Final day of conference, with some serious fatigue setting in (my hotel room was too close to, and faced, a highway. Doh!)
Discovered that I was indeed getting a reputation. Two people I met today asked about my recurrent interest in FFPE (Formalin Fixed, Paraffin Embedded) -- which is how most of the nucleic acids I want to work with are stored. FFPE is notoriously difficult for molecular studeis, with the informational macromolecules having been chemically and physically abused in the fixation process, but it is also famously stable, preserving histological features for years.
Rain Dance sponsored the breakfast & announced that their maximum primer library size has gone up to 20K. To back up, Rain Dance uses microfluidics to create libraries of very tiny (single digit picoliter) droplets in which each droplet contains a primer pair. The precise volume control & normalization of the concentrations means that each primer droplet contains about the same number of oligos, which allows each droplet in a PCR to be run to completion -- meaning that efficient PCRs and inefficient ones in theory end up both having the same number of product molecules. Another set of droplets are created which contain your template DNA, and these are cleverly merged & the whole emulsion cycled. Break up the emulsion & you have lots of PCR amplicons ready to go into a fragmentation protocol. Their movies of droplets marching around, splitting, merging, etc. are dangerously mesmerizing!
Jin Billy Li of the Church group reviewed all the really cool stuff they've done using padlock probes (and confirmed that IP conflicts are retarding indefinitely any commercialization of these). A padlock probe is a long DNA which primes on both sides of a targeted region. Filling the gap between & ligating the gap yields a circle, which can be purified away from any uncircularized DNA and then amplified with universal primers. Turns the multiplex PCR problem into a very diverse set of uniplex PCRs. Various tweaks have substantially improved uniformity, though there is still room for improvement (but the same is true for the hybridization approaches).
Nicolas Bergman presented data on transcriptomic complexity in B.anthracis. I think most of this is published, but I hadn't seen it. A very striking result is that an awful lot (~88%) of transcripts in a supposedly uniform culture are present at much less than 1 copy per cell. He mentioned that small numbers of spores are seen in log cultures, and this might explain it. Also showed that many unannotated genes -- including some that had been truly UNannotated (originally annotated but then removed from the catalogs) are clearly transcribed. Operon structures could be worked out, with 90% matching computational predictions -- and in ~30 testest experimentally by RT-PCR there was 100% concordance.
Epicentre gave an overview of their clever system for fragmenting DNA upstream of either 454 or Illumina. By hijacking a transposase in a clever way, they not only break up the DNA but add on defined sequences. For 454 you then jam on the 454 primers & just get stuck reading 19nt of transposase each time; for Illumina you must use custom sequencing primers.
Eric Wommack & Shawn Polson of University of Delaware (Go Hens!) described work on metagenomics of bacteriophages in seawater. Here's a stunning estimate: if you lined all the world's phages end-to-end, they would stretch 60 light years. Also striking is the high level of bacteriophage-driven turnover of oceanic bacteria -- in about 1/2 to 2 days there is 100% turnover. This is a huge churn of the biochemical space.
Stacey Gabriel gave an update on the Broad's Cancer Genomics effort. Some whole genomes (25 tumor+normal pairs so far) and a lot of exonic sequencing. So far, not a lot of lightning though -- in one study the only thing popping out so far is p53, which is disappointing. Using the Agilent system (developed at the Broad), they can scan 20Kgenes in 1/2 an Illumina run, with 82% of their targeted sequences having at least 14 reads covering.
Matthew Ferber at the Mayo described trying to replace Sanger assays for inherited disorders with 454 and Illumina based approaches. He underscored that this isn't for research -- these are actual diagnostic tests used to determine treatments, such as prophylactic removal of the colon if inherited colon cancer is likely. Capture of the targets on the Nimblegen chips were done and the recovered DNA split to do 454 & Illumina sequencing in parallel. The two next gen approaches came close -- but neither found enough that they could be relied on. Also, some targets are just not recoverable by array capture and would need to be backstopped by something else. One caveat: older technology was used in both cases, so it may be with longer read lengths on both platforms the higher coverage & higher mapping confidence needed would be obtained. On the other hand, some of the mutations were picked to be difficult for the platforms (small indel for Illumina, homopolymer run of >20 for 454) and might remain problems even with more coverage. PCR amplification in place of chip capture is another approach that might improve coverage and get some targets missed by the chip (this is certainly a claim RainDance made in their presentation).
The last talk I took notes on was by Michael Zody on signatures of domestication in chickens. If I had organized things, this would have been just before or after the phage talk! Alas, while the Rhode Island Red was amongst the lines sequenced (apropos the location) Blue Hens were missing -- how could that be? Seriously, the basic design was to sequence pools of DNA from either various domestic chicken lines or the Red Jungle Fowl (representing pre-domestication chicken). Some of these lines were commercial egg layer strains and others commercial broiler (meat) strains. He commented that this level of specification occured very resently (forgot to write down when, but I think it was around a century ago). Two other strains are interesting as they have been selected for about 50 years for one to be very heavy and the other lean -- apparently the heavy line will eat itself silly and the other nearly starves itself. 1 SOLiD slide on each of the 10 pools was used to call out SNPs and various strategies were used to filter out errors in the new data as well as variation due to errors in the reference sequence (in some cases, even typing the reference DNA to demonstrate the need for correction). Reduced heterozygosity was seen around BCDO3, which gives modern chickens their yellow skin (positive control) and also a bunch of other loci -- but those are still under wraps. They also looked for deletions in exons which appear to have been fixed in various lines, and found 1284 which are fixed in one or more domestic lines relative to the Red Jungle Fowl. One interesting one (which is present in the Red Jungle Fowl at low frequency) has gone homozygous (I think; my notes here show fatigue) in the high growth line but is either absent or heterozygous in the low growth line (terrible notes!). It's a 19kb deletion that clips out exons 2-5 (based on the human homolog; there isn't a good transcript sequenced for chicken) and RT-PCR confirms the gene is expressed in the hypothalamus, which has been previously implicated in controlling the feeding behavior.
I took almost no notes on the last talk, looking at dietary influences on gut microbiome (and also, regrettably, had to leave early to make sure I made school night) but it did feature some more "extreme genomics" -- microbiome studies on burmese pythons!
One last thought: sequencing techs represented were either here-and-now (the players you can actually buy) or pretty-distant-future; absent were PacBio and Oxford Nanopore and the host of other companies (save NABSys) announced in the last 3-4 years in this space. Have the others just disappeared quietly or are they in stealth mode? It's hard to imagine the conference would have deliberately snubbed them, which would be a third possibility.
Thanks for doing such a great job summarizing almost the entire conference. You've put me to shame! We hope to see you presenting next year.
To address your last question, CHI (publisher of Bio-IT World) would never *snub* a company -- both PacBio and ONT were invited to present at Providence but politely declined. PacBio's founder Steve Turner presented at our sequencing meeting last year, while ONT sponsored the poster competition 6 months ago, but has so far declined to speak (not just at our event but I believe anywhere). I did, however, interview ONT VP Research John Milton on camera at the conference and will post that link when we have it.
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