Computational protocol: Transcriptome analysis of follicles reveals the importance of autophagy and hormones in regulating broodiness of Zhedong white goose

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Protocol publication

[…] We aligned the reads of 18 samples to the UCSC (http://genome.ucsc.edu/) goose reference genome using Tophat package, which initially removed a portion of the reads based on quality information accompanying each read and then mapped the reads to the reference genome of goose. Tophat allows multiple alignments per read (up to 20 by default) and a maximum of two mismatches when mapping the reads to the reference. [...] The aligned reads were processed by Cufflinks, which use the normalized RNA-seq fragment counts to measure the relative abundances of the transcripts. The unit of measurement is fragment per kilobase of exon per million fragments mapped (FPKM). The reference GTF annotation used in Cuffinks was downloaded from the UCSC database. Cufflink was used to de novo assemble the transcriptome. The second, Cuffmerge was used to co-merge all transcripts of samples to generate unique transcripts. The downloaded UCSC GTF file was passed to Cuffdiff along with the original alignment (SAM) files produced by Tophat. Cuffdiff re-estimates the abundance of the transcripts listed in the GTF file using alignments from the SAM file and concurrently tests for different expression. Only the comparisons with “p value” less than 0.05 and status marked as “OK” in the Cuffdiff output were regarded as showing differential expression.The differentially expressed genes, including transcription factor, hormone related genes, and autophagy-related genes, were analyzed using MeV software (http://www.tm4.org/mev.html), and confirmed partially by qRT-PCR methods. Gene Ontology (GO) of differential genes was carried out using GeneOntology database (http://www.geneontology.org/). KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was analyzed by KEGG database. […]

Pipeline specifications

Software tools TopHat, Cufflinks, TM4
Databases KEGG
Application RNA-seq analysis
Chemicals Estradiol, Progesterone