Longitudinal monitoring of tumour burden with a modified Guthrie card

The ability to sequence DNA from a Guthrie card e.g. genomic or methylomic analysis (also see my post from last Summer) makes their more routine use in cancer genomics studies attractive. An area that would be great to see developed is the extraction of the cell-free DNA from a blood spot. This would be incredibly easy for patients to use and potentially allow very long term studies to be performed without visits to a clinic for a blood draw.

There are some big hurdles to overcome. A blood spot does not contain a lot of blood so the amount of cell-free DNA is likely to be very low restricting analysis to variants at high allele frequency. Also cell free DNA is likely to be contaminated with high-molecular weight genomic DNA, further diluting cell-free alleles. But for some applications this is not a problem.

Ideally cell-free DNA would be eluted/analysed separately from the genomic DNA from PBMCs for downstream mutation analysis. Current extraction methods lyse cells to extract maximum yield, but this lysis could possibly be avoided for ctDNA methods? The company Circulogene, spun out of Johns Hopkins in, is developing nucleic acid extraction products based on its proprietary Nanobind technology. Most recently their new pan-cancer NGS panel test was covered on GenomeWeb where Chen-Hsiung Yeh (CSO) described the 3,000 hotspot, 50 gene panel, which uses their ctDNA isolation technology. Technological improvements that allowed the use of dried blood spots may not be "on the cards" (sorry for the pun) for Circulogene, but I do hope someone is working on similar ideas.

Blood spot cards could be easily mailed out to patients and posted back to a central lab for processing. Adding information via 2D barcodes would allow fully automated test analysis so patients could be matched up to specific NGS panel tests.

AGBT16 it's the final countdown!

Saturday, February 13, 2016 14:45
Def Leppard are playing "Pour some sugar on me" while we wait for Nick Loman to walk on stage - not sure if this was intentional.

AGBT16 day 4: KAPA HyperDrive

Saturday, February 13, 2016 12:12:

Ross Wadsworth, KAPA Biosystems “Hyper Drive: high performance, streamlined workflows for RNA-seq and target enrichment”. KAPA Hyper (Covaris frag, fast efficient library prep), KAPA Hyper+ (enzymatic frag, fast efficient library prep), now RNA is getting the Kapa touch!

AGBT16 day 4: a new sequencing chemistry from Nanostring

Saturday, February 13, 2016 11:47:

Joe Beecham Nanostring “New optical barcode chemistry's power 3D biology & sequencing-simultaneous DNA, RNA, & protein analysis and low error-rate targeted sequencing” A sequencing system with no library prep or amplification - this is a pretty cool project. Joe is presenting something quite early on but wants to engage with the community on building out this chemistry.

AGBT16 day 4: Directed Genomics

Saturday, February 13, 2016 11:26:

Cynthia Hendrickson, Directed Genomics New England BioLabs “A novel fully customizable hybridization-based enrichment technology integrating capture and library preparation.” Cynthia was describing the new target capture chemistry she's been building with NEB. I wrote up an overview of their method in a post from last years AGBT. The method works with 10ng to 1ug of DNA from FFPE and FFZN (20-100ng inputs shown), and generates high-quality and fully automateable capture libraries in a 6-7 hour turnaround  with; 90% of reads on target, very high coverage uniformity and minimal GC bias. You can capture a single gene or many targets, allowing a single workflow  instead of having to choose between PCR or hyb-cap. The technology includes barcodes for up to 120 sample IDs at i7, and a 12bp UMI on the i5 end.

AGBT16 day 3 concurrent sessions: Nelson (C.elegans), Otto (FM ctDNA), Mungall (rearrangements)

Friday, February 12, 2016 21:10: I'm flitting between sessions tonight...first up is a talk by a users of my lab, we generated the data for several hundred C.eleganss genomes using Nextera and HiSeq 2500 - not quite a 1000 genome project yet!

Geoffrey Nelson 

Geoffrey Nelson, MRC Laboratory of Molecular Biology “A high-throughput genomics approach to efficiently link genes to phenotype”. The De Bono lab uses C.elegans as a model for how nervous systems coordinate behaviour, it's a good model as it has 20,000 genes but in a genome ust 100Mb in size.

AGBT16 10X early-access customer presentations

Stacey Gabriel

Stacey is the Director of the Genomics Platform at the Broad Institute. Gave us a brief look back to 2006: 117 instruments (GA's) producing 600,000 bases per day and 1 genome, versus 30 machines producing 12,000,000,000,000 bases per day and 32693 genomes!

AGBT16 10X Genomics workshop

10X Genomics were one of the stars of the show at AGBT15, this year they're bowling attendees over with new developments. They've obviously had an awfully busy year. They announced lots at JP Morgan (GenomeWeb, Core-Genomics). I got a sneak peak (as did Keith at OmicsOmics) to their announcements just prior to AGBT and am happy to share it with you now.

