V4 region cutadapt

User Support
1

Good evening to all,

I'm using cutadapt on 4 datasets to trim from v3-v4 regions, and keep only V4

All the data sets are in single-end format using flash.

From the 4 datasets, the 3 are normally trimmed based on the primer GTGYCAGCMGCCGCCGCGGTAA (515)

However, when I used the primer again, 1 data set, did not find it and every time I used the 5end it returned only 5, 10, 3 sequenced that have this primer and so on. I used different parameters and again no luck.

But from a paper doi: 10.1128/mSphere.01202-20, I found this primer GACTACHVGGGTATCTAATCC, which is the reverse primer when sequencing v3-v4.

And normally in my 5 end it recognized the primer and returned sequences that it has recognized 112365 times e.g. in each sample. S o it works!!!

Is it correct to assume that the sequences I have now are from the V4 region?

Thank you all for your help!!

1 Like
5

Hello @iordanis,

Just to be clear:

  • you have four sets of paired end reads, each of which has been merged and now you have four sets of merged reads
  • you are using a single primer that matches in the middle of the sequence to extract the v4 region and you want to keep everything downstream (i.e. towards the 3' end)
  • this is working as you expected for three of the sets but is failing for the fourth

Is this all correct?

Were these four sets of reads generated using precisely the same primer sets? Were they merged using precisely the same parameters to the same merging algorithm/software?

4 Likes
6

Hello @colinvwood,

Yes, i have 4 datasets, all datasets have v_region v3_v4 paired end reads, and all of them have contract with different protocols, different sequence technology like Miseq, Hiseq and so on.

All dataset merged with flash separately.

Then i use this primer GTGYCAGCMGCCGCCGCGGTAA (515), to keep the V4 region for all datasets. And it is work for 3 datasets.

But for one dataset, this primer don't recognized in 5 end of sequence.

Specially this dataset generated with:

The V3-V4 hypervariable region of the bacterial 16S ribosomal RNA (rRNA) gene was amplified from the DNA samples with the barcoded forward primers 341F (50 -CCTACGGGNBGCASCAG-30 ) and the reverse primers806R (50 -GGACTACNVGGGTWTCTAAT-30 ) using KAPA
HIFI HotStart ReadyMix (KAPA Biosystems, United States).

So when i use a reverse primer for v4, recognize it at 5 end of sequence. So i dont know what happen.

I hope to be understood!!
Jordan

1 Like
8

Hello @iordanis,

Can you share the command(s) you're running to perform the trimming on these datasets? Can you also attach the demux visualizations for each?