The number of features is normal but the number of observed_otus is too low?

Thanks, I noticed you have lost a lot after merged sequences to non-chimeric.

I think just reviewing this would be interesting (you're losing up to 75% of your reads between merged / non-chimeric.

This may explain why you los resolution. I sometimes lose up to 50% of the reads, but I think that's a lot of reads to lose on the last step.

As you can see, I don't lose a lot of sequences between merged and non-chimeric. See this:

Also try here:

Ben

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Actually, I just trouble shot this for another lab, here is my code for the V3V4 region:

qiime dada2 denoise-paired
–i-demultiplexed-seqs ~/QIIME2_2_import/paired-end-demux.qza
–p-trim-left-f 13
–p-trim-left-r 13
–p-trunc-len-f 270
–p-trunc-len-r 270
–p-n-threads 0
–o-table ~/QIIME2_3_demux/table_540.qza
–o-representative-sequences ~/QIIME2_3_demux/rep-seqs_540.qza
–o-denoising-stats ~/QIIME2_3_demux/denoising-stats_540.qza

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Hi Ben,
Super thanks for your suggestion:smiley:! Now Im trying to figure out this and I will read everything as you recommended.:muscle:

Best,
Sandro

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Hi Ben,
Your suggestion helped me sososo much!!! :blush::blush: Thank you for finding the problem in my qzv-result. And I finished reading the website you recommended, which helped me to know what I can do for the next step.
Super thanks.:green_heart::green_heart:
I will update my result.

Best,
Sandro

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Yes, let me tell you I have run against this same problem, after I fixed it I got better beta-diversity distributions. Could you confirm that your PCOAs look the same or better? Thank you. Ben

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Hi Yue,

I found someting interesting in your primers for V3-V4 region. Could you please tell me what’s the length of your amplicons or the location of the primer pair? what kind of platform did you use for NGS (Miseq or Hiseq or Nova 6000)? Thanks

Best,

Decen

Hi Decen,
Thank you for your help.:grinning: Sorry for my late reply.
The length of my amplicons is 460 and the primers are CCTACGGRRBGCASCAGKVRVGAAT(upstream) and GGACTACNVGGGTWTCTAATCC(downstream). The platform is Illumina Miseq. Is there something wrong?:face_with_monocle:

Best,
Yue

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Hi Yue,

Nothing serious.
It looks they are not “common degenerate primer”, when I try to test your primer pair in SILVA “https://www.arb-silva.de/search/testprime/”, it cannot past. I am using the communal primers, amplicon length ~470bp. If you like, Could you please share how comes for your primer pair? Thanks
Best,