinese patients diagnosed with Noonan syndrome and related disorders

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Protocol publication

[…] Peripheral blood samples were collected from the patients and their parentsafter informed consent was obtained. Genomic DNA (gDNA) was extracted using Lab-Aid Nucleic Acid (DNA) Isolation Kit (Zeesan, China) according to the manufacturer’s instructions.ClearSeq Inherited Disease or SureSelect Human All Exon V5 kit (Agilent, Santa Clara, CA, USA) were used for library preparation of targeted sequencing or whole exome sequencing, respectively. The resulting libraries were sequenced on a HiSeq 4000 platform (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions for paired-end 150-bp reads. The minimal data amount was2.5Gb per sample for TS and 8Gb per sample for WES.Fastq-format reads were aligned to the human reference genome (GRCh37/hg19) using BWA-0.7.10 []. BAM files were manipulated using Picard tools-1.124. Base calling was performed following GATK best practice version 3 []. Quality metrics were evaluated - the average depth was 80× per sample, with at least 97% of the target region covered by 10× reads or more. The vcf files were then annotated using SnpEff version 4.2 []. Variants with >1% frequency in the population variant databases -1000Genomes Project, Exome Variant Server (EVS) and Exome Aggregation Consortium (ExAC) or > 5% frequency in the local database with 150 exome datasets were filtered, and subsequentlyintergenic, intronic, and synonymous variants were filtered, except those located at canonical splice sites. Candidate variants were then evaluated in the context of clinical presentation and inheritance mode. Selected variants were validated by Sanger sequencing in the proband and parents. Paternity was confirmed for de novo variants. […]

Pipeline specifications

Software tools BWA, Picard, GATK, SnpEff
Databases Exome Variant Server
Application WES analysis
Organisms Homo sapiens
Diseases Congenital Abnormalities, Heart Defects, Congenital, Lentigo, Noonan Syndrome, Genetic Diseases, Inborn, LEOPARD Syndrome