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I have sequenced a bacterial genome and after I checked the ANI scores, the scores against similar bacterias were 79 and lower. How can I confirm if this bacteria is a novel species/strain? Is there any threshold value for ANI score? I'm pretty sure if the ANI score is above 95, it indicates the same species.

Should I double-check this result by using other methods such as GGDC?

Thanks in advance!

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Short answer:

Yes.

Long answer:

I suggest 4 methods for checking the genome.

  1. Use minimap2 or a similar aligner to perform alignments with the most similar genomes, then generate a dot plot or other visualization of the regions that align to figure out what is going on. Here is one tool, I haven't used it, here is another. I generally use minimap2 to very quickly align, and then a quick plot in R using the PAF file.
  2. Use k-mer hashing methods such as mash or sourmash to quickly compare to a large database. Large precomputed databases are available for download on the linked mash site. You can also use it for one-by-one comparisons.
  3. Use QUAST to estimate more informative summary statistics for comparisons to the more similar genomes. QUAST has a rich set of possible visualizations that you can use. It also tries to identify the most closely related public genome using rDNA sequences, though that has been hit or miss for me.
  4. Use a quality assessment tool such as CheckM or BUSCO to estimate the quality of your genome. THese also have utilities for taxonomic placement of your genome in case something bad happened (e.g. sample swap).
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