Service Description Bulk RNA sequencing is one of the most popular applications of Next-Generation Sequencing (NGS). In order to provide comprehensive support for RNAseq projects, VIPER generates output from most commonly used RNAseq analysis tools including STAR, Salmon, and DESeq2. Downstream analysis can include Gene Set Enrichment analysis (GSEA), Kegg Pathway, and Gene Ontology (GO) analysis. The primary output is a graphical summary report presenting alignment statistics, read feature distribution, rRNA removal QC, genebody coverage QC, PCA plots, sample correlation heatmaps, and volcano plots of differentially expressed genes.
Required Input Files:
Fastq.gz Files (single-end and paired-end supported)
Metasheet including sample group annotations and comparison definitions for differential expression testing.
Differential Gene Expression Testing (DESeq2 results tables with log2FC and adjusted p-values per gene)
Pathway Analysis (GSEA, Kegg Pathway, GO term)
Isoform level expression quantification*
Gene Fusion Analysis
QC Figures Include:
PCA plots
Sample correlation heatmaps
Volcano plots
GSEA dot plots (if sufficient number of differentially expressed genes detected)
Experimental Design Considerations
In order to perform statistical testing between different conditions, biological replicates are required. The minimum number of replicates required for proper statistical analysis is three (N = 3) per condition. In general, the number of replicates required depends on the amount of biological noise in the system.
Cell Line Experiments Cell line experiments tend to have minimal noise and are technically reproducible -- although not recommended, in some cases even duplicates per condition might be an acceptable approach in the context of a large cell line experiment testing different drug treatments
Mouse Models Since there tends to be more biological variation in transcriptomic profiles for RNA samples derived from mouse tissue >3 replicates are generally recommended -- ideally 4-6 replicates per condition.
Human Tissue Samples For bulk RNAseq experiments involving human tissue samples, >6 replicates per condition are recommended to account for increase natural biological variability between individuals.
FFPE There is often a high degree of variability in RNA quality and quantity for samples derived from formalin-fixed paraffin embedded tissue. Typically > 6 replicates per condition are required for statistical power and recommendations may vary significantly based on RNA quality and quantity as well as analytical goals. Please reach out to us for a consultation before proceeding.
Low Input Experiments Our low input bulk RNAseq protocols work well for high quality RNA with RIN > 6.5 (mRNAseq) or DV200 > 40% (totalRNAseq) for input amount amounts from 2-10ng of RNA. These methods may also work well with from 250pg-2ng, but risk of technical variation increases within this range. Consequently, >3 replicates are generally recommended. Please reach out to us for a consultation for low input projects to ensure experimental design to maximize data quality from inherently precious samples.