RE: Samples missing from DADA2 table but not denosing stats

[Catherine]

Samples missing from DADA2 table but not denosing stats

Hi @saatkinson, It looks like those 5 samples are the only 5 that have 0 reads post-filtering: [image] I think the feature table must be made after denoising, dropping all samples that have 0 sequences, and then chimera removal happens after that — @benjjneb is that correct? Those 5 samples (and many others) cannot be revived any way you slice it, since the input read counts are so low. However, you are losing many reads at the filtering step, which leads me to believe you might be able t…

hi，@
Nicholas_Bokulich,I have the same problem with her. I have 24 samples, but after denoising there are 18 sample left. It seems that the missing samples are all zero non-chimeric, the image as follows:

In the process of denoising, I reserved the area with good quality to the maximun according to demux result, which is shown in the follow:qiime dada2 denoise-paired --i-demultiplexed-seqs test-24.qza --p-trunc-len-f 299 --p-trunc-len-r 276 --output-dir result
ps:the qiime version 2019.1,I have 301 nts sequences

[ben (benjamin w.)]

Yikes, not sure I have any help to offer, but does this mean that all sequences failed due to quality? @benjjneb

Out of curiosity, could you post your quality scores? Ben

[Catherine]

I don't quite understand what you mean. I'd like to repeat the problem in its entirety.
First, I input my sample sequences(24 sample/48 sequences), creat and enter the absolute working path at the same time. Then the data format was been standardized. After that the step of "qiime demux summarize" was run trouble-free. The 24 samples‘ result was shown as follows:

Based on the results above, I decided to use the following codes to denoise:
time qiime dada2 denoise-paired \
--i-demultiplexed-seqs cecum.qza \
--p-trunc-len-f 299 \
--p-trunc-len-r 280 \
--output-dir result
To my confusion, 6 samples were missing from the pair-end denoise results.

Are there any mistakes in my previous steps? Did I pick the wrong criteria?
I really need those samples. Can you give me a hand?

[ben (benjamin w.)]

No, I don’t think you did anything wrong.

all 24 samples were demuxed with adequate sequencing depth.

all 24 samples lose all sequences after filtering step in DADA2/denoise.

It’s just strange to see you lose all of the sequences. Ben

[Nicholas_Bokulich]

@Catherine

–p-trunc-len-f 299
–p-trunc-len-r 280 \

I think these values are too high. Try truncating more, maybe 290 + 260 (or shorter if you can afford it)

Good luck!

[ben (benjamin w.)]

Agreed with Nicholas

• forward I would truncate @ 240-260
• reverse I would truncate @ 160-180