How to determine DADA2 trimming values for pair-end sequences?

Hi,
I am not certain on how to pick a trimming value for my 16S rRNA amplicon sequencing data. While forward sequence looks good, I am not sure about the reverse, please see the image below. From what I have read in the forum, I feel the reverse sequence should be OK without trimming as the median quality scores are pretty high throughout. What is the best way of determining a trimming value, particularly for my data. Thanks in advance for any help.

Cheers
Hasinika

Hey there @Hasinika!

Looks like your attachment didn’t work, can you please try again?

Thanks for pointing that out, I think the attachment is now successful.

Hey there @Hasinika - the file is still missing.

You will need to click this button:

50%20AM

Then click on "Browse"

Once it is done uploading there will be some indication in your edit window:

Don't delete the lines that look like this: ![16%20AM|690x252](upload://gDlqp1ISroj6iLmCXTbHzl13Otf.png), these are the attachments themselves.

Give it a shot again and then we can talk about trimming!

:qiime2: :t_rex:

Hey @Hasinika! Looks like the post needed a little clean-up on our end - you screenshot is there now. Stay tuned for someone to get back to you!

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Hey there @Hasinika!

I would highly encourage you to explore a handful of different criteria and evaluate their impact on this study, in particular taxonomic classification, differential abundance, and diversity. This will be the best way (IMO) for you to understand the impact of trim and trunc params.

Okay, so maybe it would help for us to give you some concrete starting point:

Maybe as a first pass I would use Q30 (median) as a starting point for these data. So, eyeballing from the static image you provided above:

--p-trim-left-f 0 \
--p-trim-left-r 0 \
--p-trunc-len-f 244ish \
--p-trunc-len-r 170? \

The thing to keep in mind is that DADA2 will need at least 20nts of overlap after trim/trunc in order to successfully join the pairs.

Keep us posted!
:qiime2: :t_rex:

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