Circulating RNA analysis

Analysis of circulating DNA has had a major impact in the last couple of years with 100s of publications in the last few years. I've previously posted about work at the Institute on circulating tumour DNA analysis of amplicons and exomes, but what about RNA?

Patterned flowcells: what can we expect?

There was a real buzz in the community when Illumina resurrected the idea of using patterned flowcells for SBS sequencing (Keith Robison covered lots of genomics news back in January, including patterned flowcells ). One of the big problems with clustering is the need to very carefully quantiy samples before loading onto a flowcell. Most labs use qPCR or BioAnalyser but even the best rarely achieve perfect density on every run if they are working with a diverse group of libraries. Jay Flately at a JP Morgan conference said that only 36% of clusters are useable and that the new technology should double that.

Patterned flowcells have been mentioned before in Illumina roadmaps, they'll potentially allow 1Tb or more from a standard HiSeq run and are one way we might see an end to cluster density variability. But a patterned flowcell might also allow improvement of some methods and even interesting new applications to be developed.

We'll have to wait until Illumina release the new flowcells at the end of the year (more likely in January just before AGBT) but for now I thought I'd put down some thoughts I've been having about what we might get and look through one of Illumina's more recent patents.

Mouse models of Human disease constantly need to be improved

I’ve worked on model-organisms for a long time, originally in Plant research but nowadays I'm more likely to run genomic experiments for groups using Mouse models of cancer. It is impossible to do some experiments in Human patients for a variety of reasons, so we use Mouse models instead. Genetically engineered mice (GEMs) are used in many research programs and offer us the ability to tailor a disease phenotype, as our understanding of the driving events in cancer increases we can build GEMs that carry these same driver mutations; we can even turn the specific mutations on at specific time points to try and recapitulate Human disease.



Finding your way around NGS sample prep

I'm often asked which sample prep method a user should consider for their experiments. In my lab we use a lot of Illumina TruSeq kits; we've tried other methods, and do use Rubicon's Thruplex, but Ilumina's end-to-end support is useful in a medium sized core facility. And the kits work!

I wanted to illustrate the fact that Illumina sample preps share many steps in their protocols to demonstrate that once you've mastered one protocol, you can easily move onto another. The map of sample prep below is my first try at that illustration. You can see a higher resolution image here.

Explaining how the kits work always takes time and I have always thought a strength of the Illumina technology is the flexibility given by the core of sample prep: end repair, ligate adapters & PCR. Users can think creatively about what they do to their DNA (or cDNA) before starting or during the prep to come up with novel techniques. Stranded RNA-seq, bisulfite sequencing, RRBS, exome capture are just a few of the methods developed by users of Illumina technology. Hopefully the image above shows how similar things really are with those key steps clearly highlighted as large "interchanges", along with PCR and qPCR & Bioanlayser for QT/QC.

The above image borrows heavily from the London underground maps developed by Harry Beck in 1931. Most of the Illumina protocols are listed and I've included Thruplex on its own network for comparison. I'm aiming to add more detail for some of the different RNA-seq protocols at some point as well as bisulfite sequencing. I'm also thinking about how this might be extended to include details on suggested sequencing depth; lines for Human genome vs CNV-seq or DGE vs splicing.

Let me know what you think.

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