Odds sequence in Table

[jskim]

Dear community

Hello. I am a complete beginner who has just started analyzing the stool microbiome.

Due to poor quality in the reverse sequence, I created a feature table using only the forward sequence, but I noticed a repetitive odd sequence like "TTTTT...".

I used following commands.

qiime dada2 denoise-single
--i-demultiplexed-seqs demux-filtered.qza
--p-trim-left 0
--p-trunc-len 145
--o-representative-sequences rep-seqs-dada2.qza
--o-table table-dada2.qza
--o-denoising-stats stats-dada2.qza

qiime metadata tabulate
--m-input-file stats-dada2.qza
--o-visualization stats-dada2.qzv

qiime feature-table summarize
--i-table table-dada2.qza
--o-visualization table.qzv
--m-sample-metadata-file /Forward/metadata.tsv

qiime feature-table tabulate-seqs
--i-data rep-seqs-dada2.qza
--o-visualization rep-seqs-dada2.qzv

qiime tools view rep-seqs-dada2.qzv

Any feedback is welcome.

Thanks,
JS

[colinvwood (Colin Wood)]

Hello @jskim,

Did you perform any QC steps before dada2? Do you know the details of the library prep of these sequences? Can you post the interactive quality plot from the demux visualizer?

[jskim]

Thank you for your messages.
I am not sure if this is adequate responses for your asking.

I used following command.
SingleEndFastqManifestPhred33V2
qiime tools import
--type 'SampleData[SequencesWithQuality]'
--input-path /Forward/metadata.tsv
--output-path single-end-demux.qza
--input-format SingleEndFastqManifestPhred33V2

Summarize data
qiime demux summarize
--i-data single-end-demux.qza
--o-visualization demux.qzv

qiime quality-filter q-score
--i-demux single-end-demux.qza
--o-filtered-sequences demux-filtered.qza
--o-filter-stats demux-filter-stats.qza

qiime metadata tabulate
--m-input-file demux-filter-stats.qza
--o-visualization demux-filter-stats.qzv

Generating a feature table (“table-dada2.qza”) : denoise
qiime dada2 denoise-single
--i-demultiplexed-seqs demux-filtered.qza
--p-trim-left 0
--p-trunc-len 145
--o-representative-sequences rep-seqs-dada2.qza
--o-table table-dada2.qza
--o-denoising-stats stats-dada2.qza

The sequencing company confirmed that they thoroughly removed the adapter sequences.

And I decided to use forward sequence only due to poor quality of reverse sequence

I can't guess what this means.

Thank you very much,
JS

[colinvwood (Colin Wood)]

Hello @jskim,

Is this 16S data, if so which region? Could you attach the demux.qzv, rep-seqs-dada2.qza and table-dada2.qza files if you feel comfortable doing so? By the way, a separate quality score filtering step is not usually recommended before using dada2.

[jskim]

Yes, sure. Thank you very much for your response.

These were derived from the V4 region of 16S rRNA data analyzed using fecal samples.

demux.qzv (291.2 KB)
rep-seqs-dada2.qza (156.7 KB)
table-dada2.qza (168.0 KB)

Thanks,
JS

[colinvwood (Colin Wood)]

Hello @jskim,

My guess would be that something went wrong during library preparation. Many of the sequences with the repetitive T subsequence are still primarily 16S according to blast, possibly indicating that the subsequence got ligated somehow during library prep. There are other sequences that are completely erroneous such as e768bf26ed64ea9eb0f8b5bfeb635de5 and e6db6cbc2e34fe7e75470a1faab99922.

At this point it's probably a question of how much faith you have in the results overall and whether you want to salvage what you can.

Out of curiosity do you know which Illumina model these were sequenced on? Also, could you attach the demux.qzv for the reverse sequences if possible?