10X AGBT workshop: At today's AGBT workshop 10X filled in the gaps (excuse the pun) on what they've been doing and showed their new Chromium instrument ($125,000). This is an updated version of the original GemCode platform that runs new application specific reagent kits. They also showed data from the new application platforms: Chromium Genome, Chromium Exome, Chromium Single Cell.

Sequencing at AGBT

Contrary to what you might think this post is not going to be about the sequencing technology or data being presented at AGBT this year, but what could have happened several years ago and has come to AGBT16 - real-time sequencing.

AGBT16 day 3 plenary: Bielas, Piazzan and Kohn

Jason Bielas

Jason Bielas, Fred Hutchinson Cancer Research Center “Deep profiling of complex cell populations using scalable single cell gene expression analysis”. Early adopter of 10X single-cell RNa-seq platform - find out more at the 10X workshop this afternoon. Initially running experiments on PBMCs and finding multiple clusters, the number of these is very dependant on the number of cells profiled. Changing from 4500 to 16K cells increases resolution and they went up to 68K cells. They used a mixed 293T and Jurkat population to validate the single cell separation and found only ~1% doublets. They are now looking to use this system to recover single cell transcriptomes from mixtures, enumerate and classify different immune cells, and identify novel cell types; future work will loot to count tumour infiltration, investigate cSNPs, etc, ther are lots of potential apps.

AGBT16 day 3 plenary part 2 - Anne Wojcicki

Anne Wojcicki

Anne Wojcicki, 23andMe “Making discoveries on the 23andMe platform”. Anne is going to focus on the benefit to the consumer of the Human genome. How will we decipher it, how will we cope with massive studies i.e. tens of millions of active participants.

The 23andMe research platform: 1.2 million opt-in customers using an IRB-approved broad consent with opt-out at any point, or for each survey, and all data is aggregated and anonymised. 23andME want to grow to the 10s, 100s of millions of individuals because 80% of customers are consenting to research.

AGBT16 day 3 plenary part 1 - Jay Flatley

Friday, February 12, 2016 09:40 (updated to improve the description of the chemistry and applications)

Jay Flatley, Illumina “Beyond the $1000 Genome-What’s next for NGS?”. He spent his time describing some of the key ingredients that have driven the sequencing tech at Illumina, and gave us a little historical overview of Illumina (see this 2004 presentation from Solxea).

1. the best technology, estimated about 3 million hours in development of SBS (around 350 years).
2. the best team
3. an engaged community (many applications developed by customer, ChIP-Seq, cancer genomes, single-cell genomes), showed the wonderful methods poster by Jaques Retief. An estimated 2000 analysis methods.

Jay went through some of the major breakthroughs: 2007 GA; 2010 line scan imaging (HiSeq 2000); 2011 LED optics and basespace (MiSeq); 2012 rapid run mode, faster fluidics and scanning (HiSeq 2500); patterned flowcells (HiSeq X); 2014 2 colour SBS, dry flowcells and consumer optics (NextSeq).

Firefly - What has Illlumina been doing with Avantome: Jay described this as a development plan - not available till Christmas 2017!

The original Avantome sequencing on CMOS required emulsion PCR, which was too limiting so Illumina never brought to market, Jay showed the sequencer that was built but never sold! However Illumina kept working on this technology and have implemented SBS on CMOS. "Other implementations to date have sacrificed accuracy" - I think Jay may be refering to Ion Torrent. Semindcondutor is a one channel device. He showed an image of the CMOS chip tech but no photos are allowed at AGBT so you'll have to find one somewhere else. Briefly nanowells  are fabricated over each photodiode and clusters are built directly on top of the CMOS sensors. Unlike the 4-colour HiSeq or 2-colour NextSeq the new Firefly implements an innovative 1-colour SBS  solution: A (removable fluor), T (permanently labelled), G (initially unlabelled) and C (unlabelled). After incorporation (and initial imaging) a chemistry cycle allows separation of the bases, the A fluor is removed, and G is now tagged with a fluor. A raw read accuracy of 99% is achievable and is roughly comparable to HiSeq X.

The systems uses an integrated digital fluidic library prep and sequencing instruments with a very small footprint. It uses the NeoPrep fluidics (is this going ot slow it down).

Firefly Specs: Run times are just from 3.5 to 13 hours for up to 2x150PE, 1Gb per run, 4M reads at $100 per sample and about $30,000 capital costs. The applications Illumina are aiming Firefly at are: targeted sequencing, infectious disease, inherited disease, and reproductive and genetic health.

Why is Illumina talking about it so far out from launch? The reality is that it is going to be difficult to keep this quiet with so many 3rd parties involved. Also Illumina are opening up the microfluidics apps to 3rd parties.

Recent collaborations and new ventures from Illumina: BioRad, 10X, Helix (consumer genomics from Illumina), Grail; which aims to detect cancer early by monitoring cell-free circulating tumour DNA. Screening asymptomatic individuals is challenging as you are right at the limits of detection in many cancers. may need to sequence up to 300,000 genomes at 60X by 2019.

