5hmC on Illumina 450k's: analysis without breaking the bank?

Epigenetics is one of the most exciting areas of research and promises to impact all sorts of biological systems. DNA methylation has recently been shown to come in methylcytosine (mC) and 5-hydroxymethylcytosine (5hmC) flavours. Usually analysis of mC is carried out using bisulfite conversion or enzymatic digestion. The gold standard is probably bisulfite sequncing and a few groups have made significant advances using Bis-seq methods on Human genomes.

The analysis of 5hmC using the same approach appears to be an expensive habit to pick up. There are two methods to analyse 5hmC, OxBS and TAB-seq, both require seqencing of at least two Human genome-equivalents to allow subtraction of one from the other revealing the 5hmC signature. As it currently takes about 4 HiSeq lanes to do 1 Bis-seq run then a 5hmC experiment is likely to be at least 1 flowcell per sample. And at $10,000 or more that's an expensive habit to feed!

5hmC done cheap: It should be possible to make use of methylation microarrays to reduce the costs of analysis. Illumina's 450K methylation array allows researchers to process 12 samples at a time generating quantitative methylation measurement of individual CpG's robustly and at reasonable cost. The array was developed as a collaboration by methylation experts and can be used for EWAS studies as well as individual research projects.

By using the OxBS or TAB-seq methods to generate inputs for the 450k arrays it should be possible to get robust 5hmC signatures for a reasonable portion of the genome. And we should be able to do this at a cost that allows 1000's of samples to be run. Does 5hmC regulate gene expression itself or do more than act as an intermediary between methylated or not? Large-scale 5hmC projects are going to highlight where and how 5hmC acts in a biological context.

The 450k arrays also support analysis of FFPE-samples so it may be possible to extend the analysis to huge-cohorts of patient samples with clinical follow-up. By using the Infinium FFPE DNA Restoration Solution users can QC and "rescue" samples prior to analysis.

Making the methods work is an ongoing challenge and much of that requires improvements of the underlying chemistry used in the bisulfite conversion most of us have used at some point.

However Shankar Balasubramanian's latest company is Cambridge Epigenetix and aims to commercialise the OxBS technology. If anyone can make the chemistry work better I'm sure Shankar stands a chance. I wouldn't be writing this blog and you wouldn't be reading it without his earlier work!

23and Meow

Consumer genomics is becoming easier and easier to access and a colleague recently contacted me about my experiences as some friends were planning to get tested. When I got "23andMe'd" I learnt some new stuff about me and my DNA, and I'd like to learn more about the genealogical side but have not attempted to get my data into one of the better sites for this.

But how far might we go with consumer genomics? Believe it or not there is a company offering a service to identify which pet crapped on your lawn!

Genomic tourism: I've posted before about the possibility of holidaying somewhere like India and getting your exome sequenced. And as it becomes clearer that genomic analysis can help diagnose disease an industry is likely to emerge offering that to individuals who can't get it due to financial, or legal constraints in their home country. If the NHS can't offer me an early test for Cancer what's to stop me going somewhere to have some blood taken and have my results ready when I get home from my vacation?

Heir-tracing: There are companies out there that specialise in finding people who may be beneficiaries of a will, e.g. Heir-Trace. Using DNA fingerprinting by NGS or arrays may allow descendants of an individual to be contacted more easily even if there is k=no known connection to the executors.

PooPrints vs Fingerprints: PooPrints offer a service to identify the animal responsible for making a mess on your lawn/park/pavement. Using DNA fingerprinting technology they create a database of animals in a local community and then are able to identify which animal was responsible if the owner did not clear up. There was some coverage on the BBC (from a story here) website about how a council in the UK has suggested this, although in the current financial climate I think I'd rather my library stayed open!

PooPrints charges $29.95 to get a dog registered, for just a little more they could be using one of the Canine genotyping chips to give a lot more data back to the owners.

23andMeow: If companies like PooPrints can access high-volume SNP content (Affymetrix's 130k Custom Canine array or Illumina's 130k CanineHD array) then surely there is a market for a 23andMe style service but for pets rather than people? Admittedly the market is pretty low but it could be worth someone's investment?

