We have an ongoing project sequencing the 16S rRNA gene for bacterial profiling of stool samples using Illumina MiSeq (v3 kit). The MiSeq requires base diversity to determine cluster positions. Because of the low genetic diversity our amplicon library, we have spiked in 50% phiX and aimed for cluster density of 700-800K/mm2. Using this approach, we will not utilize the full capacity of the instrument and a substantial fraction of the sequences will not be from the targeted 16S amplicons. Suggestions to improve the efficiency of our approach are most welcome.
Would the issue with low genetic diversity samples be an issue if applying another sequencing platform such as the Ion Torrent?
Which primers are you using? Some researchers have avoided the low genetic diversity problem adding few random nucleotides in the forward primer, right after the seqeuncing primer binding and before the in-line barcode and the 16S primer, although the 16S amplicon libraries are spiked with phiX. In my case I use my own metagenoemes instead so I won't throw away data. Have a look to this paper:
Eren et al. 2013 A filtering method to generate high quality short reads using illumina paired-end technology. PLoSONE 8(6): e66643
A recent review article by Claudia Knief in Frontiers in Plant Science goes over all of the different next-generation sequencing methods, giving the pros and cons of each. This would be a good read if you are considering different sequencing options: Front. Plant Sci., 21 May 2014 | doi: 10.3389/fpls.2014.00216.
There are many biologist that would be happy to sequence there strain together with your sample for 0 cost instead of sequencing PhiX again !
We are using the primers described in Kozich et al. 2013 (doi: 10.1128/AEM.01043-13) and the latest update of Illumina software. Our experience has so far been that we need to spike in more external libraries than the Illumina recommendations.
I was wondering if sequencing low diversity libraries also is challenging using the Ion Torrent platform?
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