Align.seq and filter problems

Hi, Im a new beginner,and I’m using mothur v.1.45.2. I followed the SOP for my samples that generated from illumina iseq as fastq, everything was good until the align.seq step where i got
I get a bit confused as I read if the start and end at same position then the alignment is weak. and when I complete I found that the length of the sequences was decreased to 46 bp after filter.seq commands.
Can anyone tell me what is the problem?

Hi - can you tell us what you are sequencing? I suspect you don’t have V4 sequences since not much is aligned. Do you maybe have V4-V5 data instead?


Stool sample
One of my colleagues tried my data but she had an old version, the process moved smooth and the results were good… I don’t know does this version has a problem or should I add any necessary step.

What region are you sequencing?

We amplify V3- V4 region.

If you’re following the MiSeq SOP, the reference alignment you are using is for the V4 region. See Customize your reference alignment for your favorite region to learn how to generate the positions for the V3-V4 region. I also wonder if your sequences aren’t backwards. You might try flip=T in align.seqs to try both orientations. Since you are sequencing the longer region, you’l also want to be aware of this blog post…

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