How to analyse single end and paired end sequences together?

[Shreya (panchkarma)]

I'm using QIIME2- 2023.5. I looked over some other forum responses to the same subject and became even more confused. I have paired-end sequences of v3-v4 region (Illumina primers) (232 bp) and publicly available single end data (Primer pair Probio_Uni and Probio_Rev were used to amplify the V3 region. To target the V4 region of the 16S rRNA gene, a primer pair of 520F and 802R was used) (631 bp) .

1. How should I proceed to trim my data?
2. After trimming how to analyze the single end and paired end reads together?
   
   
   
   
   

Thank you in advance.

[colinbrislawn (Colin J Brislawn)]

Hello @Shreya,

EDIT: I've rewritten this post. I hope it's easier to follow.

@Shreya

1. How should I proceed to trim my data?

If you choose to use DADA2, trim where the quality drops to maximize total reads passing through the DADA2 pipeline!

This thread has some good advice.

I usually run DADA2 with a few different truncating settings to see what works best.

@Shreya

2. After trimming how to analyze the single end and paired end reads together?

Because DADA2 builds an error profile for each sequencing run, we recommend processing each run separately.

Once you have DADA2 results for each run, you can choose how to combine them. This is pretty easy if all your reads are from the same region!

Because you have data from different regions, this may be tricky...
Do both runs fully cover the same part of V3?

Or it may be impossible!

Multiple Regions of 16s Sequenced

Hi @ChaseR, You’ll need to analyze those separately for most QIIME 2 analyses to make sense. The issue is that you can have V8/V9 reads from the same organism as a V4/V5 read, but (outside of taxonomy assignment, or closed-reference OTU picking which isn’t yet supported in QIIME 2) QIIME wouldn’t know how to relate those sequences to one another. Most of the analyses that you’ll run in QIIME are not dependent on taxonomy (e.g., alpha and beta diversity), so for those analyses a V8/V9 read from …

Working with multiple regions is a big challenge, so let us know if you have more questions!

[Shreya (panchkarma)]

@Shreya

How should I proceed to trim my data?

Let me rephrase my question: As both the data have been processed in different systems (Illumina/Ion Torrent), having different base pair length, how shal i trim them to the same length, because the 600 bp region sequences may differ from the 250bp region sequences as far as ASV determination is concerned. How to proceed? Thanks

[colinbrislawn (Colin J Brislawn)]

You can trim the read length when running DADA2.

For IonTorrent use denoise-pyro: Denoise and dereplicate single-end pyrosequences — QIIME 2 2024.2.0 documentation
For Illumina paired-end use denoise-paired: Denoise and dereplicate paired-end sequences — QIIME 2 2024.2.0 documentation

@Shreya

the 600 bp region sequences may differ from the 250bp region sequences as far as ASV determination is concerned

That's correct!

Are you asking how to trim the V3-V4 to match the shorter V3?