Computational protocol: MSTO1 is a cytoplasmic pro‐mitochondrial fusion protein, whose mutation induces myopathy and ataxia in humans

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[…] DNA was extracted from blood and skeletal muscle using QIAamp DNA blood and tissue kits, according to the instructions of manufacturer (QIAGEN, Hilden, Germany).The total coding region of mtDNA was screened by Sanger sequencing using ABI Prism 3500 DNA Sequencer (Applied Biosystems, Foster City, USA); the obtained sequences were compared with the mitomap databases using NCBI's Blast® application, while mtDNA deletion was investigated with long PCRs using Phusion High‐Fidelity DNA Polymerase (Finnzyme, Vanta, Finland).Exome Capture was performed in two steps according to the manufacturer's protocol. Genomic DNA library preparation was performed by using TruSeq® DNA Sample Prep Kit v2‐Set A (Illumina), followed by NimbleGen SeqCap EZ Human Exome Library v3.0 Kit exome enrichment (Roche). First pre‐capture genomic library was prepared using 1 μg of highly purified genomic DNA. Crude pre‐captured genomic library was analyzed by Agilent 2100 Bioanalyzer 1000 DNA chip to assess library quality. Next, during exome enrichment step, individual pre‐captured libraries were hybridized to biotinylated NimbleGen SeqCap EZ Human Exome Library for 68–70 h at 47°C. Hybridized libraries were captured by magnetic Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific). Streptavidin beads were washed, and the captured DNA was eluted. Eluted DNA was PCR amplified by 18 cycles. Crude target‐captured libraries were gel purified on 2% E‐Gel (Life Technologies) and cleaned up with QIAquick Gel Extraction Kit (QIAgen, Hilden, Germany). Target‐captured, gel‐purified libraries were analyzed by Agilent 2100 Bioanalyzer 1000 DNA chip to assess libraries quality. The final library concentration was determined by manual integration of molecular profile by Agilent 2100 Bioanalyzer 1000 DNA chip. Illumina adaptor‐specific qPCR was also performed for each library to measure the library template concentration containing adaptor sequences on both ends which will subsequently form clusters on the Illumina flow cell. All library pools were sequenced on the HiScanSQ Illumina sequencing platform, using 2 × 95‐bp pair‐end sequencing protocol, with an extra 9‐bp index sequencing run. Final exome‐captured sequencing libraries were diluted to 10 nM. Reads were filtered according to the Q30 standard. 95‐bp paired‐reads were aligned to the human reference genome (hg19). The alignment was performed using Burrows–Wheeler aligner (BWA) software. For variation calling, Samtools software was used (Li et al, ; Li, ). After identification of variants, we focused only on non‐synonymous variants, splice acceptor and donor site mutations, and short, frame shift coding insertions or deletions (indel) using Genomes Management Application (GEM.app) software (Department of Human Genetics, University of Miami Health System, and Miami, FL, USA).The WES filtering procedure was the following: GATK (QUAL) > 50, GQ > 40, RD > 4 quality scores; autosomal dominant inheritance; only the missense, nonsense, indels, and splice‐site rare variants with MAF < 0.01 were selected. All SNVs higher than three occurrences in GEMapp were excluded.For crosschecking, the mitochondrial genetic alterations were investigated by targeted screening for both autosomal dominant and recessive inheritance performed in genes, which is responsible for mitochondrial function. The rare variants were filtered out based on mitochondrial gene function and disease association used a gene list, which was created by UNIPROT, NextProt, MitoMiner, and NCBI databases.As large‐scale genomic data are not available from the Hungarian population, a mutation with low minor allele frequency may be population specific. Finally, we prioritized mutations based on consequence. Exonic frameshift and stop mutations were considered as damaging. Missense mutations were prioritized, which was based on the protein prediction score annotations given by ANNOVAR (Wang et al, ). Further narrowing was based on protein prediction scores (Polyphen2 score > 0.5, Mutation taster: disease causing, SIFT < 0.1, PHASTCONS > 0.5; GERP > 3). Using ACMG guideline (Richards et al, ), the pathogenic and likely pathogenic variants were confirmed by Sanger sequencing. After identification of candidate alterations, the segregation analysis was performed in all affected family members.For the validation of the exome capture result COL5A1 Exon 3, total coding region of MSTO1 from genomic and cDNA, RELN Exon 62, RYR2 Exon 16, were analyzed with bidirectional Sanger sequencing (the primers listed in ). The obtained sequences were compared with the human reference genome (ENST00000371817, NM_000093.4; ENST00000245564, NM_018116.3; ENST00000343529, NM_173054.2; ENST00000366574, NM_001035.2) using NCBI's Blast® application.The analysis of copy number variation of all exons of MSTO1 gene was performed by quantitative real‐time RT–PCR with SYBG methodology in the real‐time PCR StepOnePlus system according the manufacturer's instructions (Life Technology). For quantification, the ddCt method was used. […]

Pipeline specifications

Software tools BWA, SAMtools, GATK, ANNOVAR, PolyPhen, PHAST
Databases neXtProt MitoMiner
Application WES analysis
Organisms Homo sapiens
Diseases Muscular Diseases, Mitochondrial Diseases
Chemicals Lactic Acid