Computational protocol: Congenital myasthenic syndromes due to mutations in ALG2 and ALG14

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Protocol publication

[…] For Family 1, whole-exome capture was performed from 3 µg of genomic DNA using Agilent SureSelect Human All Exon Kit v2. The captured libraries were sequenced on Illumina HiSeq platforms using 51 bp paired end reads. Sequence data were mapped to human genome build hg19 using Novoalign software (Novocraft Technologies). To avoid artefacts, we filtered out the duplicate reads generated as a result of PCR amplification. Only reads that mapped uniquely to the genome were used for further analysis. Visualization of sequence data was performed using GBrowse () and the UCSC genome browser (). Variants were called using the Samtools program (). To exclude common variants, we filtered the data against the dbSNP132 database () (unless they were annotated as medically-associated single nucleotide polymorphisms). This filtering narrowed the list of variants to 1511 or 1548 variants per exome (Supplementary Table 1). We then used ANNOVAR software () to functionally annotate the variants and thereby identify non-synonymous substitutions, splicing mutations or mutations in 3′UTR or 5′UTR.For Family 2, genomic DNA was fragmented by adaptive focused acoustics (Covaris). Libraries were constructed using the TruSeqExome enrichment capture technology (Illumina) as per the manufacturer’s protocol in a technical service provided by Eurofins MWG Operon ( Sequencing was performed on the Illumina HiSeq2000 platform as 2 × 100 bp paired end reads. Raw sequencing reads were aligned to the consensus genome (hg19), sorted and variants called using SAMtools and optimized for indel calling using Dindel. The resulting list of variants were visualized and assessed using the UCSC Genome Browser. All sequencing was performed using bi-directional fluorescent sequencing on an ABI 3730 XL 96 capillary sequencer, with BigDye Version 3.1 chemistry. [...] Whole-exome sequencing was performed for the index case from Family 3. Detailed methods of the sample preparation, sequencing and analysis are provided in the online Supplementary material. Briefly, 2 µg of DNA was fragmented, libraries constructed and adaptors ligated. Size-selected ligated libraries were amplified by PCR, and sequencing was performed on aHiSeq2000 as 100 paired end. Whole-genome sequencing reads were mapped to the human reference genome (GRCh37d5/hg19) using STAMPY () and duplicate reads removed using Picard. Identification of variant sites and alleles was performed with in-house software Platypus (), which can detect single nucleotide polymorphisms and short (<50 bp) indels. The variants were then processed with a functional annotation pipeline based on the ANNOVAR software package (Wang et al., 2010). […]

Pipeline specifications

Software tools NovoAlign, GBrowse, SAMtools, ANNOVAR, Dindel, Stampy, Picard, Platypus
Databases dbSNP UCSC Genome Browser
Applications WGS analysis, WES analysis
Organisms Homo sapiens, Saccharomyces cerevisiae
Diseases Congenital Disorders of Glycosylation, Myasthenic Syndromes, Congenital, Genetic Diseases, Inborn, Muscular Dystrophies, Limb-Girdle, Cryopyrin-Associated Periodic Syndromes
Chemicals Acetylcholine