Short sequences after align.seqs

Commands in mothur
1

Hello, I am having issues with the align.seqs command. Everything is ok until I run it, which results in the same number of sequences but all with very few Nbases. This is the summary.seqs before aligning.

Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	1	301	301	0	3	1
2.5%-tile:	1	327	327	0	4	5481
25%-tile:	1	332	332	0	5	54803
Median: 	1	333	333	0	5	109606
75%-tile:	1	334	334	0	5	164409
97.5%-tile:	1	341	341	0	6	213731
Maximum:	1	500	500	0	8	219211
Mean:	1	334	334	0	5
Number  of unique seqs:	113403
total # of seqs:	219211

This is the summary.seqs after align.seqs

Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	0	0	0	0	1	1
2.5%-tile:	8633	9583	4	0	2	5481
25%-tile:	8633	9583	15	0	3	54803
Median: 	8633	9583	60	0	3	109606
75%-tile:	8688	9583	62	0	4	164409
97.5%-tile:	9578	9583	63	0	5	213731
Maximum:	9583	9583	254	0	8	219211
Mean:	8710	9437	46	0	3
Number of unique seqs:	113403
total # of seqs:	219211

I followed MiSeqSOP and used the coordinates without the primers. I’ve been running the same analysis on a total of 6 datasets, and for three I can get to the classify.otu but for three I have to stop here because of the shortness of the sequences. Let me know if there is anything I can do, thanks!

2

Hi - what region of the 16S rRNA gene did you sequence? Are you using the V4 alignment that we use in the SOP or something else?

pat

3

Hi! I was working with data provided by another lab member and assumed it was V4, and it was not! Thank you for making me double check, I’ll follow the post on how to generate a database on a different region.

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