Computational protocol: Thyroid transcriptome analysis reveals different adaptive responses to cold environmental conditions between two chicken breeds

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Protocol publication

[…] Read quality was evaluated using the FastQC suite version 0.10.1 (, and quality trimming was performed using Trimmomatic-0.36 [] with parameters set as HEADCROP:19 SLIDINGWINDOW:4:15 MINLEN:60. After quality filtering, paired reads were extracted and mapped to the ENSEMBL genome build Gallus_gallus-5.0 (GCA_000002315.3) using HISAT2 version 2.0.5 []. StringTie version 1.3.3b [, ] was used to assemble the alignments into potential transcripts. Sorting and indexing of the read alignment files were implemented with SAMtools version 1.4 []. Mapping statistics were calculated with SAMtools and RSeQC (version 2.6.3) []. We compared the assembled transcripts to the reference genome using gffcompare version 0.10.1 (available from Transcripts coded with “u”, “i”, or “x” were recognized as new loci, and those coded with “j”, “o”, or “e” were treated as new transcripts of known loci from the annotation database.A Python script ( provided by the StringTie maintainer (available at was used to generate a gene count matrix based on the StringTie outputs. The R (version 3.4.0) [] package edgeR version 3.18.0 [] was used to detect differentially expressed genes (DEGs) using TMM normalization and quasi-likelihood (QL) F-test, which provides a more robust and reliable error rate control [, ]. Raw gene counts were filtered with counts per million (cpm > 2) to eliminate genes with low counts across multiple samples (< 3 samples), and 13,678 genes passed the filtering. Multidimensional scaling (MDS) analysis was performed using the function plotMDS within edgeR package to investigate the overall gene expression patterns, and 3 samples (BS_Cold2, RIR_Cold1, and BS_Warm4) recognized as outliers were removed from differential gene expression analysis. DEGs were identified at FDR < 0.05 (Benjamini-Hochberg correction []) and absolute fold change ≥ 1.5 (|log2FC| ≥ 0.58). Venn diagrams were created using the online tool Venny 2.1.0 ( FPKM (fragments per kilobase million) values of gene expression were also extracted from the StringTie outputs. R studio ( was used to run custom scripts to construct heat maps, box plots, and density plots. Unless otherwise stated above, all the programs were run with the default parameter settings. […]

Pipeline specifications

Software tools FastQC, Trimmomatic, HISAT2, StringTie, SAMtools, RSeQC, GFF utilities, edgeR, VENNY
Application RNA-seq analysis
Organisms Gallus gallus