How to proceed with pooled R1 and R2 reads?

Commands in mothur
1

Hi, Below are the files I received from a sequencing vendor.

  1. sample1-35_r1.fastq.gz file
  2. sample1-35_r2.fastq.gz file
  3. Map file with sample_id, barcodesequence and linkerprimer sequence.
    R1 and R2 are both in the 5’-3’ orientation as raw files.
    Forward primer format BARCODE-FORWARD PRIMER
    Reverse primer format REVERSE PRIMER

After unzipping fastq.gz files, I am using make.contigs to get the demultiplexed contigs. (mothur > make.contigs(ffastq=sample1-35_r1.fastq, rfastq=sample1-35_r2.fastq, oligos=test.oligos))

But, I am getting the same error,
[ERROR]: cannot mix paired primers and barcodes with non paired or linkers and spacers, quitting.
Making contigs…
It took 0 secs to process 0 sequences.
[WARNING]: your sequence names contained ‘:’. I changed them to ‘_’ to avoid problems in your downstream analysis.

I am not sure if the step is wrong or the oligos file. Any help in this regard is greatly appreciated.

Oligos file looks like this
primer CCTACGGGNGGCWGCAG
barcode CATTCCGT sample1
barcode CATTCGTG sample2
barcode CATGGAAG sample3
barcode CATGTTGT sample4
barcode CATCACCG sample5
barcode CATGCGCA sample6
barcode ATGATTAG sample7

Thanks,
-RP

2

You want to use the paired primer and paired barcode format, https://mothur.org/wiki/Oligos_File#Paired_Primers and https://mothur.org/wiki/Oligos_File#Paired_Barcodes


primer CCTACGGGNGGCWGCAG YourReversePrimer barcode CATTCCGT NONE sample1 barcode CATTCGTG NONE sample2 barcode CATGGAAG NONE sample3 barcode CATGTTGT NONE sample4 barcode CATCACCG NONE sample5 barcode CATGCGCA NONE sample6 barcode ATGATTAG NONE sample7
3

Thanks Ms.Westcott. Unfortunately, It doesn’t seem to work.
After running Make.contigs It picks only few 100 sequences.

Oligos file looks like this:

primer CCTACGGGNGGCWGCAG GACTACHVGGGTATCTAATCC
barcode CATTCCGT NONE sample1
barcode CATTCGTG NONE sample2
barcode CATGGAAG NONE sample3
barcode CATGTTGT NONE sample4
barcode CATCACCG NONE sample5
barcode CATGCGCA NONE sample6
barcode ATGATTAG NONE sample7


I contacted the vendor and their suggestion is to use their software FASTq Processor to separate files into individual samples and then proceed with MOTHUR.

Best
RP