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OTU reads in no template control (NTC)

I have set of few samples which are no template control(NTC) , it was analyzed using Illumina DRAGEN Metagenomics Pipeline which resulted in these NTC samples having reads assigned which start from 2 OTUs to as high as 500-600 range, ideally there shouldn't be any OTUs since there are no samples.

Now when I ran a fastqc there are illumina universal adaptors in the NTC fastq files which were used in the analysis.

My question is:

  1. Is it because of the adaptor there are OTU being assigned to the NTC samples ? or it has no role even if adaptor are trimmed there will be no change
  2. The tape station data which we got show clean reading for the NTC samples.

So what steps should I follow to troubleshoot the issue.

Any suggestion or methods would be really appreciated.

illumina 16s
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updated by GenoMax GenoMax • written by 1769mkc 1769mkc

1 answer

there are illumina universal adaptors in the NTC fastq files

Are they just adapter dimers or there is actual sequence that is assigned to OTU.

The tape station data which we got show clean reading for the NTC samples.

Illumina sequencing can be very sensitive to presence of any trace contamination and perhaps that is what you are seeing.

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written by GenoMax GenoMax

"Are they just adapter dimers or there is actual sequence that is assigned to OTU." in the kraken output there are OTUs assigned at genus level data what we received not much maximum are having 1 or 2 and few are in range of 100-600 that how the OTUs are assigned

"adapter dimers" what is the way to determine this in data? I would like to know to verify this.

"Illumina sequencing can be very sensitive to presence of any trace contamination and perhaps that is what you are seeing." mean it can be introduced during the library prep it self if I get that correctly

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written by 1769mkc 1769mkc

"adapter dimers" what is the way to determine this in data? I would like to know to verify this.

You could scan/trim the data with any of standard tools and the reads that are adapter dimers will be go away, if they are not dimers you should still retain some sequence.

mean it can be introduced during the library prep it self if I get that correctly

Correct. Dirty pipette tip/contaminated reagent/water etc. If the contaminant is common you may see it in other/all samples.

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written by GenoMax GenoMax

"If the contaminant is common you may see it in other/all samples." this will verify and thanks for your insights

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written by 1769mkc 1769mkc

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