We are trying to get started analyzing 16S RNA using the primers and protocols posted by the Human Microbiome Project (see here : V1-V3 and V3-V5 primer sets). Our problem is that we are getting the majority of our reads removed by the "short quality" filter. We started with 2.5M reads passing the key sequence, but about 1.8M are failing the short quality filter, and we only get 295,000 passing all filters. This is with the 2.5.3 version of the 454 software, which was actually an improvement on the previous version, which only gave ~100,000 passing filters.
On the theory that there might be short fragments in the sample, we repurified everything on Ampure and ran 2/8s of a plate but didn't see any improvement.
Is anyone else using these HMP primers? Have you seen this short quality filter problem? Are there adjustments to the sample prep or the analysis software that we can make? Could it be something with the density of beads, are we loading too many? Any help would be appreciated, thanks.
On the theory that there might be short fragments in the sample, we repurified everything on Ampure and ran 2/8s of a plate but didn't see any improvement.
Is anyone else using these HMP primers? Have you seen this short quality filter problem? Are there adjustments to the sample prep or the analysis software that we can make? Could it be something with the density of beads, are we loading too many? Any help would be appreciated, thanks.
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