High sequences merged but low non-chimera sequences

User Support
1

Hi, I'm running Qiime dada2 denoise-paired and have some weird results.

First, my sequence quality is good. The 25th percentile at every position is > Q30. I only have some outliers at Q20.

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Then, I run a script and ensure I don't want to truncate or trim any sequence.

qiime dada2 denoise-paired
--i-demultiplexed-seqs demultiplexed-sequences.qza
--p-trunc-len-f 0
--p-trunc-len-r 0
--p-n-threads 20
--o-representative-sequences asv-sequences-0.qza
--o-table feature-table-0.qza
--o-denoising-stats dada2-stats.qza

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Some sequences are still being filtered. But I still have a lot of merged sequences. Anw, the sequences of non-chimera is only 2%.

I don't know any problem with the --p-chimera-method?

2

Hello!
Did you try to remove primers first? You can use q2-cutadapt plugin to remove them. Primers in sequence may increase the number of false positives at chimera removal step.
Still I am surprised with the number of chimeras you are getting.
I would try to remove primers as the first option and see if it will work or not.

2 Likes
3

Thank you for your advice.
It really works after I remove the primers.
Yeah, I'm surprised with the number of non-chimera because I thought that it's biological sequences and it would not highly impact with my data.

2 Likes
4

Glad that it worked out!
Degenerative primers may mess with the chimera detection algorithm, so I guess it was a case with your data.