How To Remove Macrophage Contamination From A Rna-Seq Experiment?

Question

[joaslucas]

I have an RNA-seq data from bacteria and macrophages. They are mixed, I couldn't get completely rid of macrophages RNA in this experiment. So, now I need to get rid of it to analyse the the bacterial transcriptome. Does any one suggest a program where I can do it? The RNA-seq comes from a HScan illumina platform, single read and 100 bp.

Thanks.

Answer 1

[Leonor Palmeira]

This will depend on whether your macrophage's genome is "known" or not. Do note that "known" is always a quite relative thing, but here I mean that there is a correctly annotated genome available.

If this is the case, it should be fairly easy:

• get the transcriptome data from the genome using its annotations.
• map your RNA-seq reads to the macrophage transcriptome (using a mapping tool like SOAP or BWA)
• remove all reads mapping to the macrophage transcriptome

If your macrophage's genome is not "known", is there a large amount of data on it that is available, or from a very closely related species? If so, you should adapt the above procedure (namely lowering mapping thresholds) to remove all reads mapping to the sequence information you have. If not, you are in front of an open problem in bioinformatics, congratulations :-) If you are willing to share some more details on your project, we might then still be able to help by providing some ideas on how to proceed.

[joaslucas]

Hi Leonor Palmeira, thanks for the valuable information. We know the macrophage genome and transcriptome. I will do it and let yuo know what we got.

[joaslucas]

Will it also work fine if I do it the other way around? Map the RNA-seq reads straight to the bacteria genome and pick the mapped reads? Thanks.

[Leonor Palmeira]

I would prefer to have a stringent removable of contamination by first mapping the reads against the contaminant genome and then removing them. Why? Because removing a few bacteria reads by mistake (because they map quite well to the contaminant) sounds better than leaving contaminant reads in your assembly process (because they map quite well to the bacteria). Also, depending on the assembly algorithm, these contaminant reads might be interpreted as repeats and interfere with the assembly in an uncontrolled way.