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How to Demultiplex dual indexed Illumina reads

Hello Friends,

I have Illumina Miseq amplicon sequences that I need to demultiplex for use in qiime2. My library prep used a dual indexed method that allowed me to multiplex 4 separate gene markers (ie. 16S, 18S...) per sample. Each individual sample number has a tag that I can easily demultiplex, but I am having trouble demultiplexing the separate genes out of each sample. I figure I can use the primer pairs that were used in the original amplification step to parse out the 4 genes within each sample. Unfortunately, Qiime does not offer this in their demultiplexing tools. Can anyone point me in the right direction or have any tips on the best way to demultiplex my samples by both sample name and then by gene.

Thanks, Danny

next-gen sequencing gene
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updated by Liam Thompson Liam Thompson • written by dw845316 • dw845316

could you post an example of what your data looks like?

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written by Gabriel R. Gabriel R.

I created a subset of one of my sample files that was created after demultiplexing using the illumina indexs. Within the forward and reverse files are amplicons of 16S, 18S, UPA, and rbcL. I want to use the primers that I amplified each of these genes with to parse them out. Below are forward reads taken from the same sample, the first two are 18S reads that you can see the forward primer (GTACACACCGCCCGTC). The third read is from the UPA amplicon and starts with a separate primer that was used to amplify them (GGACAGAAAGACCCTATGAA). Does this make more sense?

BBCDBFFFFFFCGGGGGGGGGGHGGGGGGGGHGHGGGGGGHHGGGGGHGHHHHHGGGHHGHHGHGGGGGGGHGGGGGHHHHHHHHHHHGGGGGHHHHHGGGGGGGHGHHGGGGGGGGHHHHGGGA;CGHHHHHHHHHHHHHGGGGFFGGGGGGGGGGGGGGCGGGFGGGGGFFFFFFFFFFFFFF.DFFFFFFFFFFFFFFFFFFFFFFFFFFFDFFFFFFFFFFFFFFFFFFFFFFFFFFFFDFFFFFFF @M00348:26:000000000-D2NFB:1:1101:16189:1725 1:N:0:GGAGCTAC+ACTGCATA GTACACACCGCCCGTCGCTACTACCGAGTGAATTATGTCATGATGCCCTGGGACTGGACGTTGAACGGGTGTCAAAGCCTGTTCGATGCTAGAATCAGCGTAAAATGGCGCAATTTCGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCAGAAGGATCAA + ABBBBFFFFFFFFGGGGFGGGGHHGGGGGGGGGHHHGGGGHHHHHGGGHHHHHHHHGGGGHHHGGGGGGHHGHGGGGDHHGHHHHHHHHHGGHHHHHHHHGGGGGHHHHHHHHGGGGGHHHGHHHGHHHHHFHCCGHHHHHHHHHHHGDFGGGGFFGGGGGGGGGGGGAFEGFFGFFFFFFFFFFFFFFFFFFFFEFFFEDFFFFFFEFFFFFFFAFFFB@@CFBFFFBBFFFFFFFFFFFFFFDFFFBA. @M00348:26:000000000-D2NFB:1:1101:12525:1946 1:N:0:GGAGCTAC+ACTGCATA GTACACACCGCCCGTCGCTACTACCGATTGAATGAATTAGTGAGCTTCAGAGATCGAGCTGTTTCGGGCAACCGGGTCAGTTTGAGAACCGAATCAAACTTGCTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCAGAAGGATCAT + EEDEEFFFFFFEGGGGGGGGGGHGGGGGEFGHHHHGGGGGHHGGGGGHHHHHHHHGGGGHHHGGGGGGHHHHGGGGGGHHHHHHHHHHHHHHHHHHHHHGHGGGGHGHHGGEFGGHGHHFGGG.CGHHHHHHHHGGGHHGGGHHHHGGGGGGGGGGGFGGGGGGGGGGGDFFFFHFFFFFFFHFHFFFFFFFHFFFFFFFFFFFFFFFFFDFFFFFFFFFFFFFFFHFFFFFFFFFFFFFFFFFFFFFFFF @M00348:27:000000000-D2TDJ:1:1101:8422:19699 1:N:0:GGAGCTAC+ACTGCATA GGACAGAAAGACCCTATGAAGCTTTACTATAGCCTGGAATTGTGTTCGGGCTTCGCTTACGCAGGATAGGTGGGAGGCTGTGAAGTTCTGCTTGTGGGCAGGATGGAGCCAACGGTGAGATACCACTTTAGCGAGGCTAGAATTCTAACCCCTGCCCGTCATCCGGGAGGGAGACAGTTTCAGGGGGGTAGTTTGACTGGGGCGGTCGCCTCCTAAAAGGTAACGGAGGCGCGCAAAGGTTCCCTCAGG

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written by dw845316 • dw845316

1 answer

You can check out a beta python script on a repo at Github. I had a similar problem a few years back, and used it. Let me know if it sorts out your problem.

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written by Liam Thompson Liam Thompson

Hello Liam,

Thanks for showing me this script! When I run it though I am receiving this error... Any idea what Im doing wrong? 

$ python2.7 find_primers_demultiplex_and_cut.py --m
    MetaData_Primers_corrected.txt --f DW2_S12_L001_R1_001.fastq.gz --r
    DW2_S12_L001_R2_001.fastq.gz --t True --o
    Output_findprimersdemultiplexandcut/ --l False
        - Checking the output path Output_findprimersdemultiplexandcut/
        Path was found - Output_findprimersdemultiplexandcut/
        Traceback (most recent call last):
          File "find_primers_demultiplex_and_cut.py", line 919, in <module>
            start_run = Demultiplex()
        TypeError: __init__() takes at least 2 arguments (1 given)
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written by dw845316 • dw845316

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