RNA-seq differential expression analysis (Sciuridae)

Knowledge graph centered on RNA-seq differential expression analysis (Sciuridae) with 33 nodes and 113 connections. Top connected: Marmota flaviventris, Atriplex canescens, mark-recapture (Sciuridae), Bacteria, Parus major.

Method synopsis

Bioinformatics pipeline for processing RNA-seq reads including quality control, mapping to reference genome, and statistical analysis of differential gene expression using mixed models.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Gene expression shifts in yellow-bellied marmots prior to natal dispersal (2019), Behavioral Ecology. Implementations in other papers (listed below) may differ.

1. Map reads and quantify expression
   Trimmed adapters and removed short (<20 base pairs) and low-quality reads using Trim Galore. Resulting reads were mapped to the 13-lined ground squirrel genome using TopHat2 v2.1.0. Allowed 8 mismatches, a 10-base pair gap length, and a 20 bp edit distance between read sequences and the reference genome.
   Quantities: Generated 29 million reads per individual with 62% (18 million) uniquely mapped to the squirrel genomeDuration: Computational processing timeConditions: Computational analysis
   Equipment: Trim Galore, TopHat2 v2.1.0
2. Statistical analysis of gene expression
   Used linear mixed models in EMMERML with dispersal status as a fixed predictor variable and kinship as a random predictor variable. Again, dependent variables were the residuals of the gene expression counts after filtering, normalizing, and regressing batch effect variance.
   Quantities: 22,389 protein-coding genes analyzed, 11,381 (50.8%) detectableDuration: Computational analysisConditions: Statistical analysis using q < 0.1 significance threshold
   Equipment: EMMERML, LIMMA package, R version 3.3.1

Typical Equipment

• Trim Galore
• TopHat2 v2.1.0
• EMMERML
• LIMMA package
• R version 3.3.1

Output Measurements

• differentially expressed genes
• fold changes
• q-values

Papers Using This Protocol (1)

Species Studied With This Protocol