DADA2 Denoise-Paired

[Aditya_Ragil_Suharto]

Hi all,
i am confuse with my dada2 result, you can see it in the picture. am i doing something wrong when i do that? because the results is very low.

Here is my script for denoising

(qiime2-2019.1) aditya@biokomws9-BK135AA-AR6-s5389d:~/Baru$ qiime dada2 denoise-paired --i-demultiplexed-seqs Feses-demux.qza --p-trunc-len-f 244 --p-trunc-len-r 200 --o-table Feses_Table.qza --o-representative-sequences Feses_reps-seqs.qza --o-denoising-stats Feses_Denoise-stats.qza --verbose

and here is the result

[Nicholas_Bokulich]

Hi @Aditya_Ragil_Suharto,
You are probably losing sequences because the trimmed paired-end reads are not long enough to join. Check your Feses_Denoise-stats.qza file to see where sequences are being dropped, and check your expected amplicon lengths and your truncation parameters:

@Aditya_Ragil_Suharto

--p-trunc-len-f 244 --p-trunc-len-r 200

There are many discussions already on this forum about how to solve truncation/joining issues with dada2. I recommend reading those posts to gain insight if you are still having trouble.

[Aditya_Ragil_Suharto]

Thank you for the fast reply Mr. @Nicholas_Bokulich
i already check my denoise stats, but i dont know if it good or not. I also check my demux.qzv and i think i already truncate in the right point, sorry if iam wrong. or maybe i need to remove the adapter/primer first?

Here is my denoise stats

and this is my demux.qzv

[Nicholas_Bokulich]

@Aditya_Ragil_Suharto

i already check my denoise stats, but i dont know if it good or not.

See how the read counts drop off dramatically at the "merged" (a.k.a. joining) stage. This is clearly the issue.

@Aditya_Ragil_Suharto

I also check my demux.qzv and i think i already truncate in the right point

So you are choosing appropriate quality cutoff points, but this leaves too little sequence to successfully join the forward and reverse reads. You must calculate the expected amplicon length and how long the forward and reverse reads must be to achieve ≥ 20 nt of overlap. Check out other posts in this forum for more detailed examples and description of troubleshooting this.

Good luck!

[Aditya_Ragil_Suharto]

Thanks very much Mr. @Nicholas_Bokulich, i will try with –p-trunc-len-f 265 --p-trunc-len-r 219 and inform you the result later

[Aditya_Ragil_Suharto]

Hello again, god bless iam already finished my dada2 and the table.qzv data is better than before. But my denoise stats didn't look different than before. My answer is can i increase the table.qzv result? and did i do something wrong because my denoise stats is not different than before?.
Thank You..

This is my new Table.qzv result

and this is my latest denoise stats result

.

[Nicholas_Bokulich]

You must be looking at the wrong stats file, because the table summary indicates that you have many sequences, but the stats indicate that you have almost none.

[Aditya_Ragil_Suharto]

Thank you for the quick reply, my apologize that was right, iam input the wrong file, thank you very much for your help Mr. @Nicholas_Bokulich