Mismatch detected after align

Commands in mothur
1

Hi everyone!
I’ve an incompatibility problem after aligning with reference Silva. I would like to remind you that I’ve already done 4 times the commands to create the file names, then count_table, but the problem continues.
Can you help me?

1
CREATE GROUPS (8 groups)
mothur > make.group(fasta=S59.fasta-S60.fasta-S61.fasta-S62.fasta-S63.fasta-S64.fasta-S65.fasta-S66fasta, groups=T11-T12-T13-T14-T15-T16-T17-T18)
Output File Names: merge.groups
Rename [1merge.groups]

mothur > make.group(fasta=S67.fasta-S68.fasta-S69.fasta-S70.fasta-S71.fasta-S72.fasta-S73.fasta-S74fasta, groups=T21-T22-T23-T24-T25-T26-T27-T28)
Output File Names: merge.groups
Rename [2merge.groups]

2
REGROUP GROUPS
mothur > merge.files(input=1merge.groups-2merge.groups-3merge.groups-4merge.grups-5merge.groups-6merge.groups-7merge.groups-8merge.groups, output=caiomerge.groups)
Output File Names: caiomerge.groups

3
CREATE ARCHIVE (.fasta)
mothur > merge.files(input=S59.fasta-S60.fasta-S61.fasta-S62.fasta-S63.fasta-S4.fasta-S65.fasta-S66.fasta-S67.fasta-S68.fasta-S69.fasta-S70.fasta-S71.fasta-S72.fasta-S73.fasta-S74.fasta-S75.fasta-S76.fasta-S77.fasta-S78.fasta-S79.fasta-S80.fasta-S81.fasta-S82.fasta-S83.fasta-S84.fasta-S85.fasta-S86.fasta-S87.fasta-S88.fasta-S89.fasta-S90.fasta-S91.fasta-S92.fasta-S93.fasta-S94.fasta-S95.fasta-S96.fasta-S97.fasta-S98.fasta-S99.fasta-S100.fasta-S101.fasta-S102.fasta-S103.fasta-S104.fasta-S105.fasta-S106.fasta-S107.fasta-S108.fasta-S109.fasta-S110.fasta-S111.fasta-S112.fasta-S113.fasta-S114.fasta-S115.fasta-S116.fasta-S117.fasta-S118.fasta-S119.fasta-S120.fasta-S121.fasta-S122.fasta, output=caio)
Output File Names:caio

4
SUMMARY (caio.fasta)
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5
SCREEN SEQS
mothur > screen.seqs(fasta=caio, group=caiogroups, summary=caiosummary, maxambg=0, maxlength=305)
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6
UNIQUE SEQS
mothur > unique.seqs(fasta=caiogood)
22

7
COUNT SEQS
mothur > count.seqs(name=caiogoodnames, group=caiomerge.good.groups)
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8
SUMMARY SEQS
mothur > summary.seqs(fasta=caiogoodunique)
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9
PCR.SEQS
mothur > pcr.seqs(fasta=silva.bacteria.fasta, start=11894, end=25319, keepdots=F, processors=8)
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10
RENAME
mothur > rename.file(input=silva.bacteria.pcr.fasta, new=silva.v4.fasta)
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11
SUMMARY SEQS
mothur > summary.seqs(fasta=silva.v4.fasta)
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ALIGN
mothur > align.seqs(fasta=caiogoodunique, reference=silva.v4.fasta)
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13
SUMMARY SEQS
mothur > summary.seqs(fasta=caiogooduniquealign, count=caiogoodnamescount_table)

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1__#$!@%!#__Pasted Graphic 4.png723x88 4.18 KB

These were the commands I made.
Thanks a lot.

2

what is your amplicon? How did you generate the files that you put into make.groups? I’m surprised that all of your sequences are exactly 305 long???

3

Is this the same as your other thread - Summary.seqs = 25% of the base to be ambiguous? If so, could you close one so we aren’t doubling our efforts or working with incomplete information?

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