Question: How to Demultiplex dual indexed Illumina reads
1
gravatar for dw845316
11 days ago by
dw84531610
dw84531610 wrote:

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

sequencing next-gen gene • 137 views
ADD COMMENTlink modified 10 days ago by Liam Thompson110 • written 11 days ago by dw84531610

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

ADD REPLYlink written 10 days ago by Gabriel R.2.1k

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

ADD REPLYlink modified 10 days ago • written 10 days ago by dw84531610
0
gravatar for Liam Thompson
10 days ago by
Liam Thompson110
Gothenburg, Sweden
Liam Thompson110 wrote:

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.

ADD COMMENTlink written 10 days ago by Liam Thompson110

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)
ADD REPLYlink modified 10 days ago • written 10 days ago by dw84531610
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