Danielle Perrin, from the genome centre at Broad presents an interesting webinar demonstrating what the Broad intends to do with MiSeq and lastly a 300bp single read dataset. Watch the seminar here. I thought I'd summarise the webinar as it neatly follows on from a previous post, MiSeq growth potential. Where I speculated that MiSeq might be adapted for dual surface, larger tiles to get up to 25Gb. There is still along way to go to get near this but if the SE300 data presented by Broad holds up for PE runs then we jump to 3.2Gb per run.
Apparently this follows up from an ASHG presentation but as I was not there I missed it.
Apparently Broad has six MiSeq's. Now I understand why mine has yet to turn up! I must add these t the Google map of NGS.
MiSeq intro: The webinar starts with an intro to MiSeq if you have not seen one and goes through cycle time, chemistry and interface. They have run 50 flowcells on 2 instruments since August. They are now up to 6 boxes running well. No chemistry or hardware problems yet and software is being developed.
What will broad do with it? At the Broad they intend to run many applications on MiSeq: Bacterial assembly, library QC, TruSeq Custom Amplicon, Nextera Sample Prep and Metagenomics. So far they have run 1x8 to 2x151 runs and one 1x300 run.
2x150 metrics look good at 89% Q30, 1.7Gb, 5.5M reads 0.24% error rate.
Getting cluster density right is still hard even at Broad (something for another blog?). They use Illumina's Eco qPCR system for this.
Bacterial Assembly: The Broad has a standard method for bacterial genomics which uses a mix of libraries; 100x coverage of 3-5KB libs, 100x coverage of 180bp libs, 5ug DNA input and AllPaths assembly. They saw very good concordance from MiSeq to HiSeq. And the MiSeq assembly was actually higher but Danielle did not say why (read quality perhaps).
library QC: 8bp index in all samples by ligation (they are not using TruSeq library prep at Broad) 96well library prep, pool all libraries and run it son the number of lanes required based on estimated coverage. QC of these libraries and evenness of pools is important. They run the index first and if the pool is too uneven they will kill the flow cell and start again. They use a positive control in every plate and run as a 2x25bp run to check the quality of the plate. Thinking of moving this QC to MiSeq to improve QC turnaround. They aim to run the same denatured pool onto HiSeq after MiSeq QCC. This will avoid a time delay requiring the denaturation to be repeated. Very important in ultra low input libraries where you can run out if flow cells need to be repeated. All QC metrics seem to correlate well between MiSeq and HiSeq.
Amplicons: They presented Nextera validation of 600bp amplicons: 8 amplicons, pool, Nextera, MiSeq workflow (very similar to the workflow I discussed in a recent Illumina interview). And TruSeq Custom Amplicon (see here), Illumina's GoldenGate extension:ligation and PCR system. After PCR samples are normalised using a bead based method, pooled, run on MiSeq (without quantification) and analysed. Danielle showed a slide (#34) with the variation seen in read numbers per sample after bead based normalisation and a CV of only 15%. I wonder if the bead normalisation method will be adopted for other library types?
SE300bp run: The Broad took a standard kit and ran it as a 300bp single end run. They have done this once and first time round achieved 1.6Gb, 5.29M read, 65%Q30, 0.4% error. Pretty good to start with and hopefully demonstrating the future possibilities.
How long can you go, 550bp amplicons (PE300) anyone? Another goodbye 454 perhaps?