No stability.trim.contigs.fasta as output of make.contigs

Commands in mothur
1

Hi
I am trying to use make.contigs on my Miseq data but didnt get stability.trim.contigs.fasta file as output.
My input was F_L001_R1.fastq and F_L001_R2.fastq through stability.files, in which i have 176 sample which are demultiplexed here. As mention in Miseq SOP i didnt get separate R1 + R2 file for each sample from the company. hence i need to demultiplex them using Trim.seqs afterwards. I guesss it is OK
As output i got F_L001_R1.0rqualtemp, F_L001_R1.0rfastatemp and F_L001_R2.0rqualtemp, F_L001_R2.0rfastatemp, and i am supposed to have stability.trim.contigs.fasta which i can process further. It looks my data didnt merged here.
I am using MOTHUR version 1.31.2 at cluster (www.abel.uio.no).
Can someone solve this problem.
Regards
Sunil

2

Hello,
Looking forward to have feedback here, than i can proceed further with my analysis
Regards
Sunil :slight_smile:

3

The F_L001_R1.0rqualtemp, F_L001_R1.0rfastatemp and F_L001_R2.0rqualtemp, F_L001_R2.0rfastatemp files are temporary files mothur makes while working on running the command. Mothur will use these to assemble your reads. Did the command crash or is it still running?

4

It was crashed.
I used following function.
mothur > make.contigs(ffastq=test_1.fastq, rfastq=test_2.fastq, oligos=test.oligos)
and my oligos file looks like this. i put forward and reverse primer sequence in first row and barcodes afterwards, which are same at both end.

primer GTGAATCATCGAATCTTTG TCCTCCGCTTATTGATATGC ITS
BARCODE TGCATCT TGCATCT W1C7c.2
BARCODE ACTATCT ACTATCT W1C8c.2
BARCODE CAGATCT CAGATCT W1C9c.2
BARCODE AACTTCT AACTTCT W1C7.2
BARCODE GCGTTCT GCGTTCT W1C8.2
BARCODE CGATTCT CGATTCT W1C9.2
BARCODE GTAATCT GTAATCT W1D10c.2
BARCODE CGCTTTCT CGCTTTCT W1D11c.2

I have few more general question as well.

  1. In some sequences before Barcode_F_Primer there is single N, and also after R_Primer_Barcode there is single N as well. how we can retrieve those sequence, as i didnt mention here in oligos file?
    2.barcode are of variable lenths, is it OK for MOTHUR?
  2. In my data each fastq file shows a mixture of orientated reads:
    Fastq1
    BARCODE–F. PRIMER–NNNN
    BARCODE–R.PRIMER–NNNN
    Fastq 2
    BARCODE–R.PRIMER–NNNN
    BARCODE–F. PRIMER–NNNN
    I guess that make_contigs script designed to join overlapping PE reads will not be able to take into consideration the mixture of Fwd and Rev sequences in each file?
    is this normal, or should I be seeing all Fwd and Rev primer sequences in separate Fastqs?
    is there any way to analyses such data by MOTHUR?
    Can i run make contigs twice, first using F primer first and than using R primer first and than reverse complement the R primer file and finally merge it with F primer file?
    so that i will have all sequences in same orientation in same file?
    Looking forward to hear from you…
    Regards
    Sunil
5

Hi Sunil,

  1. In some sequences before Barcode_F_Primer there is single N, and also after R_Primer_Barcode there is single N as well. how we can retrieve those sequence, as i didnt mention here in oligos file?

Using the bdiffs and pdiffs parameters should take care of this. This allows for differences in the barcodes and primer. Mothur will assume the N is a diff.

2.barcode are of variable lenths, is it OK for MOTHUR?

Yes, mothur does not require barcodes to be the same length.

  1. In my data each fastq file shows a mixture of orientated reads:
    Fastq1
    BARCODE–F. PRIMER–NNNN
    BARCODE–R.PRIMER–NNNN
    Fastq 2
    BARCODE–R.PRIMER–NNNN
    BARCODE–F. PRIMER–NNNN
    I guess that make_contigs script designed to join overlapping PE reads will not be able to take into consideration the mixture of Fwd and Rev sequences in each file?
    is this normal, or should I be seeing all Fwd and Rev primer sequences in separate Fastqs?
    is there any way to analyses such data by MOTHUR?
    Can i run make contigs twice, first using F primer first and than using R primer first and than reverse complement the R primer file and finally merge it with F primer file?
    so that i will have all sequences in same orientation in same file?

In our next version we will add the checkorient parameter to the make.contigs command which will resolve this issue for you. When checkorient=t, if mothur does not find the barcodes and primers in the forward direction, it will look for the reverse compliments of the reverse on the left end and the reverse compliments of the forwards on the right end. If found, the sequence is flipped for you. For now, as a work around you could run the forwards, then rerun your scrapped files with the reverse, and merge the good results.

Kindly,
Sarah

6

Hi
That sounds good, hope to have new version soon.
and what about qualitu filtering in make.contigs, as output of this is only the fasta file, which we cannot used in trim.seqs for quality filtering.
How to solve this problem?
Regards
Sunil