Differential gene expression on metatranscriptome data

[Tka]

Hi,

I am new to RNA-seq analysis and wanted to ask for some advice. I'm working on several soil metagenomes taken at different temperatures. My plan is to map the reads from each sample against a reference genome and subsequently use DeSeq and Edger to screen for differentially expressed genes.
I was wondering if this will give reasonable results as I am only working with those metagenome reads that map to the reference. What do you think?

Regards,
Tka

[severin]

Originally Posted by Tka

Hi,

I am new to RNA-seq analysis and wanted to ask for some advice. I'm working on several soil metagenomes taken at different temperatures. My plan is to map the reads from each sample against a reference genome and subsequently use DeSeq and Edger to screen for differentially expressed genes.
I was wondering if this will give reasonable results as I am only working with those metagenome reads that map to the reference. What do you think?

Regards,
Tka

To get a proper answer you will need to provide a lot more information about how deep the sequences was, if this is an environmental sample or sterilized a with reference bacteria added, and how many biological replicates do you have?

Let's assume these are environmental samples, then I would recommend doing de novo assembly and mapping back to the assembly to get count data.

Andrew

[Tka]

Thank you for the reply and I will try to be a little more specific.

The experiment design included 3 biological replicates for different temperature points. It's biological soil samples but the coverage is unknown. Only thing I know is that the number of reads (Illumina) for each sample is the same. I have a reference that is either included in the sample or has multiple very closely related strains in it.

By the way: sorry for asking real basic questions. Just started with transcriptomics and didn't think about de novo assembly, yet. Could you hint me to some tools that map de novo assembled transcripts to a reference genome?

Thanke you very much,
Tka

[severin]

Originally Posted by Tka

Thank you for the reply and I will try to be a little more specific.

The experiment design included 3 biological replicates for different temperature points. It's biological soil samples but the coverage is unknown. Only thing I know is that the number of reads (Illumina) for each sample is the same. I have a reference that is either included in the sample or has multiple very closely related strains in it.

By the way: sorry for asking real basic questions. Just started with transcriptomics and didn't think about de novo assembly, yet. Could you hint me to some tools that map de novo assembled transcripts to a reference genome?

Thanke you very much,

Tka

In diagram form.

Take all raw read files -> Trinity assembly program -> assembled scaffolds
take each raw reads -> align using GSNAP alignment program to assembled scaffolds -> SAM output
take assembled scaffolds -> metageneMark -> gene models gff file
use aligned file, gff file and htseq-count --stranded=no to generate count information on a per gene basis for your denovo assembled transcripts
blast gene models against NR to determine function of genes.

Good luck!