mothur

Why am I getting this error message "HM-1_Analysis/fileList.paired.trim.contigs.good.fasta is blank, aborting"?

Hi,
I am new to bioinformatics.Please help me.
When i try the below command, I get error message saying HM-1_Analysis/fileList.paired.trim.contigs.good.fasta is blank, aborting.
What does it mean? is there any issue with sequencing data quality?

mothur > summary.seqs(fasta=fileList.paired.trim.contigs.good.fasta)

Unable to open HM-1/fileList.paired.trim.contigs.good.fasta. Trying default /usr/local/bin/fileList.paired.trim.contigs.good.fasta

Unable to open /usr/local/bin/fileList.paired.trim.contigs.good.fasta. Trying output directory HM-1_Analysis/fileList.paired.trim.contigs.good.fasta

[ERROR]: HM-1_Analysis/fileList.paired.trim.contigs.good.fasta is blank, aborting.

Using HM-1_Analysis/fileList.paired.trim.contigs.good.fasta as input file for the fasta parameter.

Using 12 processors.

[ERROR]: HM-1_Analysis/fileList.paired.trim.contigs.good.fasta is blank. Please correct.

Error in reading your fastafile, at position -1. Blank name.
Your reply is highly appreciated.
thanks

Welcome to the mothur community! When the screen.seqs command removes all the reads you can get a blank file error in commands that follow. What do the summary results look like for the fileList.paired.trim.contigs.fasta file? What inputs did you use with the screen.seqs command?

thanks for your reply.
I rectified that issue by changing few parameters.

Now, I am facing issue with the below command:
mothur > screen.seqs(fasta=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.align, count=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.count_table, summary=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.summary, start=1, optimize=end)
error message:
IC1 because all sequences have been removed.

Removing group: IC10 because all sequences have been removed.

Removing group: IC11 because all sequences have been removed.

Removing group: IC12 because all sequences have been removed.

Removing group: IC13 because all sequences have been removed.

Removing group: IC14 because all sequences have been removed.

Removing group: IC15 because all sequences have been removed.

Removing group: IC16 because all sequences have been removed.

Removing group: IC17 because all sequences have been removed.

Removing group: IC18 because all sequences have been removed.

Removing group: IC19 because all sequences have been removed.

Removing group: IC2 because all sequences have been removed.

Removing group: IC20 because all sequences have been removed.

Removing group: IC21 because all sequences have been removed.

Removing group: IC22 because all sequences have been removed.

Removing group: IC23 because all sequences have been removed.

Removing group: IC24 because all sequences have been removed.

Removing group: IC25 because all sequences have been removed.

Removing group: IC26 because all sequences have been removed.

Removing group: IC27 because all sequences have been removed.

Removing group: IC28 because all sequences have been removed.

filter.seqs(fasta=fileList.paired.trim.contigs.good.unique.good.align, vertical=T, trump=.)

mothur > filter.seqs(fasta=fileunique.good.align, vertical=T, trump=.)

Using 18 processors.

[ERROR]:
mothur> fileList.paired.trim.contigs.good.unique.good.align is blank. Please correct.

Error in reading your fastafile, at position -1. Blank name.

Creating Filter…

[ERROR]:
mothur> fileList.paired.trim.contigs.good.unique.good.align is blank. Please correct.
kindly help in this case.
i don’t know how to proceed further if the file is blank.
thanks!
SS

The start and end parameters in the screen.seqs command are likely what is causing the samples to be removed. Can you post the results of running this command?

mothur > summary.seqs(fasta=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.align)

hello,
This is the result of mothur> summary.seqs(fasta=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.align)

image
Start End NBases Ambigs Polymer NumSeqs
Minimum: 0 0 0 0 1 1
2.5% tile 1043 1061 4 0 1 76444
25% tile 1043 11891 433 0 4 764432
median 1043 11891 464 1 5 128864
75% tile 1043 11891 465 3 5 2293295
97.5% tile 10368 11891 478 7 7 2981283
Maximum 11891 11892 517 8 8 3057726

of unique seqs:2468833

total #of seq:3057726

Output File Names:

/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.summary

Next command given was…

mothur>screen.seqs(fasta=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.align, count=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.count_table, summary=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.summary, start=1, optimize=end)

This command removed all 96 samples…
Removing group: IC1 because all sequences have been removed.

Removing group: IC10 because all sequences have been removed.

Removing group: IC11 because all sequences have been removed.

Removing group: IC12 because all sequences have been removed…

Next command was…
mothur > filter.seqs(fasta=/scratch/sathya/HM2_Analysis/fileunique.good.align, vertical=T, trump=.)

Using 18 processors.

[ERROR]: /scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.align is blank. Please correct.

Error in reading your fastafile, at position -1. Blank name.

Creating Filter…

[ERROR]: /scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.align is blank. Please correct.

mothur> summary.seqs(fasta=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.fasta, processors=18)

Start End NBases Ambigs Polymer NumSeqs

Minimum: 1 291 291 0 3 1

2.5%-tile: 1 301 301 0 4 293503

25%-tile: 1 505 505 2 5 2935029

Median: 1 522 522 5 5 5870057

75%-tile: 1 534 534 11 6 8805085

97.5%-tile: 1 570 570 35 35 11446611

Maximum: 1 602 602 299 301 11740113

Mean: 1 499.528 499.528 7.73476 6.84619

of Seqs: 11740113

The start parameter is used to set a location that “good” sequences must start by. The end parameter is used to set a location that “good” sequences must end before. You want to select start and end values that screen sequences for good overlap. This helps reduce spurious OTUs downstream.

image

With a start value of 1, all reads that start after position 1 are removed (most if not all reads). Have you tried setting start=1043, end=11891?

Hello Westcott,
I did as you said. , it worked. thank you… ut showing error in chimera.uchime…please help
mothur> screen.seqs(fasta=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.align, count=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.count_table, summary=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.summary, start=1043, end=11891, processors=18)

Output File Names:

/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.summary

/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.align

/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.bad.accnos

/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.good.count_table

mothur> filter.seqs(fasta=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.align, vertical=T, trump=.)

Length of filtered alignment: 1560

Number of columns removed: 10333

Length of the original alignment: 11893

Number of sequences used to construct filter: 1975899

mothur>unique.seqs(fasta=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.filter.fasta, count=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.good.count_table)
out put generated…

mothur>pre.cluster(fasta=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.filter.unique.fasta, count=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.filter.count_table, diffs=2),this command was ok…

mothur>chimera.uchime(fasta=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.filter.unique.precluster.fasta, count=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.filter.unique.precluster.count_table, processors=12, dereplicate=t)
chimera command showed error
mothur > chimera.uchime(fasta=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.filter.unique.precluster.fasta, count=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.filter.unique.precluster.count_table, processors=1, dereplicate=t)

Using 1 processors.

uchime by Robert C. Edgar

http://drive5.com/uchime

This code is donated to the public domain.

Checking sequences from /scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.filter.unique.precluster.fasta …

packet_write_wait: Connection to 142.103.74.203 port 22: Broken pipe

tried several times, it says the same…
it was running for the first time, it was running for long time, so i quit the programme and rerun the chimera command with more processors, it did not run at all.
what is the reason? is it because i stopped the programme in the middle?
kindly help…
SS

Are you seeing the error with chimera.vsearch?

I used chimera.uchime.

Could you try this?

mothur > chimera.vsearch(fasta=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.filter.unique.precluster.fasta, count=/scratch/sathya/HM2_Analysis/fileList.paired.trim.contigs.good.unique.good.filter.unique.precluster.count_table, processors=1, dereplicate=t)

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