We have just started to sequence 16S on the MiSeq and there were a couple of points that I wanted to get feedback on.
One is that we followed the Illumina publication "16S Metagenomic Library Prep Guide", substituting V1V3 primers for the V3V4. That says if you use the latest software you can get by with 5% PhiX but after we submitted the library our core informed us they would have to put in 50% PhiX, and that we really should have used an alternative protocol from Ravel's lab with variable length spacers.
The other thing is that when we do amplifications with universal 16S primers we get a band of expected size with a smear above it. We're fairly sure these are heteroduplexes. When we have sequenced preps like this on 454 there is no evidence of longer fragments or non-16S amplicons. There are multiple publications on 16S heteroduplexes, it's a recognized phenomenon. Nonetheless, our sequencing core wants to size select the library on a Pippen prep, which I don't want to do because I really think its unnecessary and may introduce bias. They are telling me if I don't let them size select they won't guarantee the results, if they fail (for any reason) we are on the hook.
I would appreciate any input:
--does that Illumina protocol not work as advertised?
--Are some cores still using the old software for whatever reason?
--Do you regularly size select 16S preps for MiSeq?
--Have you compared size selected to non-sized to look for bias?
One is that we followed the Illumina publication "16S Metagenomic Library Prep Guide", substituting V1V3 primers for the V3V4. That says if you use the latest software you can get by with 5% PhiX but after we submitted the library our core informed us they would have to put in 50% PhiX, and that we really should have used an alternative protocol from Ravel's lab with variable length spacers.
The other thing is that when we do amplifications with universal 16S primers we get a band of expected size with a smear above it. We're fairly sure these are heteroduplexes. When we have sequenced preps like this on 454 there is no evidence of longer fragments or non-16S amplicons. There are multiple publications on 16S heteroduplexes, it's a recognized phenomenon. Nonetheless, our sequencing core wants to size select the library on a Pippen prep, which I don't want to do because I really think its unnecessary and may introduce bias. They are telling me if I don't let them size select they won't guarantee the results, if they fail (for any reason) we are on the hook.
I would appreciate any input:
--does that Illumina protocol not work as advertised?
--Are some cores still using the old software for whatever reason?
--Do you regularly size select 16S preps for MiSeq?
--Have you compared size selected to non-sized to look for bias?
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