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Old 06-08-2016, 09:41 AM   #1
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Location: Canada

Join Date: May 2016
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Default Unclassified 16SrRNA using green gene taxonomy

Hello Fellow NGS users,
I recently sequenced my micro biome (Five body sites) using the Illumina 16SrRNA V4 metagenomics platform. The paired reads were analyzed on base space with sample information showing 100% quality filtering. From the classification statistics, one of the samples had over 50% as unclassified at Kingdom level, phylum, class, order, family, genus and species levels. What could be responsible for this and is there a way of extracting the sequence reads that were reported as unclassified.
Uzobiogene is offline   Reply With Quote
Old 06-09-2016, 01:37 AM   #2
Location: Leeds, UK

Join Date: Apr 2010
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Have you looked at the distribution of read lengths in the badly behaved sample. I was having this issue a while back and found that many more reads in poor samples were the wrong length. If I'm expecting a 240bp PCR product, then getting rid of any read which is more than 5bp away from 240bp massively reduces the number of unclassified reads. I suspect that it is some kind of PCR artefact, caused by a poor initial DNA sample.
You could play around with read lengths, seeing what kind of filtering works best for you.
henry.wood is offline   Reply With Quote
Old 06-09-2016, 08:15 AM   #3
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The basespace applications are just a very basic first pass. I'd suggest your run your sequences through mothur (my preferred software for amplicons) or qiime to really see what's going on.
thermophile is offline   Reply With Quote

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