Screen.seqs removed all sequence

Dear Researcher,
I am working on Southern Ocean Bacterial samples, Mothur 1.48.0. The sequence is V3-V4 region. Apparently all my sequence were removed by screen.seqs command.

mothur > make.file(inputdir=., type=fastq, prefix=akshay)
Setting input directories to: 
	/home/NCAOR/bhaskar/2022_2/


Output File Names: 
/home/NCAOR/bhaskar/2022_2/akshay.files


mothur > make.contigs(file=akshay.files, processors=36)

Using 36 processors.

>>>>>	Processing file pair 10_R1.fastq - 10_R2.fastq (files 1 of 12)	<<<<<
Making contigs...
Done.

It took 71 secs to assemble 669312 reads.


>>>>>	Processing file pair 11_R1.fastq - 11_R2.fastq (files 2 of 12)	<<<<<
Making contigs...
Done.

It took 16 secs to assemble 137554 reads.


>>>>>	Processing file pair 12_R1.fastq - 12_R2.fastq (files 3 of 12)	<<<<<
Making contigs...
Done.

It took 121 secs to assemble 1129375 reads.


>>>>>	Processing file pair 1_R1.fastq - 1_R2.fastq (files 4 of 12)	<<<<<
Making contigs...
Done.

It took 61 secs to assemble 566924 reads.


>>>>>	Processing file pair 2_R1.fastq - 2_R2.fastq (files 5 of 12)	<<<<<
Making contigs...
Done.

It took 24 secs to assemble 229411 reads.


>>>>>	Processing file pair 3_R1.fastq - 3_R2.fastq (files 6 of 12)	<<<<<
Making contigs...
Done.

It took 64 secs to assemble 627868 reads.


>>>>>	Processing file pair 4_R1.fastq - 4_R2.fastq (files 7 of 12)	<<<<<
Making contigs...
Done.

It took 96 secs to assemble 951887 reads.


>>>>>	Processing file pair 5_R1.fastq - 5_R2.fastq (files 8 of 12)	<<<<<
Making contigs...
Done.

It took 18 secs to assemble 165689 reads.


>>>>>	Processing file pair 6_R1.fastq - 6_R2.fastq (files 9 of 12)	<<<<<
Making contigs...
Done.

It took 56 secs to assemble 536747 reads.


>>>>>	Processing file pair 7_R1.fastq - 7_R2.fastq (files 10 of 12)	<<<<<
Making contigs...
Done.

It took 56 secs to assemble 542875 reads.


>>>>>	Processing file pair 8_R1.fastq - 8_R2.fastq (files 11 of 12)	<<<<<
Making contigs...
Done.

It took 94 secs to assemble 727186 reads.


>>>>>	Processing file pair 9_R1.fastq - 9_R2.fastq (files 12 of 12)	<<<<<
Making contigs...
Done.

It took 64 secs to assemble 559834 reads.


Group count: 
1	566924
10	669312
11	137554
12	1129375
2	229411
3	627868
4	951887
5	165689
6	536747
7	542875
8	727186
9	559834

Total of all groups is 6844662

It took 814 secs to process 6844662 sequences.

Output File Names: 
akshay.trim.contigs.fasta
akshay.scrap.contigs.fasta
akshay.contigs_report
akshay.contigs.count_table


mothur > summary.seqs(fasta=current, count=current)
Using akshay.contigs.count_table as input file for the count parameter.
Using akshay.trim.contigs.fasta as input file for the fasta parameter.

Using 36 processors.

		Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	1	249	249	0	1	1
2.5%-tile:	1	281	281	0	4	171117
25%-tile:	1	434	434	0	5	1711166
Median: 	1	441	441	0	6	3422332
75%-tile:	1	465	465	0	6	5133497
97.5%-tile:	1	473	473	7	17	6673546
Maximum:	1	500	500	282	250	6844662
Mean:	1	431	431	1	6
# of unique seqs:	6844662
total # of seqs:	6844662

It took 188 secs to summarize 6844662 sequences.

Output File Names:
akshay.trim.contigs.summary


mothur > unique.seqs(fasta=current, count=current)
Using akshay.contigs.count_table as input file for the count parameter.
Using akshay.trim.contigs.fasta as input file for the fasta parameter.
6844662	4483761

Output File Names: 
akshay.trim.contigs.unique.fasta
akshay.trim.contigs.count_table


mothur > summary.seqs(count=current)
Using akshay.trim.contigs.count_table as input file for the count parameter.
Using akshay.trim.contigs.unique.fasta as input file for the fasta parameter.

Using 36 processors.

		Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	1	249	249	0	1	1
2.5%-tile:	1	281	281	0	4	171117
25%-tile:	1	434	434	0	5	1711166
Median: 	1	441	441	0	6	3422332
75%-tile:	1	465	465	0	6	5133497
97.5%-tile:	1	473	473	7	17	6673546
Maximum:	1	500	500	282	250	6844662
Mean:	1	431	431	1	6
# of unique seqs:	4483761
total # of seqs:	6844662

It took 214 secs to summarize 6844662 sequences.

Output File Names:
akshay.trim.contigs.unique.summary


mothur > align.seqs(fasta=current, reference=silva.nr_v138_1.align)
Using akshay.trim.contigs.unique.fasta as input file for the fasta parameter.

Using 36 processors.

Reading in the silva.nr_v138_1.align template sequences...	DONE.
It took 156 to read  146601 sequences.

Aligning sequences from akshay.trim.contigs.unique.fasta ...
It took 6180 secs to align 4483761 sequences.

[WARNING]: 2409733 of your sequences generated alignments that eliminated too many bases, a list is provided in akshay.trim.contigs.unique.flip.accnos.
[NOTE]: 1994476 of your sequences were reversed to produce a better alignment.

It took 6181 seconds to align 4483761 sequences.

Output File Names: 
akshay.trim.contigs.unique.align
akshay.trim.contigs.unique.align_report
akshay.trim.contigs.unique.flip.accnos


mothur > summary.seqs(fasta=current, count=current)
Using akshay.trim.contigs.count_table as input file for the count parameter.
Using akshay.trim.contigs.unique.align as input file for the fasta parameter.

Using 36 processors.

		Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	0	0	0	0	1	1
2.5%-tile:	1044	1060	5	0	1	171117
25%-tile:	6388	25316	423	0	5	1711166
Median: 	6388	25316	440	0	5	3422332
75%-tile:	6388	25316	462	0	6	5133497
97.5%-tile:	43102	43116	465	1	13	6673546
Maximum:	43116	43117	500	250	250	6844662
Mean:	8824	24778	384	0	5
# of unique seqs:	4483761
total # of seqs:	6844662

It took 408 secs to summarize 6844662 sequences.

Output File Names:
akshay.trim.contigs.unique.summary


mothur > screen.seqs(fasta=current, count=current, summary=current, start=6388, end=43116)
Using akshay.trim.contigs.count_table as input file for the count parameter.
Using akshay.trim.contigs.unique.align as input file for the fasta parameter.
Using akshay.trim.contigs.unique.summary as input file for the summary parameter.

Using 36 processors.

It took 517 secs to screen 4483761 sequences, removed 4483761.

/******************************************/
Running command: remove.seqs(accnos=akshay.trim.contigs.unique.bad.accnos.temp, count=akshay.trim.contigs.count_table)
Removed 6844662 sequences from akshay.trim.contigs.count_table.
[WARNING]: akshay.trim.contigs.count_table contains only sequences from the .accnos file.

Output File Names:
akshay.trim.contigs.pick.count_table

/******************************************/

Output File Names:
akshay.trim.contigs.unique.good.summary
akshay.trim.contigs.unique.good.align
akshay.trim.contigs.unique.bad.accnos
akshay.trim.contigs.good.count_table


It took 825 secs to screen 4483761 sequences.

mothur > summary.seqs(fasta=current, count=current)
Using akshay.trim.contigs.good.count_table as input file for the count parameter.
Using akshay.trim.contigs.unique.good.align as input file for the fasta parameter.
[ERROR]: akshay.trim.contigs.unique.good.align is blank, aborting.
Using akshay.trim.contigs.unique.good.align as input file for the fasta parameter.
[ERROR]: akshay.trim.contigs.good.count_table is blank, aborting.

Using 36 processors.
[ERROR]: akshay.trim.contigs.unique.good.align is blank. Please correct.
Error in reading your fastafile, at position -1. Blank name.

mothur > quit()

Hi there -

You have this…

		Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	0	0	0	0	1	1
2.5%-tile:	1044	1060	5	0	1	171117
25%-tile:	6388	25316	423	0	5	1711166
Median: 	6388	25316	440	0	5	3422332
75%-tile:	6388	25316	462	0	6	5133497
97.5%-tile:	43102	43116	465	1	13	6673546
Maximum:	43116	43117	500	250	250	6844662
Mean:	8824	24778	384	0	5
# of unique seqs:	4483761
total # of seqs:	6844662

And you used start=6388, end=43116. This means your sequences need to start at or before 6388 and end at or after 43116. Evidently, nothing satisfies that criteria. I’d suggest using start=6388 and end=25316. Also, you need to remove the sequences with ambiguous base calls using maxambig=0 in screen.seqs

Finally, since you sequenced the V3-V4 regions, I’d recommend reading this:

Take care,
Pat

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