Highly Multiplexed 16S Sequencing on MiSeq

16S amplicon sequencing has proven to be an important tool for identifying and quantifying microbes present in metagenomic samples. We have several researchers here at IGS who have used this to analyze organismal and environmental communities for several years.

Together with these researchers, the GRC has been working over the past year to transition high-throughput sequencing of 16S rRNA regions amplified from metagenomic samples from the 454 platform to the Illumina platform. With the increased read length (2x250bp) on the MiSeq, it is now well suited to generate 16S data for a fraction of the cost of generating data on the 454 FLX.

A typical 16S amplicon run on the 454 produces ~1M reads with an average read length of ~500 bp, which enables deep profiling of 100-200 samples. A paired-end MiSeq run generates 500 bp of sequence per amplicon and produces an average of 12M read pairs per run. We are now routinely profiling a minimum of 400 samples per run with even greater depth than possible on 454 for less than half the per-sample cost.

Please contact us for more information about our 16S profiling service using the Illumina MiSeq.

MiSeq: More reads and longer read lengths are now available

Over the past couple of months we have been evaluating the MiSeq upgrade. This upgrade includes the ability to sequence longer reads (250nt from each end, so 500 nt per library fragment) and to collect data from more clusters (both the top and bottom of each channel are imaged). We just had a 250 PE run that exceeded 30 million reads – that is just over 8 Gbases of data! This is a nice jump up from the ~13M reads (~2 Gbases) per run we were getting before.

Disclaimer: We are still in data-gathering mode to determine what the average expectation should be for each run- it would be nice to get 30 million reads from every run, but that may not happen.

Here are quality plots of a 250 PE run with genomic PE libraries. We are working to maximize quality as the read lengths increase.

Read 1:

Read 2:

We can now combine the benefits of Illumina’s high read counts with the benefits of longer reads.

Up next is to see how MiSeq/Pac Bio hybrid assemblies measure up to HiSeq/454 hybrid assemblies, and a comparison of assemblies using HiSeq 100bp PE reads vs MiSeq 250 bp PE reads.