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Help understanding best binning practices

Hi all,

I am working on a virus discovery and virome profiling project. I am interested in quantifying viruses in my samples. So far, my idea was to... 1) Assemble reads into contigs 2) Use CD-HIT to group together contigs with >=95% identity 3) Use metabat2 to bin this "nonredundant" pool of contigs, in order to have a binned and nonredundant list of putative viruses to which I can map my raw reads using a program like Salmon. 4) Normalize read counts after using metagenomeseq, visualize in MicroViz.

Basically, I thought it would be best to create this non redudundant grouping of contigs for accurate quantification (if there were 10 contigs all belonging to one virus, and Salmon tried to quantify all these, I would have to manually add all these counts together downstream).

I am a bit confused what the best practice would be after binning my contigs though, for 2 reasons:

  1. My first idea was to simply combine all contigs in a single bin into one very long contig, so I was capturing the most genomic information. Or is it best to simply select the longest contig of this bin?
  2. How is it best to handle separate bins that come from the same organism? For example, I have one bin that corresponds to virus 1's RNA polymerase, and one bin that corresponds to virus 1's coat protein. Would I have to manually combine these counts later to get the "total counts"?

Thank you

binning viruses metagenome contigs

Many assembled contigs may represent complete viral genomes. At least I have recovered complete viral genomes from metagenomic assemblies, no binning or anything. They were just contigs..

1 answer

Hi,

I hope I am not too late.

  • MetaBAT2 is not suitable for viral data
  • You shouldn't combine contigs into a single bin
  • You shouldn't try to manually stitch them together. avoid the binning step entirely.

Hope it helps.

MetaBAT2 is not suitable for viral data

What is the basis for this statement? MetaBAT2 doesn't know anything about the DNA source. It uses tetranucleotide frequencies and abundance information, so it will work with any set of DNA sequences. If their TNF signatures are distinct, they will separate into bins regardless of the DNA source.

Hi,

Thanks for raising that point. You are right that MetaBAT2 is composition and coverage based and does not explicitly distinguish DNA origin.

My concern comes from the biological assumptions behind those signals. Viral genomes tend to be shorter, more mosaic and can show inconsistent coverage patterns. Which can weaken the reliability of tetranucleotide frequencies and abundance based clustering.

I may be missing something though.

Any short contig with poor read mapping will not be appropriate for binning, regardless of its source. Once they are over 2-3 kb, it shouldn't matter whether they are viral or not. I have successfully binned viral contigs using custom programs even without abundance information, so MetaBAT2 should not have any problem with viruses either.

Out of curiosity, what do you use for viral binning if MetaBAT2 is not appropriate?

Any short contig with poor read mapping will not be appropriate for binning, regardless of its source. Once they are over 2-3 kb, it shouldn't matter whether they are viral or not.

That's fair point about poor read mapping on short contigs. My worry is around 2-3kb, sometimes it may be the entire virus genome size itself. If we drop shorter sequences, we might just drop the entire populations of small viruses from our dataset altogether.

Out of curiosity, what do you use for viral binning if MetaBAT2 is not appropriate?

I use vRhyme. It uses coverage and protein-sharing networks to link viral fragments. (https://doi.org/10.1093/nar/gkac341)

Seems like a good tool that probably works better for small viruses. The smallest viruses I know are in the 5-7 kb range, but maybe there are even smaller ones.

Still, there is nothing fundamentally lacking in common binning tools such as MetaBAT2 when it comes to binning viruses that are 20+ kb. I have binned hundreds of viruses in that size range, many of them 100% complete. It goes without saying that giant viruses (500+ kb) bin like any other MAGs. Granted, this was with a custom tool, but it is conceptually the same as MetaBAT2 and other binners.

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