Training classifier against V4 region

User Support
1

Hi,

16s V4 region was amplified by using the PCR primers (F515/R806), and sequencing read length for both pair end data is 250bp. so in case of training classifier should I use

–p-min-length 200
–p-max-length 300 \

KInd Regards
Yogesh

2

Hi! I think if you have paired reads you should increase max length up to 500. Please correct me if I am wrong.

3

Hi @timanix,

Actually, I am not able to understand this if we amplifying the region which is present between F 515 and R 806 (F515/R806) then why merged read length is more than 300 bp.

4

Sorry, I misread your message. I was thinking about V3-V4 at the same time.

5

Hi @timanix,

Is there any way to see the maximum and minimum read length from dada2 output?

6

Yes, try to run with your filenames

qiime feature-table tabulate-seqs \
    --i-data rep-seqs.qza \
    --o-visualization rep-seqs.qzv
7

@timanix,

Thanks, it says min. is 229 and max is 436. I am not able to understand it when fragment length between forward and reverse primer is 291, how it can be 436.

Does this length between forward and reverse primer vary between bacterial phyla?

8

It can be up to 390 - 400. There is also a possibility of chimeras but they should be removed. Maybe you should use 200 as min and 400 as max. Let’s wait for the answer of more advanced users.

9

Hi @Nicholas_Bokulich,

Could you please suggest what min. and max length should I use in training classifier:

dada2 output shows min read length is 229 and max is 436.

Kind Regards
Yogesh

10

do as @timanix advised.

1 Like
11

Hi,

I do have query regarding dada2 output file rep-seqs.qza:

16s V4 region was amplified by using the PCR primers (F515/R806), and sequencing read length for both pair end read is 250bp.

dada2 rep-seqs.qzv file gives min. seq. length is 229 and max is 436. I am not able to understand it when fragment length between forward and reverse primer is 291, how it can be 436

Kind Regards
Yogesh

12

You already answered your own question above, and a quick perusal of the literature would give you more hard data to confirm this:

Yes.

13

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