RNA-seq of homo sapiens: healthy adult muscle
RNA was extracted from patient and control skeletal muscle biopsies (~15-50 mg) using the RNeasy Fibrous Tissue Mini Kit (QIAGEN #74704). RNA purity was assessed by Nanodrop, followed by PCR amplification and 1% agarose gel electrophoresis to confirm the absence of DNA. Sample sequencing was performed at Genomics WA (Harry Perkins Institute of Medical Research, Perth, Australia). Stranded Poly A RNAseq libraries were prepared from extracted RNA using Agilent Sureselect XT library preparation kit. The protocol includes poly A enrichment followed by fragmentation, reverse transcription, ligation with adaptors and amplification for indexing. QC was performed using TapeStation 4200 and Qubit, and QC sequencing on the Illumina iSeq 100 flow cell. Paired-end sequencing (strand-specific reverse) was performed on the Illumina NovaSeq 6000 instrument using a 2 x 150 cycle configuration to a depth of 50 million read pairs per sample. Adaptor sequences were removed and demultiplexed FASTQ files were provided by Genomics WA for download and further analysis.Processing of FASTQ files, including read quality control and alignment, was performed using the nf-core/rnaseq pipeline (https://nf-co.re/rnaseq/1.3), version 3.8.1. Raw reads were aligned to the GRCh38 human reference genome using STAR (version 2.7.10a). Detection of aberrant expression was achieved using DROP (version 1.3.3),13 as previously described.14 DROP leverages OUTRIDER,15 a method for detecting aberrant RNAseq read counts, which uses a denoising autoencoder to control for latent effects and returns multiple-testing correctedp-values (FDR) for each gene and sample. The cohort consisted of 130 skeletal muscle RNA-seq from rare neuromuscular disease patients, including three OPDM patients and five unaffected controls.