Computational protocol: Transcriptome analysis of the biofilm formed by methicillin-susceptible Staphylococcus aureus

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Protocol publication

[…] Due to lack of the reference genome of S. aureus ATCC25923, we used Trinity de novo assembler (version 20140413) to assemble these RNA-seq sequences. The raw reads were filtered using NGS QC ToolKit software. The low-quality reads (which contained adaptor contamination, the pair-end reads satisfied N > 10%, and low quality reads (quality value < 20)>50%) were removed.Coding sequences (CDSs) were identified using TransDecoder (http://transdecoder.sourceforge.net/). These CDSs were used for BLAST searches against the NCBI Nr protein database (NCBI non-redundant sequence database) (version 2014-5) with an E-Value cut-off of 1e-5. CDSs were further aligned by BLASTX to protein database including Swiss-Prot (version 2014-5), KEGG (version 2013-10), COG (version 20090331), and GO (version 2014-5) to retrieve their functional annotations. If results of different database conflicted, a priority order of Nr, Swiss-Prot, KEGG, COG and GO was followed. For transcripts that were not identified by TransDecoder software but were annotated by above protein databases, they were still regarded as CDSs.The Blast2GO program was used to obtain GO annotation for CDSs, as well as for KEGG and COG analyses. The WEGO software was used to perform GO functional classification. This analysis mapped all of the annotated CDSs to the GO terms in the database and calculated the number of CDSs associated with every GO term. COG and KEGG pathway annotations were performed using Blastall software (version 2.2.25) against the COG and KEGG databases. [...] The RNA-seq reads were mapped to our transcriptome reference database, and transcript abundances were quantified by RSEM. Genes that differentially expressed among these nine samples were identified using the numbers of mapped reads as EdgeR inputs. Genes with an adjusted P value ≤ 0.05, FDR ≤ 0.001 and fold change ≥ 2 were identified as being differentially expressed. Differentially expressed genes were regarded as up-regulated if their expression levels in treated samples were significantly higher than those in the control samples. Similarly, they were down-regulated if their expression levels in treated samples were significantly lower than those in the control samples. […]

Pipeline specifications

Software tools Trinity, NGS QC Toolkit, TransDecoder, BLASTX, Blast2GO, WEGO, RSEM, edgeR
Databases UniProt KEGG KEGG PATHWAY
Application RNA-seq analysis
Organisms Staphylococcus aureus, Epipremnum aureum
Chemicals Methicillin, Vancomycin