Computational protocol: Listeria monocytogenes Induces a Virulence-Dependent microRNA Signature That Regulates the Immune Response in Galleria mellonella

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Protocol publication

[…] Publically available Illumina and 454 RNA-seq reads and ESTs from G. mellonella were retrieved from NCBI (SRR1021612, SRR1272440, ERR031115, ERR031116, ERR031117, ERR031118, ERR031119, ERR031120, ERR031121, and ERR031122). Additionally 18,690 pre-assembled contigs from an additional study (Vogel et al., ) were included. The read quality was checked using FastQC (Andrews, ) and trimmed accordingly (parameter used for Illumina reads: HEADCROP:15 ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10 MAXINFO:30:0.5 MINLEN:50, parameter used for 454 reads: HEADCROP:40 SLIDINGWINDOW:10:21 MINLEN:50 CROP:200 TOPHRED33) using Trimmomatic (Bolger et al., ). All reads were pooled and digitally normalized using the k-mer coverage approach implemented in Trinity (Haas et al., ). Multiple de novo assemblies were performed. The reads were assembled using the Trinity (Grabherr et al., ) assembler. The Velvet/Oases (Schulz et al., ) assembler was applied to assemble reads including the ESTs and pre-assembled contigs using the—conserveLong option to preserve the EST and pre-assembled contigs. To take into account the heterogeneity of the data, multiple Velvet/Oases assemblies were computed with varying k-mer parameters ranging from 19 to 75. The sequences from all de novo assemblies, the ESTs and pre-assembled contigs were screened for potential coding regions with Trans Decoder (transdecoder.github.io). The predicted amino acid sequences were clustered using cd-hit (Li and Godzik, ) with 98% global identity. For each cluster, the sequence with the longest 3′ UTR and a CDS length of at least 75% of the longest CDS in the cluster were selected as final transcripts. The transcripts were uploaded into SAMS (Rupp et al., ) and an automatic functional annotation was performed.For miRNA target prediction only the 3′ UTR parts of the transcripts were used from the above prepared database. Target sites were predicted using miRanda (Enright et al., ) with -strict option to get only exact matching seed sequences. Gene ontology analysis was performed with GOstats (Falcon and Gentleman, ) applying default parameters. Using cytoscape, we created miRNA-mRNA network including target genes with known function. In addition, the minimum free energy level of miRNA-mRNA duplex structure was determined by RNAhybrid tool provided by Bielefeld Bioinformatics server (Rehmsmeier et al., ). [...] Statistical analysis of experiments was performed with SigmaPlot 11 (Systat Software, San Jose, CA, USA). Student's t-test was used determine the significance of microRNA/mRNA expression levels. Survival data was analyzed using log rank test. […]

Pipeline specifications

Software tools FastQC, MaxInfo, Trimmomatic, Trinity, Oases, TransDecoder, CD-HIT, GOstats, RNAhybrid, SigmaPlot
Applications Miscellaneous, RNA-seq analysis
Organisms Listeria monocytogenes, Galleria mellonella, Caenorhabditis elegans, Homo sapiens, Listeria innocua
Diseases Bacterial Infections