Amplicon RNA-Seq of DM1-036_amp
RNA was extracted from biopsies via a TRIzol-chloroform extraction supplemented with a bead-based tissue homogenization (Benchmark Scientific) and purified using the Zymo Clean and Concentrator  5 kit following the standard protocol for on-column DNase treatment. RNA quality and abundance was assessed using fragment analysis (Agilent Fragment Analyzer 5200, DNF-472 (15 nt RNA kit) and Qubit 4 Fluorometer (Invitrogen). Most RNA samples had RIN values >7, but samples were sequenced if they had RIN scores > 5.5.A protocol for multiplexed alternative splice sequencing was originally developed as described previously (Thornton 2024) for targeted RNA-sequencing of the 22 events of interest and modified for low volume, high coverage sequencing using the iSeq 100 (Illumina). cDNA was synthesized from 10 ng of high-quality RNA using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara). Target amplicons of inclusion and exclusion isoforms were created in two separate multiplex reactions and combined for each sample. Amplicons were subsequently indexed using Illumina-adapted barcodes for sequencing using Nextera chemistry. Deviations from the protocol include separate bead-based purification, quality assessment, and normalization of each individual library prior to pooling and sequencing. Libraries were individually normalized based on concentrations assessed using the Qubit 4 Fluorometer (Invitrogen) and average library size as determined by smear analysis (Agilent Fragment Analyzer 5200, DNF-474 HS NGS kit). Libraries were paired-end sequenced on the iSeq 100 using an i1 Reagent v2 (300-cycle) kit at a loading concentration of 125 pM with a 10% PhiX spike-in. Samples were sequenced at 1000X coverageAmplicon library reads in raw FASTQ format were aligned to a custom reference sequence set of exon inclusion and exclusion isoforms using HISAT2 v2.1.0 and filtered reads with the -no-spliced-alignment flags and -no-softclip run parameters. The custom reference FASTA file is composed of hg38 derived exon inclusion and exclusion sequences for all 22 events in the composite panel. Primary aligned reads spanning splice junctions were subsetted from the resulting bam files using a combination of Samtools v1.7 and Bash CLI. Isoform-specific reads with unambiguous alignment were then counted and further processed in a custom python script to derive  and the Splicing Index (SI) score