minimum start and end -1

Commands in mothur
1

Hi there,
I’m analyzing illumina data, and my summary after alignment produces a minimum start and end of both -1. I can’t seem to find anything on the forum regarding this… I know this is probably resulting from my screening parameters, and also leading to only unique taxonomy levels of taxonomy further down the pipeline… What’s causing this? What can I do to change this?

Thanks!
Jessica

2

That’s generally a sign that the alignment didn’t work, perhaps because the reads were in the wrong orientation. If you post all of your commands we can help you figure this out.

pat

3

I am having the same issue. My commands so far have been:

#make.contigs(file=RPSeasonal_group_16S.txt, processors=4)
#summary.seqs(fasta=RPSeasonal_group_16S.trim.contigs.fasta)
#screen.seqs(fasta=RPSeasonal_group_16S.trim.contigs.fasta, group=RPSeasonal_group_16S.contigs.groups, maxambig=0, minlength=250, maxlength=600, processors=4)
#unique.seqs(fasta=RPSeasonal_group_16S.trim.contigs.good.fasta)
#count.seqs(name=RPSeasonal_group_16S.trim.contigs.good.names, group=RPSeasonal_group_16S.contigs.good.groups)
#summary.seqs(fasta=current, count=current)
#align.seqs(fasta=RPSeasonal_group_16S.trim.contigs.good.unique.fasta, reference=~/data/clw224/mothur/silva.nr_v119.align, flip=T, processors=1)
#summary.seqs(fasta=current, count=current)

Thanks!
Candace

4

What percentage of your reads have start and end values of -1? Non-specific amplicons will do this when you align them and we generally just chuck them when we run screen.seqs in the next step and move on…

5

I did remove them with screen.seqs, but when I moved on to filter.seqs, it gave an error ([ERROR]: Sequences are not all the same length, please correct)
and then with pre.cluster ([ERROR]: your sequences are not all the same length. pre.cluster requires sequences to be aligned.), so I fear that something went awry with my alignment.

#screen.seqs(fasta=RPSeasonal_group_16S.trim.contigs.good.unique.align, count=RPSeasonal_group_16S.trim.contigs.good.count_table, summary=RPSeasonal_group_16S.trim.contigs.good.unique.summary, start=6388, end=25316, maxhomop=8, processors=4)
#summary.seqs(fasta=current, count=current)
#filter.seqs(fasta=RPSeasonal_group_16S.trim.contigs.good.unique.good.align, vertical=T, trump=., processors=4)
#unique.seqs(fasta=RPSeasonal_group_16S.trim.contigs.good.unique.good.filter.fasta, count=RPSeasonal_group_16S.trim.contigs.good.good.count_table)
#pre.cluster(fasta=RPSeasonal_group_16S.trim.contigs.good.unique.good.filter.unique.fasta, count=RPSeasonal_group_16S.trim.contigs.good.unique.good.filter.count_table, diffs=2)

6

Could you send your input files and log file to mothur.bugs@gmail.com?

RPSeasonal_group_16S.trim.contigs.good.unique.align
RPSeasonal_group_16S.trim.contigs.good.count_table
RPSeasonal_group_16S.trim.contigs.good.unique.summary
mothur.XXXX.logfile

7

I re-ran my alignment, and it must have been a glitch. I’m not getting those errors anymore downstream. Thanks for your help!