make.contigs questions

Commands in mothur
1

Hello!

We just received our sequencing data from Argonne which were forward barcoded and given the following files:

Undetermined_S0_L001_I1_001.fastq.gz
Undetermined_S0_L001_R1_001.fastq.gz
Undetermined_S0_L001_R2_001.fastq.gz

and the mapping file (which is currently in .txt format) which contains the information below:

#SampleID BarcodeSequence BarcodePlate Well LinkerPrimerSequence Description
BG165stl AGCCTTCGTCGC 1 A1 GTGTGYCAGCMGCCGCGGTAA BG165stl

So a couple of questions:

  1. Is our mapping file considered the “oligo” file?
  2. Do we use all four files in the make.contigs command?
  3. Does it make sense to use the following:

mothur > make.contigs(ffastq=forward.fastq, rfastq=reverse.fastq, findex=forwardIndex.fastq, none)
##this should generate a four column stability file correct?

then use:

mothur > make.contigs(file=sequences.stability , oligos=sequences.oligos)
##Alternatively is there any way to do this all in one command instead of splitting it into two?

Thank you in advance and sorry if my questions seem naive as we are new to mothur.

2

You don’t need to run make.contigs twice (you can feed it either the fastq OR the file files)

qiime mapfile:
#SampleID BarcodeSequence BarcodePlate Well LinkerPrimerSequence Description
BG165stl AGCCTTCGTCGC 1 A1 GTGTGYCAGCMGCCGCGGTAA BG165stl

mothur oligo (no header):
barcode AGCCTTCGTCGC BG165stl

I can’t remember if the barcode has already been RC’d for the qiime mapfile. If this throws all your sequences in the scrap, RC the barcodes and try again. Pat posted R code to do just that for an oligo file sometime in the past, you could search for “argonne” to find it

3

Hello!

Thank you for your response. We have made the oligo file from the mapping file. Which looks like this now:

forward GTGTGYCAGCMGCCGCGGTAA
barcode AGCCTTCGTCGC BG165stl


When I run the following code:

make.contigs(ffastq=boonedata/Undetermined_S0_L001_R1_001.fastq.gz, rfastq=boonedata/Undetermined_S0_L001_R2_001.fastq.gz, findex=boonedata/Undetermined_S0_L001_I1_001.fastq.gz, oligos=boonedata/oligosG.txt, bdiffs=1, pdiffs=2, processors=4)

I get back this result:

Using 4 processors.
d?ԙYOK??-PD?EK???L?f?͡?;RP;??z???,oBcƚ?V is in your forward fastq file and not in your reverse file, please remove it using the remove.seqs command before proceeding.
m_0???ŧ&.?1F7XL?5??Ɨ??q.1??Y?EH#EpUe??wa???lLni"???^ is in your forward fastq file and not in your reverse file, please remove it using the remove.seqs command before proceeding.
is in your forward fastq file and not in your reverse file, please remove it using the remove.seqs command before proceeding.
??+]???<?_¬~P??Q-?sM?U/|??<?G??eo)?[?I%,???`~?O4*%???L?%,??1???}???* is in your forward fastq file and not in your reverse file, please remove it using the remove.seqs command before proceeding.
[ERROR]: cannot mix paired primers and barcodes with non paired or linkers and spacers, quitting.
Making contigs…
Segmentation fault (core dumped)

I am not sure how to proceed. . . . .


Thank you

amco

4

not sure about the error. But you shouldn’t have your forward primer in your oligos because it isn’t sequenced using either the Caporasso or Kozich primer set up.

5

Mothur expects the barcodes and primers to be paired for make.contigs. You can indicate that they are not using the keyword NONE. Here is a link to the oligos wiki page, http://www.mothur.org/wiki/Oligos_File, and an example:

Oligos File: forward primer with no reverse, barcode in forward index file.

primer GTGTGYCAGCMGCCGCGGTAA NONE
barcode AGCCTTCGTCGC NONE BG165stl

make.contigs(ffastq=boonedata/Undetermined_S0_L001_R1_001.fastq.gz, rfastq=boonedata/Undetermined_S0_L001_R2_001.fastq.gz, findex=boonedata/Undetermined_S0_L001_I1_001.fastq.gz, oligos=boonedata/oligosG.txt, bdiffs=1, pdiffs=2, processors=4)

The strange output looks like mothur is misreading the compressed file. Are you running our Windows version? The windows version does not support reading from compressed files.