Hi, I am analyzing MiSeq data of nifH gene with a nifH databasein fasta format http://www.css.cornell.edu/faculty/buckley/nifh.htm. But, Mothur removing all sequences in the groups with following message— “Removing group: CB1_NP because all sequences have been removed” while using screen.seqs command.
Please suggest.
The message would appear to indicate that your screen.seqs command is too harsh, and is removing all of your sequences. Perhaps you need to alter some of the parameters to be more relaxed?
Yes, i am just running command without any parameters. Further, while using filter.seqs and unique.seqs commands, Mothur removing all data and making blank output file for chimera.uchime command.
Let me show the process------------
summary.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table)
Using 1 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 0 0 0 0 1 1
2.5%-tile: 247 247 1 0 1 184
25%-tile: 358 1097 8 0 2 1837
Median: 364 1103 367 0 6 3673
75%-tile: 534 1103 386 0 7 5509
97.5%-tile: 1686 1772 392 0 7 7161
Maximum: 1772 1772 427 0 12 7344
Mean: 635.301 1109.05 237.035 0 4.57979
of unique seqs: 6840
total # of seqs: 7344
screen.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table, summary=stability.trim.contigs.good.unique.summary)
Output File Names:
stability.trim.contigs.good.unique.good.summary
stability.trim.contigs.good.unique.good.align
stability.trim.contigs.good.unique.bad.accnos
stability.trim.contigs.good.good.count_table
It took 2 secs to screen 6840 sequences.
filter.seqs(fasta=stability.trim.contigs.good.unique.good.align, vertical=T, trump=.)
Length of filtered alignment: 0
Number of columns removed: 1772
Length of the original alignment: 1772
Number of sequences used to construct filter: 4808
Output File Names:
stability.filter
stability.trim.contigs.good.unique.good.filter.fasta
unique.seqs(fasta=stability.trim.contigs.good.unique.good.filter.fasta, count=stability.trim.contigs.good.good.count_table)
Output File Names:
stability.trim.contigs.good.unique.good.filter.count_table
stability.trim.contigs.good.unique.good.filter.unique.fasta
pre.cluster(fasta=stability.trim.contigs.good.unique.good.filter.unique.fasta, count=stability.trim.contigs.good.unique.good.filter.count_table, diffs=0)
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.W1_100.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.W4_100.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.W5_100.map
chimera.uchime(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table, dereplicate=t)
[ERROR]: stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.chimerasW5_100 is blank. Please correct.
You have to select start and end positions in screen.seqs. you might try start=534 / end=1103. This will keep anything that starts before 534 and ends after 1103.
Pat
Thanks, I got understand. 