Sample Table

Alignment Summary

Total Aligned Fragment Summary: Stacked bar plots of the total number of reads aligned stratified by mapping quality.

Base Trimming Summary: Grouped bar plots of the expected paired-end read length and the average observed paired-end read length after adapter trimming and soft-clipping to find the best local alignment.

EndBias Plot: A plot of the 3 prime and 5 prime bias averaged across all known genes for a given sample. Ideally, the plot should show high and consistent coverage from the 3 prime to the 5 prime ends that appears as a large plateau over the normalized 100bp gene body. High coverage at one end and low coverage at the opposite end indicates end bias during sample and library preparation. Inconsistent spikes in coverage across the gene body with low coverage in between spikes indicates sample degradation or library preparation artifacts that are typical with non-strand displacing random priming library preparation such as the Sigma kit for low input or degraded FFPE samples.

Junction Saturation Plot: A plot of the coverage over known and novel splicing junctions. When the known/novel junctions plateau, the plot indicates that the sensitivity for known/novel junctions has been maximized and continued sequencing would only increase coverage rather than sensitivity. If the plot shows no signs of plateauing for the known/novel junctions, the plot indicates that low and low-medium expressors are likely under-sampled and the abundances measured will not reflect the true distribution of expressed genes or transcripts. Continued sequencing will increase sensitivity and would likely prove beneficial for all downstream analysis such as gene or transcript level differential expression.

Gene Expression Summary

Gene Library Complexity: Line plot per sample illustrating the fraction of counts taken up by what fraction of genes. Adapted from the R/CRAN RNASeqQC package. The semi-interactive version of this plot is located in the QC_FILES folder.

Gene featureCount Summary

Total featureCount Summary: Stacked bar plots of the total number of fragments stratified by featureCount assignment.

Gene Biotype Table: Summed counts-per-million of each annotated gene biotype.

Gene Biotype Plot: Stacked bar plot of the total sum of counts-per-million of all observed gene biotypes per sample.

Gene Model Plot: Stacked bar plot showing ratios of reads aligned to CDS exons, UTR exons, Introns, and neighboring intergenic regions within 10Kb of the nearest gene.

Gene Spearman Correlation Matrix

Gene Pearson Correlation Matrix

Isoform Summary

Isoform Biotype Table: Summed counts-per-million of each annotated transcript biotype.

Isoform Biotype Plot: Stacked bar plot of the total sum of counts-per-million of all observed transcript biotypes per sample.

Isoform Spearman Correlation Matrix

Isoform Pearson Correlation Matrix

Methods

Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 500ng to 1ug of total RNA. Ribosomal RNA was removed by an RNase-H method using RiboErase kits (Kapa Biosystems). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq X Plus using paired end reads extending 150 bases.Basecalls and demultiplexing were performed with Illumina’s DRAGEN BCLconvert version 4.3.13.  RNA-seq reads were then aligned to the Ensembl GRCm39.113 primary assembly with STAR version 2.7.11b.  Gene counts were derived from the number of uniquely aligned unambiguous fragments by Subread:featureCount version 2.0.8. Isoform expression of known Ensembl transcripts were quantified with Salmon version 1.10.0.  Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected.  The ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified with RSeQC version 5.04.