regarding the barcodes

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1

Hello everyone

i am sorry to asking this silly question . I have single end reads v3-v4 16S region. My question is it mandatory to remove barcodes from the sequence? and if it is mandatory then how can we checked whether barcodes is present or not?

2

Hello again,

This is a good question. Not silly at all.

Yes, if your reads contain barcodes, then you should remove them. Some primer and sequencing methods do not contain the barcode in the main read (like the EMP protocol).

Did you do Illumina sequencing?

Did you get

  • Read 1 forward, Read 2 reverse, Index
  • Read 1 forward, Read 2 reverse
  • something else?
3

i got only single read which is in sff format then i have converted into fastq file.
The illumina sequencing has done with only barcodes with 3 replicates with each biological event. there is no primers has given and the region which is amplifies that is V3- V4 region.

So how can i proceed this major steps in qiime2

5

Ah OK. I was not sure if you had a new data set, or if this was the data set from your last thread:

My advice is the same. Use cutadapt, which will remove barcodes for you!
https://docs.qiime2.org/2020.2/plugins/available/cutadapt/

Given that you have forward reads, I would suggest
qiime cutadapt demux-single

Let me know if you have any questions!
Colin

10

then again i have question that how can i make the multiplexed sequence…
this is the data from the last thread and again i am doing with new start up. because there are so many consequences which is not able to clear so i am doing again with fresh up.

11

The output of qiime cutadapt demux-single is the demultiplexed sequences (you could call it demux.qza) that is mentioned in this step of the Moving Pictures tutorial.

Once you have made your demux.qza file using qiime cutadapt demux-single, you can follow the next steps in the Moving Pictures tutorial.

12

so sir again sorry for ask silly question…
Now i have to follow the emp protocol rather than casava

13

and i have question that how can we described the raw file is emp or casava

16

Take a look at this tutorial:

17

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