16S Amplicon Experiments: Which Platform to Choose?
Since 2010 several studies have been published that analyze microbial community composition by amplicon sequencing on the Illumina Genome Analyzer (GA). However, direct adaption of these protocols for sequencing on the HiSeq 2000 – the currently predominant Illumina sequencer – is not possible as both systems use different basecalling pipelines. Therefore amplicon sequencing on Illumina HiSeq 2000 is still left to the very experienced users and only a few publications can be studied on this.
In the meanwhile Illumina has introduced the MiSeq as the optimal platform for this kind of projects. In this context they have published an application note presenting sequencing of the V4 region of 16S rRNA genes on the MiSeq system.
And I totally agree that the MiSeq is a very good tool for these studies. For me, the most important advantages of the MiSeq layout in comparison to the sequencing on Illumina HiSeq 2000 are as follows:
- Shorter turnaround time: The sequencing run itself takes a bit more than one full day, while a HiSeq 2000 run takes up to 12 days.
- More informational content: By overlapping two paired end reads of 150 bp, full-length reads of about 250 bp can be generated
- Potential for even longer reads: Illumina has announced read length of 250 bp for the end of the year. Then reads of up to 450 bp should be possible.
Nevertheless Roche GS FLX+ sequencing is still able to generate much longer reads with an average of up to 500-600 bp. And the long read length will provide a deeper insight into the microbiome of interest or more precisely higher classification efficiency down to species level. However Roche sequencing goes along with higher costs per base, so it will always be a decision based on the individual experiment, whether read length or sequencing depth is the most important factor.