How do you get DNA from your pet dog: Take a look at the image below from DNA Genotek's blog. Looks easy but mind your fingers!



Perhaps if we can successfully genotype mummified cats from the Pyramids then you might just find out your kitty is a descendant of Tutankhamen's favourite pet!

DNA barcoding for cnidarians with Oxford Nanopore

I read an interesting post over at boingboing.net on the use of DNA barcodes and the possible appearance of Dr Spocks Tricorder.

Mouse models of Crohn's Disease

At the Illumina Scientific Summit in Berlin last month Prof. Philip Rosenstiel from the Christian-Albrechts-University in Kiel spoke about his work on inflammatory bowel disorder (IBD). He presented a fabulous series of experiments where mouse-models of the disease were used to show the impact of maternal phenotype on litter genotype.

Where are all the "hot" female scientists

Science Watch have published their list of "Hot" scientists. Their website front page has 21 researchers on the hot-list from the "Genomics and Biomedicine" section and 8 of the 21 hottest scientific researchers are involved in Genomics. However only two of these are female.

The report aims to highlight the work of those scientist that are trailblazers shaping tomorrow's world. The hot-list is generated by analysis of citations mainly during 2012. The reports authors used InCites to rank hot papers as those published in the last two years at a level notabaly higher than similar papers, as well as looking at citation indexes on Thomson Reuters Web of Science (excluding reviews).

Top of the hot-list is Rick Wilson from WashU, there are three other WashU scientists giving WashU 20% of the hot-list work. Sounds like WashU is the place to be!

A "driver" paper was A map of human genome variation from population-scale sequencing (1000 Genomes Project, Nature, 467(7319): 1061-73, 2010), which also appeared in the 2011 rankings and is still one of the most highly-cited papers. Several of the hot-list researchers were authors on this highly cited paper. It did not feature in the list of papers in the ScienceWatch report though.

New England Journal of Medicine and Nature had three each of the seven most highly cited "Genomics and Biomedicine" papers. I guess that's why we all aim to publish in those journals.

Illumina Scientific Summit "Cancer Genomics" breakout

This years Illumina Scientific Summit was a great meeting, lots of great talks and great people to talk to at dinner and the bar. I already posted about a collaboration Geoff Smith presented and this post is some rough notes on the "Cancer Genomics" breakout that took part today.

Moleculo data presented at Illumina Scientific Summit

Geoff Smith from Illumina presented a great piece of work his group have been collaborating on with Jill Banfield’s research group at the University of California Berkely, Department of Earth and Planetary sciences. Geoff included a discussion on their use of Moleculo.

NGS forensics might require unique barcodes

The use of NGS in forensics is likely to supplant current STR profiling. One issue that has been discussed is the need to robustly determine that the correct sample has been analysed and reported on. With NGS almost all sequencing today is being done in a multiplex fashion using indexing barcode reads of generally around 6-10bp.

Current barcoding uses sets of up to 96 or 384 barcodes which is fine for many research applications. However there are reports of carry-over of barcodes from one run to another  on the kinds of instruments that are likely to be used in forensic labs. This is likely to make courts nervous about bringing this technology in.

One way to remove some of the problems would be to create significantly longer index oligos that are unique. I can see a market for a consumable kit where each is truly unique. The indexes included would be single-use only and no-one else in the world would get the same index oligo. Oligos can be made in very small synthesis scales today and in high-throughput. Given a long enough read even lower quality synthesis should allow confident discrimination of contamination allowing users to be confident about the final results.

A 25bp barcode read should be as unique as we might ever need and on HiSeq today would not add much time to a sequencing run.

Using SPRI beads to improve Nextera

We have been running Nextera in my lab for a while now and had some great success e.g. 600x C. elegans genomes. With the release of Illumina's rapid exome kits Nextera is proving to be a versatile and useful addition to our lab tools. Unfortunately it has at least one significant weakness; the need to accurately quantify dilute DNA.

Automating Illumina library preparation

Illumina's just announced a partnership with major automation vendors so their s-bot looks like it has been killed as a project. We've got a Tecan Evo in our lab and would like to use it for library prep but a major headache has been the rapid development of library prep methods. There just does not seem time to program and validate a method before the next one comes out.