AGBT16 Day 2 Genome Technology Concurrent Session

Thursday, February 11, 2016 21:35

Mariateresa de Cesare
Mariateresa de Cesare, University of Oxford “Unlocking the heterogeneity of the human transcriptome utilizing the ONT MinION TM”. The lab has been working with the ONT MinION for about two years. Mariateresa presented some of the Pros of RNA-seq, as well as the Cons, mainly the need to reconstruct transcripts from short reads. Ideally we'd be reading through the whole transcript to get direct counts of isoforms rather than inferring them from short-reads.

AGBT16 day 2 afternoon

Thursday, February 11, 2016 17:08:

Beth Shapiro: 
Beth Shapiro (@bonesandbugs) talked about "the rise and fall of passenger pigeons", which went extinct in 1914, prior to this rapid decline there were lots of them, described as "an infite number of them" in 1534, but "massive flocks" were efficiently trapped/shot and the species rapidly declined to zero.

Beth posed the question "How did that happen?" seems to be the same answer for other recent extinctions or near-misses - massive over hunting by us.

AGBT16 day 1 plenary session

Wednesday, February 10, 2016 21:00: you can follow my official blog coverage on the AGBT blog and see posts from the rest of the AGBT team.

Sean Eddy
Sean Eddy kicks off the first plenary session “Genomics I” (chaired by Mike Zody from NYGC) his talk title was “Genome evolution: the future of deciphering the past” but has been changed to "A statistical test of RNA base pair variation applied to lncRNA structure". Sean’s lab in the HHMI at Harvard University focuses on developing computational methods for genome sequence analysis, particularly interested in methods to identify how genomes evolve. Sean was the 2007 Benjamin Franklin Awardee for his open access distribution of HMMER, and co-creation of the Pfam database.

Get ready for AGBT

Wednesday, February 10, 2016 11:26 Welcome to AGBT 2016 (or #AGBT16 if you are on Twitter) in sunny Orlando. We’re not going to be able to walk to the beach and take a dip in the Gulf this year, but I’m certainly hoping the Marriott Grande Lakes can live up to what attendees have come to expect from the Marco Island Marriott. Getting here was easy enough, but I’ve come to enjoy the drive across the Everglades on Route 41 from Miami.

What does my MP have to say about personalised medicine?

Quite a lot actually...In a New Statesman article, sponsored by Merck, George Freeman (my local MP) was asked for his thoughts on future of Personalised Medicine by Merck’s Head of Public Affairs, Larushka Mellor.

George described himself as one of the “early evangelists for the new landscape of “translational” medical research”. George Freeman has a 15-year career in biotech/biomed behind him; Merlin Biosciences, Amedis Pharmaceuticals and 4D Biomedical, so his evangelicism for personalised medicine is unlikely to be purely political spin. Just five years after his election he was appointed Government Life Science Advisor, and three years later in 2014, the first Minister for Life Sciences. His portfolio is pretty big - including the NHS drugs budget, data and digital health, the NIHR, genomics, NICE and industry engagement.

He describes the promise of personalised medicine as “the Holy Grail for 21st-century healthcare” and “as game-changing as…penicillin”. I’m not quite so sure personalised medicine will have the same impact as penicillin, but I agree wholeheartedly that it is a part of a medical revolution, particularly, and most obviously in oncology, but one that will have implications in many (all) fields of medicine. George hopes to cement the UK as a world-leader in this revolution, and wants to drive big changes in the NHS. And Genomics England’s 100,000 genomes project is the most obvious example driving this agenda for change. Launched in 2011 (with the PM visiting my lab and starting a MiSeq run during the launch) the £250 million project is starting to get traction, patients are being recruited very rapidly, genomes are starting to come out of the Illumina pipeline now based at the Hinxton campus next door to Sanger and EBI.

International competition: But George says that no other country in the world is investing in genomics in the same way, I’d disagree. Many countries are running or starting genomic medicine projects (Estonia, Sweden, Iceland, Saudi Arabia, etc), and even private funding is getting in on population genomics e.g. Human Longevity Inc.. The world is marching towards personalised medicine fast. It is great that the UK is leading the world in this, but being the leader also means discovering many of the problems that lie in the path of success, education (of doctors and patients), analysis (from sample collection to results) and interpretation (from bioinformtics to clinical reports). All of these areas are likely to produce growth in the science economy that the UK Government is looking for, but with other countries certain to compete for what is a big prize the UK needs to demonstrate it can be successful. It is a little ironic that a UK funded £250M sequencing project is being processed by a multi-billion dollar US business built on UK science and technology (Solexa), although it is not clear we'd have the $1000 genome without Illumina's drive. For the personalised medicine push lets hope the UK goverment can retain much of the innovation coming out of UK academia as UK businesses.