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misclassification of Melissococcus plutonius #2
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Hi @wapoga Thanks for bringing this up. Melissococcus plutonius is a member of Bacilli at the 'class' rank, and also Lactobacillales at the 'order' rank, so this would appear correct for the higher rank levels of the original taxonomy string you got. But I agree, it seems quite odd that it came back as unclassified at the family level and lower ranks. I did a quick validation check with the 'IDTAXA___BEEx-V3V4-TS.RData' classifier database (v2021.0.2) using a 16S rRNA gene sequence from a confirmed Melissococcus plutonius isolate (NCBI ref NR_074098.1 - https://www.ncbi.nlm.nih.gov/nuccore/NR_074098.1) and was able to get the correct species-level identification as follows: Bacteria(100);Firmicutes(100);Bacilli(100);Lactobacillales(100);Enterococcaceae(98.90);Melissococcus(98.90);Melissococcus_plutonius(98.90) If you don't mind, could you please provide the sequence you were trying to classify (copy and paste here or email is fine) as well as the the following details so that I may reproduce the result you got and address this issue more thoroughly:
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i'll get these to you in the next day |
Maybe there's an explanation for this in our protocol: Here is the representative sequence for the Melissococcus that is coming up as: AGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAATAGGGGAAAGAGTAACTGTTTTCCTCGTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACA |
Okay, so I believe I figured out what was going wrong here. Replicating the steps you indicated, I ran the
However, when I ran the
To see if this could be a formating issue with the RDP classifier implemented in mothur, I tested the misclassified_seq using another RDP classifier algorithm implemented in the dada2 R package. The sequence was classified correctly:
Based on this, I constructed a single seed subset reference databse from the full-length BEExact database for use with mothur. The respective files are available here: To format the taxa headers and make a mothur-specific classification databse, I ran the following:
I've uploaded this formatted database to the classifier databases directory, available here: To confirm these changes solved the issue, I ran the following in mothur:
Everything looks to be in order now. I would suggest re-classifying the rest of your sequences using the mothur-formatted database, as it is likely other taxa were also incorrectly classified. Let me know if this solves the problem on your end. ***Additional info: For clarity to others who may read this, before running |
Thanks Brendan, you are awesome!! |
Hello and thanks for this tool. Recently I applied the BEExact database to a dataset from an EFB outbreak. The number one OTU came up as:
Bacteria(100);Firmicutes(100);Bacilli(100);Lactobacillales(100);Lactobacillales_unclassified(100);Lactobacillales_unclassified(100);Lactobacillales_unclassified(100);
When I BLAST this sequence in the database it indeed comes up as Melissococcus plutonius
Not sure why this hit would come up under Bacilli, but I hope this helps to improve this awesome tool.
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