How to choose start and End point for screen.seq command

Commands in mothur
1

Hi guys,

After Align.sequence step, my sequence summary looks like this

Summary.seq_output
Summary.seq_output839×368 12.2 KB

I am finding it difficult to choose start-end position for screen.seqs command, since start end position is varying all over the place in the sequence data. Does this mean, sequences were not aligned properly? or having some issues with in it. how do i go about further in this scenario.

The sequence data sets were generated by V3-V4 primers.
and i am using Mothur 1.44.0 version

Many thanks in advance

Venkat

1 Like
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For MiSeq sequence data, that table looks weird. You should see the same start/end position number in the 25th, median, and 75th percentiles. If you took E. coli’s 16S rRNA gene sequence and trimmed it to the region amplified by typical V3-V4 primers, you would get a start position of 6428 and and end position of 23444 (you can see how to do this at https://mothur.org/blog/2016/Customization-for-your-region/

I wonder whether you trimmed your sequences before running make.contigs or if you assembled your reads elsewhere before starting in mothur.

3

HI pat,
Thanks for your reply. Yes, your right. This time, i have used an external s/w afterQC which does automated trimming, error corrections in the raw sequence data and then proceed further with Make.contigs. So, i presume it might be the reason for the same.

Venkat

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Can you start over with the raw fastq files within mothur?

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HI Pat,

Thanks, the problem is solved, after starting over all the steps in mothur. However, i am having another issue, which i will post separately

Thanks again

Venkat

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