Tuesday 29 September 2015

Do you have a HiSeq 2500 V4 to sell

My lab is looking to put an additional HiSeq 2500 v4 in place, Illumina do not have any (refurbs) to sell in the US or Europe so I thought I'd post here to see if any Core Genomics readers have an instrument they are looking to retire? If we can offer you more than Illumina's trade-in on a HiSeq 4000 then we might be able to come to some arrangement!

Drop me an email at James.Hadfield@cruk.cam.ac.uk if you might be able to help.

Friday 25 September 2015

Plagiarism alert via Twitter

I built several TwitterBots last year to scrape papers on PubMed (thanks again to Casey), these have turned out to be really useful in alerting me to new work but last night I got a very interesting Tweet from @MattiasAine...

Wednesday 23 September 2015

Rubicon Genomics webinar on making cfDNA/ctDNA NGS libraries

Rubicon Genomics have a webinar on Creating Cell-free DNA Libraries with ThruPLEX Plasma-seq that was broadcast on September 17th 2015. The new PlasmaSeq kit has a very simple, 3-step, 15 minutes hands on time, single-tube workflow.

Dr. Kamran Shazand, Rubicon Genomics' Director of Applications presented an intro to cell-free DNA - generally 170bp fragmented DNA, which is the result of degradation by apoptocic enzymes, and that can appear in dimeric and trimeric nucleosomal fragments. Rubicon's earlier chemistry ThruPLEX was use in 2012/12 to sequence foetal and tumour genomes and it has a recommended input of 1-30ng of DNA.

The new PlasmaSeq kit seems to make very nice libraries. Internal data from Rubicon showed better performance against NEBnext and KAPA Hyper, with high diversity, low PCR duplication rates and good GC coverage. Most interestingly Rubicon have built a target capture pipeline using their PlasmaSeq kits, Agilent SureSelect and IDT blockers allowing exome sequencing from as little as 1ng of input DNA.

Methods: optimised DNA repair and ligation of stem-loop adapters, PCR to extend, cleave and amplify the library. You should end up with a major peak at 310bp for cfDNA libraries.

Rubicon also talked about their work looking into how molecular indexes night be added to allow removal of PCR duplicates to PlasmaSeq - -send them your comments if you are interested.

Disclosure: I am a user of Rubicon library prep technology; but also Illumina, NEB, Enzymatics, and many others too! I am an author on the ctDNA paper from 2012 mentioned above and in their webinar. I am on the SAB of Rubicon.

Research Professional: making finding funding easier

I'm incredibly lucky because I don't usually need to write grants. My lab is core funded by CRUK to provide genomics services to the CRUK- Cambridge Institute. However I do like to keep an eye out for opportunities to bring funding in for specific projects.

Our very good grants team pointed me to a tool that the University subscribes to *Research Professional. Once logged in I can easily search for new funding opportunities, if you've never used it it could become one of your favourite websites.

Research Professional began as a (print) “newspaper for the research world" in 1994, and moved to the web in 2001. It make accessible the opportunities and information on research grants to registered users and may institutions have purchased access. If your host Institute does subscribe then you should certainly take a look! You can search very easily and follow links directly to funders websites. The site generally provides a good summary of funding criteria so you can quickly weed out opportunities that you will not be eligible for.

Monday 21 September 2015

Better ChIP, requires better antibodies

ChIP-seq can only really work if you have a good antibody for the protein of interest. Any non-specific binding is going to add noise to your data making motif finding, differential binding analysis difficult or even impossible. Back in 2008 Mathias Uhlén's group at the Royal Institute of Technology, Sweden, published a study that showed how less than 50% of antibodies tested were specific to the target. 

Wednesday 9 September 2015

#GenSci15 plenary session and wrap up

Sorry about the accidental posting...here's the final version.

Innes Barroso, Head of Human Genetics at Sanger Institute "Genomics and Metabolic Disease – what have we learned so far"

Type 2 diabetes, many patients controlled by table some on insulin, it is a very complex disorder, both genetic and environmental effects (particularly obesity, diet, exercise). Incomplete understanding of Type2 diabetes genetics, extended families are hard to obtain due to generally late onset. Progress has been slow, early studies failed to find robust loci that replicated across studies. However as power of GWAS studies improved six loci were found outside previously suspected candidate genes, but with small effect. Analysis of extremes can be very powerful i.e. young, lean patients vs old obese controls (similar approach being pursued in smoking with some success). Development of genotype imputation and meta-analysis had big impact on how GWAS could impact Type2 diabetes genetics. But still lots of "hidden heritability", drove development of custom GT chips e.g. Metabochip and UK Biobank Axiom array.

