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metagenome binning with MetaBAT2, and Maxbin2 created bins for some samples but not others.

Hi all,

Thank you for helping me here. I have been analyzing 96 samples including four control samples and the remaining 92 samples represent decaying wood, which are divided to 10 categories based on the fungus that was identified at the time of sampling. Out of the 10 fungi, eight have 10 biological replicates while two have six replicates.

  • The goal: To understand bacterial-fungal interactions by looking at the bacterial MAGs, and possibly, fungal as well.
  • Short reads metagenomic data
  • NovaSeq X 25B Single Lane - 300
  • Assembly: metaSPADES
  • Binning with MetaBAT2 and Maxbin2
  • I used scaffolds.fasta instead of contigs.fasta

Results:

  • For some samples I got bins but others I didn't.

Questions:

  • Could this be due to the starting DNA concentration (wood is a little hard to get better yield than other samples, particularly when wood is not completely decomposed)?
  • Given that these samples were collected from positions on the wood where the fungal mushroom was protruding from, naturally, fungal DNA would be more abundant. I wonder if that could be another factor that I couldn't get bins for some of these samples?
  • Are there any better approaches that might result in robust and thorough analysis, or at least explicitly determine what might be going on here?
  • Should I use contigs.fasta instead of scaffolds.fasta?

Again, thank you for any suggestions, advice, and help.

bacteria fungi metagenome binning

1 answer

Could this be due to the starting DNA concentration (wood is a little hard to get better yield than other samples, particularly when wood is not completely decomposed)?

I am singling out this question, but this is an answer to just about all your questions. Binning is done with no regard to how the data is collected, what types of organisms are there. The only thing that matters is that there are contigs of sufficiently large size (larger than 1000 nucleotides for MetaBAT2, don't know about Maxbin2) and that these contigs have a tetranucleotide signal that clearly separates them into groups. You can rationalize that through sample collecting any way you want, but for our purposes it is the quality of your assembly that is relevant rather than your collection method. We don't know anything about the former, for example the number of contigs, average contig size, the N50 value, etc.

With the information you provided, an educated guess is that some of your assemblies are of poor quality - likely fragmented.

Should I use contigs.fasta instead of scaffolds.fasta?

It should make no difference, as in most cases those two are identical. Again, if you do the statistics on both files, you will probably arrive at the same conclusion.

Thank your for your answers. I think I will have to revisit my assemblies before moving ahead with my analysis. Do you think the following approach is reasonable?

  1. I am going to correct errors with bfc before assembly. I will run this on my fastp trimmed reads.
  2. Use megahit for assembly with --presets meta-large. This will allow me to have both spades and megahit assemblies, which could reveal potential discrepancies.

Do you have any suggestion of the -s parameter and -1 flag in bfc?

Again, thank you for your time.

This is a reasonable strategy on both counts. I don't use -s in bfc but I do use -1.

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