Computational protocol: Whole genome sequencing reveals a 7 base-pair deletion in DMD exon 42 in a dog with muscular dystrophy

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Protocol publication

[…] To test for presence of the previously reported CKCS intron 50 donor splice site mutation, we used primers developed in the earlier study (Walmsley et al. ). Total RNA was isolated from myoblasts extracted from the vastus lateralis (VL) muscle, as detailed previously (Nghiem et al. ). Genomic DNA was extracted from muscle tissue with Qiagen DNA Blood and Tissue Kit. Reverse transcriptase (RT)-PCR of total RNA was performed to make cDNA using the Applied Biosystems High Capacity cDNA kit (Foster City, CA), also described previously (Walmsley et al. ; Nghiem et al. ). PCR was performed with AccuPrime Supermix (Thermo Scientific) on a Thermocycler, under the following conditions: 94 °C for 5 min; 94 °C for 30 s, 58 °C for 30 s, 68 °C for 60 s (cycle repeated 35 times); and 68 °C for 5 min. The PCR product was submitted for Sanger sequencing and restriction fragment length polymorphism (RFLP) mutational analysis, as previously reported (Walmsley et al. ). A separate primer design for exons 45–53 can be found in supplemental methods (Supplemental Table 2). PCR was performed with identical conditions, as detailed above.Nested PCR of gDNA was performed with primers flanking the targeted site at DMD exon 42. Outside sense and anti-sense primers were as follows: 5′-GTGGTTTAGGAATTCCACATGTACG-3′ and 5′-TGTCTATACCAGCACACTGTCC-3′, respectively. Inside sense and anti-sense primers were as follows: 5′-CTAAGTCAATCATTGTACTGCG-3′ and 5′-CGATGACTGAAGACATGCCTTTGG-3′, respectively. Reaction products were subjected to gel electrophoresis for quality control. Target bands were cut from the agarose gel and the DNA was extracted and processed. Transformation of the PCR product and cloning into the pCR 2.1 Vector was completed using the Invitrogen TA Cloning Kit, as recommended by the manufacturer. PCR products were submitted to the Texas A&M Gene Technology Lab for Sanger sequencing. Sequence chromatograms were viewed with Sequencher (Gene Codes Corporation; Ann Harbor, MI). [...] Genomic DNA was extracted from muscle using the Qiagen DNA Blood and Tissue Kit (Hilden, Germany). 2 µg of gDNA was prepared and sent to Macrogen (Rockville, MD), where it was clustered and sequenced on the Illumina HiSeq X Ten (San Diego, CA). Briefly, the gDNA was further prepped according to the Illumina TruSeq DNA PCR-free library preparation guide. DNA was fragmented and a final library of 300–400 base pair (bp) average insert size was created. Using an End Repair Mix, the double-stranded DNA 3′ or 5′ overhang fragments were converted into blunt ends, while the 3′–5′ exonuclease removed the 3′ overhangs and the polymerase filled the 5′ overhangs. After end repair, the appropriate library size was selected using Sample Purification Beads. 3′ end adenylation was performed by adding a single ‘A’ nucleotide to the blunted fragments along with a corresponding ‘T’ nucleotide to provide complementary overhang for adapter ligation to the fragment. Multiple indexing adapters were ligated to DNA fragment ends and prepared for flow cell hybridization. Library validation, including a quality control analysis of the sample library and quantification of DNA library templates, was performed by Macrogen. The flow cell containing unique clusters was loaded into the HiSeq X Ten for automated cycles of sequencing by synthesis (extension and imaging). WGS data were aligned with Isaac aligner (Raczy et al. ). Isaac Variant Caller was used to identify and genotype single-nucleotide polymorphisms (SNPs) and small insertions/deletions (indels). A variant call format file was produced with the probability of consensus difference from genotype and probability of the called genotype. Mapping quality was performed by Phred-scaled probability of all samples and reported in −log. National Center for Biotechnology Information’s (NCBI) Genome Workbench software (Bethesda, MD) was used to evaluate mutations, SNPs, and indels within the DMD gene (compared to the CanFam3.1 whole genome shotgun sequence used as the reference) (Hoeppner et al. ). […]

Pipeline specifications

Software tools MUSCLE, Sequencher, Isaac Genome Alignment Software, IVC, NCBI Genome Workbench
Applications WGS analysis, Nucleotide sequence alignment
Organisms Canis lupus familiaris, Mus musculus, Homo sapiens
Diseases Muscular Dystrophies, Muscular Dystrophy, Duchenne