Input oligos to make.contigs

Commands in mothur
1

Hello all,

I tested the mothur with oligos for my raw sample fastq (without trimming my adapter using cutadapt tool). Here i used only two samples in order to test how this work.

Doing so, I have some queries:

  1. What makes difference with and without oligos file to make.contigs command, because the command works fine without too as tutorial of MiSeq_SOP described. I mean, is there any default oligos (once we user dont use his oligos) that mothur uses and so trim the reads as we got output name with *trim.contigs.

  2. Considering using oligos file for paired reads, i got warning in each of my sample, why so ?? what is the groups for here?? (please find logfile created by mohur).

[WARNING]: your oligos file does not contain any group names. mothur will not create a groupfile.

  1. Further, If i conisder oligos for my reads, should i have create this line
    primer ATTAGAWACCCBDGTAGTCC CCCGTCAATTCMTTTRAGT
    for my each paired sample ?? like i got 52 paired sample fastq file (104 fastq individual). Or this should work with only 1 line of primer ATTAGAWACCCBDGTAGTCC CCCGTCAATTCMTTTRAGT
2
  1. What makes difference while with and without oligos file to make.contigs command, because this command works fine without too as you have described on MiSeq_SOP. I mean is there any default oligos (once we user dont use his oligos) that mothur uses and so trim the reads as we got output name with *trim.contigs.

There is no default oligos file. The oligos parameter is optional. Some companies remove the primers and barcodes for you and split the reads into files by sample, other do not. The oligos option is used to trim the primers and barcodes from reads that were not trimmed by the sequencing company. It is a necessary step if the barcodes and primers are still attached.


2. Considering using oligos file for paired reads, i got warning in each of my sample, why so ?? what is the groups for here?? (please find logfile created by mohur)

For the make.contigs command you must use paired primers. The oligos file allows you to set either the forward or reverse pair to NONE if needed. For example if you don’t have a reverse barcode or primer you could do this:

primer yourForwardPrimer NONE optionalGroupName
barcode yourForwardBarcode NONE yourGroupName.

The warnings about group names are because you oligos file does not contain group names so mothur can’t create a group file.

  1. Further, If i conisder oligos for my reads, should i have create this line
    primer ATTAGAWACCCBDGTAGTCC CCCGTCAATTCMTTTRAGT
    for my each paired sample ?? like i got 52 paired sample fastq file (104 fastq individual).

Do you have 52 primer sets or 52 barcode pairs? Here’s a sample of a typical oligos file:

primer CCTACGGGAGGCAGCAG ATTACCGCGGCTGCTGG V3
primer ATTAGAWACCCBDGTAGTCC CCCGTCAATTCMTTTRAGT V5
primer ACTYAAAKGAATTGACGGG ACRACACGAGCTGACGAC V6
BARCODE ccaac cactg F01R2A
BARCODE ccaac aacca F01R2B
BARCODE ccaac tgtca F01R2C
BARCODE ccaac aaacc F01R2D
BARCODE ccaac cccaa F01R2E
BARCODE ccaac gggtt F01R2F
BARCODE ccaac tttaa F01R2G
BARCODE ccaac gaggt F01R3H
BARCODE ggttg cactg F02R2A
BARCODE ggttg aacca F02R2B
BARCODE ggttg tgtca F02R2C
…

Kindly,
Sarah

3

Hi, sorry to intrude into the conversation! But I have a question…
I realised after running almost all SOP that my sequences still had the primers… so this is what I did and just want to know if it is correct.

So I have ran the the make.contigs again adding this time an oligos file as follows
mothur > make.contigs(file=fileList.paired.file, oligos=Pn_bac.oligos)

the fileList.paired.file is as follows:
KLS1X1TF1 KLS1X1TF1_CTACTA_L001_R1.fastq KLS1X1TF1_CTACTA_L001_R2.fastq
KLS1X1TF2 KLS1X1TF2_GGCTTG_L001_R1.fastq KLS1X1TF2_GGCTTG_L001_R2.fastq
KLS1X1TF3 KLS1X1TF3_GCCGCG_L001_R1.fastq KLS1X1TF3_GCCGCG_L001_R2.fastq
KLS1X2TF1 KLS1X2TF1_TCATGT_L001_R1.fastq KLS1X2TF1_TCATGT_L001_R2.fastq
KLS1X2TF2 KLS1X2TF2_GAACAC_L001_R1.fastq KLS1X2TF2_GAACAC_L001_R2.fastq
KLS1X2TF3 KLS1X2TF3_CGCACA_L001_R1.fastq KLS1X2TF3_CGCACA_L001_R2.fastq
KLS1X9TF1 KLS1X9TF1_AATGAA_L001_R1.fastq KLS1X9TF1_AATGAA_L001_R2.fastq
KLS1X9TF2 KLS1X9TF2_AACTTA_L001_R1.fastq KLS1X9TF2_AACTTA_L001_R2.fastq
KLS1X9TF3 KLS1X9TF3_GGAGGT_L001_R1.fastq KLS1X9TF3_GGAGGT_L001_R2.fastq
KLS2X1TF1 KLS2X1TF1_TGCGCT_L001_R1.fastq KLS2X1TF1_TGCGCT_L001_R2.fastq
ETC…

An my oligos file:

primer GTGYCAGCMGCCGCGGTA CCCCGYCAATTCMTTTRAGT

This time I have about 300000 less sequences and I have and extension to the name with fbddiffs, rbdiffs etc which I didn’t have when I did not use the oligos option the first time.

M00629_39_000000000-APEBN_1_2107_9597_1632 fbdiffs=0(match), rbdiffs=0(match) fpdiffs=0(match), rpdiffs=0(match)

So I am wondering if it is a problem to have this name extension and why I am getting less sequences… if we can improve this somehow?

Thanks