Computational protocol: STUB1 mutations in autosomal recessive ataxias – evidence for mutation-specific clinical heterogeneity

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Protocol publication

[…] Genome wide SNP genotyping was performed with the Genome Wide Human SNP array 6.0 (Affymetrix, Santa Clara). Whole genome homozygozity mapping was performed using PLINK v1.07 [,] searching for any region >2 Mb, with minimum of 30 SNPs and less than four heterozygous calls. Whole exome capture and paired-end 100 nt sequencing was performed at HudsonAlpha Institute for Biotechnology (Huntsville,AL) as described in (Haugarvoll 2013). The 8.7 Giga-bases of aligned sequence data resulted in 55X median coverage of the target capture regions, with more than 96% of target bases covered a minimum of 8X. PCR duplicates were removed with PICARD ( and the Genome analysis toolkit [] was used for base quality recalibration and variant calling using a minimum threshold of 8X sequencing depth and quality score ≤ 30. Annovar [] was used for variant annotation. Variant prioritization was performed as described in [] based on an autosomal recessive model, filtering against variants identified in more than 100 Norwegian exome-resequencing samples (obtained using the same whole exome sequencing pipe-line) and variants present at >0.5% allele frequency in the 1000 Genomes database. Variants were verified by Sanger sequencing using the BigDye terminator kit and the ABI7900 Genetic Analyzer. For the proband in Family 2, all exons and intron/exon boundaries in STUB1 were sequenced by Sanger sequencing (primers and conditions available upon request). To test whether the mutations found in Patient 2 were located on different strands we used the TOPO® TA Cloning® Kit (Invitrogen, Life technologies, 11329-H07E-25, California) to clone PCR-products spanning both mutations, followed by Sanger sequencing of the clones. STUB1 reference sequence (RefSeq) used: NM_005861.2 […]

Pipeline specifications

Software tools PLINK, Picard, GATK, ANNOVAR
Applications WES analysis, GWAS
Organisms Homo sapiens