Help required for Bacterial RNAseq data

Question

[bioinforesearchquestions]

Hi friends,

I am going to 8 bacterial bulk RNAseq samples. I am interested in doing following bioinformatics analyses for the 12 samples.

a) Trimming of the reads

b) Alignment of the RNA-seq reads to the reference bacterial genome

c) ID and construction of splice-junctions

d) Reports of known transcripts with annotation and abundance

e) Report of novel transcripts and abundance

f) Testing differential expression

g) GO and Kegg annotation and enrichment analysis

h) Also, DeNovo Assembly

1) For mammalian, I used the following softwares Tophat, cufflinks, cuffmerge, cuffquant, and cuffdiff? 2) Is there any separate tools/pipelines for bacterial RNAseq data? 3) What are tools for GO, Kegg and enrichment analysis? 4) Which denovo assembly tool is recommended for bacteria?

[liz.marjory]

Hi, I just begin in the RNA seq analysis field and I am working with bacteria as well. Did you find the right pipeline? I am looking for the better tool for aligment step

[ATpoint]

Hey, how about starting with this guide: https://www.annualreviews.org/doi/abs/10.1146/annurev-biodatasci-072018-021255 Written by some of the more well-known names in the RNA-seq field. It contains alot of background any helpful suggestions. This workflow is also helpful. https://bioconductor.org/packages/release/workflows/vignettes/rnaseqGene/inst/doc/rnaseqGene.html The principles of RNA-seq are basically the same for bacteria just that you do not need to use a special software that is splice-aware for alignment or quantification.

[liz.marjory]

Thanks Atpoint for the advices! Was really useful I´ve been reading and I found the SPARTA pipeline. I will try.

Answer 1

[GenoMax]

For # 1 since you would not expect splicing to be an issue you could use any aligner (I recommend BBMap suite for trimming/alignment).
#2 there are dedicated pipelines available for bacterial RNAseq. Rockhopper is a web based one. Search Entrez for others.
#3 You should be able to use GeneSCF (Gene Set Clustering based on Functional annotation (GeneSCF) ).
#4 SPAdes is about the best bacterial genome assembler.

[bioinforesearchquestions]

Thanks, Genomax for your recommendations. I checked SPAdes website and I found there is a separate "De Novo RNAseq Assembler" called "rnaSPAdes".

For mammalian RNAseq data, I started with FASTQ files --> BAM/SAM from Tophat --> raw read count from HTseq-count or GTF file from Cufflinks --> merged GTF from Cuffmerge --> differential outpu from Cuffdiff.

For bacterial RNAseq data, when I don't have reference genome. Using rnaSPAdes assembler, I will get the output file in FASTA format. But with that FASTA file how do I address (d, e, f, g, h)?

[Kevin Blighe]

Take a look at Trinity, which does everything that you want: https://github.com/trinityrnaseq/trinityrnaseq/wiki

Rockhopper, mentioned by genomax is good. It runs from your local disk as a JAVA jar file, too. Afterwards, you can infer functionality of the identified RNA transcripts via BLASTx

[bioinforesearchquestions]

Thanks, Kevin. I will first run trinity and see what are all the output files I am going to get.