Hello!
Please tell me why in some of my samples a large number of readings are in groups unique-reads-missed-reference and reads-missed-reference? How to fix it? With taxonomic classification, the remaining sequences are identified as 2 organisms, although there should be a large variety.
Thanks for your reply!
Hello Yulia,
Welcome to the forums! :qiime2: Thank you for your patience.
The Deblur pipeline relies on a reference, and it looks like most of the reads in samples S4-16 and S1-16 did not match the reference. There's a bunch of reasons this could happen...
What region did you amplify? What database are you using for reference? What settings did you use to preprocess you data and run deblur?
I amplified V3-V4 region and used Greengenes Database, also used qiime vsearch join-pairs, qiime quality-filter q-score and qiime deblur denoise-16S.
qiime vsearch join-pairs
--i-demultiplexed-seqs /the path/
--o-joined-sequences /the path/
qiime quality-filter q-score
--i-demux /the path/
--o-filtered-sequences /the path/
--o-filter-stats /the path/
qiime deblur denoise-16S
--i-demultiplexed-seqs /the path/
--p-trim-length 0
--p-sample-stats
--p-jobs-to-start 4
--o-stats /the path/
--o-representative-sequences /the path/
--o-table /the path/
Hello Yulia,
That pipeline looks good to me! My only thought is that the default vsearch join-pairs settings might not work well for your read length and V3-V4. (Did you use 300 or 600 cycle kits?)
I feel like this under-classification is an important clue, but I'm not sure what to make of it.
Let's see if @thermokarst has any ideas about deblur's performance.
Hello!
I used 600 cycle kits. Previously, for other samples obtained in a similar way, I also used this pipeline and there were no problems.
I had the idea that the sample was contaminated during the amplification step and therefore the wrong sequences were obtained. Is it possible?
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It's totally possible! I was approaching this with the assumption that the data was fine and something went wrong with the pipeline. However, these could be low quality / contaminated samples.
Would you be will to post the artifact so we could see the provenance?
Yes, sure!
I hope this helps to avoid this problem in the future.
demux-joined-filter-stats.qza (16.3 KB)
I'm going to step in for @colinbrislawn, if it's okay with him, and try to do some trouble shooting as well.
I have two or three potential thoughts.
- Did you trim your primers? Did someone else remove your primers? You might try using cutadapt to trim and discard as a quality check.
- I'm assuming you quality filtered ahead of time?
- What taxa are they hitting? Is there a possibility of mislabeling anywhere along the pipeline (its more common than you might think)
Best,
Justine
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