Metagenomic quantification (abundance of reads on assembled reference)

Question

[emiliomastriani]

Hello, I need to estimate the relative abundance by viral genus or family in my samples after assembly. I have found the following reference https://github.com/edamame-course/Metagenome/blob/master/2017-08-09-counting-abundance-with-mapped-reads.md , but 1. some step is still not clear, 2. data are not available to reproduce/test the procedure. Can someone suggest me an alternative source to learn about Metagenomic Quantification? Something like a tutorial/step-by-step resource?

Thank you very much

Answer 1

[Istvan Albert]

Use a tool like Kraken2 or another metagenomics classification tool.

https://ccb.jhu.edu/software/kraken2/

The method described in your reference seems somewhat ill-suited for the process, short read aligners were not designed to distinguish between potentially very similar sequences or to resolve these into a taxonomic tree.

[emiliomastriani]

Hello Istvan, thank you for your reply. I know kraken2 and we already used it for the taxonomic classification in couple with krona. Now we would like to accomplish a more detailed analysis already having identified the viral genus/family of interest and its contigs. Some different suggestion?

EM

[Istvan Albert]

a short read aligner won't be more accurate as it was not designed for that purpose.

what is it that you are looking to find that Kraken/Krona is unsuited for? As far as I know, it already quantifies the read abundances

what does that "detailed analysis" consist of?

[emiliomastriani]

I would not rely on the kraken classification result for abundance. There are risks of false positives and in a publication one would be criticized with good reason. Abundance must be done after confirmation of the taxonomic assignment of each read/contig classified as viral sequence. Therefore, it is necessary either to perform the confirmation by blast, which is an option, or to perform the confirmation by contigs obtained. I am looking for a practical session or tutorial where this kind of strategy has been already applied.