#GenSci15 day3 clinical genomics session

Andrew Beggs is chairing the clinical genomics session – this is why I am here, cool stuff!

Interesting that the Tolstoy quote from Pippa Thomson's talk (see below) "Happy families are all alike; every unhappy family is unhappy in its own way" came up, as it could be argued that the Anna Karenina principle probably applies to the clinical use of genomics right now: a deficiency in any one of a number of factors dooms it to failure. Clinical genomics can only have a successful impact if every possible deficiency in the study has been avoided i.e. clinical and family history, quality of exome coverage, issues with alignment etc, etc, etc,

Tuesday 8 September 2015

#GenSci15 day 2

Day 2
Single cell was popular, but were all crammed over in the Arts building somewhere close to Solihull. Nick forgot to organise a coach so we had to walk, I guess we're building up an appetite for the street food tonight! Bill Hanage asked if Nick could sequence some of the street food just in case - perhaps next years conference pack can come with a stool collection kit for some crowd-sourced science: "the differential impact of conference attendance on gut microbiota of PhD students, post-docs and PIs"?

#GenSci15 day 1

I'm at the 6th UK Genome Science conference in beautiful and vibrant Birmingham. There is a good attendance and the place is full with a real buzz about new technologies (more about that in a sec), the work people are doing and the freebies on the stands!

I'm not going to attempt to round up each and every day but I'll probably post tomorrow and Wednesday on my favourite bits. Here's what happened yesterday...

Daniel MacArthur: Mass Gen, Broad, Harvard spoke about the lessons from analysis of 60,000 human exomes. He was making the point that "making sense of one genome requires thes of thousands of genomes." In the ExAC (exome aggregation consortium) they've gathered almost 100,000 exomes which have then been consistently analysed as a single batch to produce a single VCF. Dan showed that by doing this they were able to remove noise from rare disease analysis - and although I'm not sure about the impact on cancer exomes it is likely to make it easier to filter variants from those datasets too.

Tuesday 1 September 2015

S is for Sequencer: the new instrument from Ion Torrent

Today Ion Torrent launched their newest sequencers the S5 ($65k or $150k with the S5 XL, which includes more compute). Ion Torrent was exciting technology when we first heard about it and I'm disappointed it never quite lived up to the promise, and that it never competed against Illumina in the space I cared about. The new systems do not change my disappointment, but they may increase the competition for Illumina in the amplicon sequencing space. In my lab we're still running lots of amplicons using Fluidigm and HiSeq and I don't think we'll be buying an S5 anytime soon. However labs looking to set up clinical (or other) tests on standard panels might see the S5 as a realistic alternative to a MiSeq. But Ion need to deliver on this visibly and believably, I know lots of labs that have stopped using their PGMs, although I know a few that have stopped using their MiSeq's too!

The YouTube video (above) offers a very marketing-heavy overview (where is the S5 vs MiSeq video). Ion have focused very clearly on amplicons and delivering results as quickly as possible at reasonable cost. The S5 XL includes additional compute for processing to speed up this data delivery in an automated manner.

Like PGM and Proton before, S5 offers multiple chip configurations that allow you to run fewer samples without sacrificing per sample costs (too badly). The 520/530 chips are equivalent to the PGM offering 200-400bp runs with 5 or 20M reads per run. The 540 chip only offers 200bp runs but with 80M reads. All with 2 to 4 hour run times and analysis completed in as little as 5 hours.

S5 and Ampliseq: An open letter from Mark Gardner (Ion's GM) at the Behind the Bench blog explains the desire to continue the democratisation of sequencing. The aim was to  make targeted NGS easy for anyone to work with, and to deliver the best value benchtop sequencer. As the S5 system is very obviously targeted to amplicons I thought I'd highlight what Ion say in their literature about the numbers of samples per run - using the Ion AmpliSeq Cancer Hotspot Panel v2 (50 genes), 16, 48 or 96 samples can be run on 520, 530 or 540 chips respectively.

You'll need the Ion Chef in your lab to go from DNA to data with less than 45 minutes of hands-on time; otherwise you'll have to manually prepare libraries and templates for chip loading.

Data analysis can be completed automatically within 5 hours.

This does offer a tempting solution for people sequencing panels, but it may be limited in what else is realistically possible on other (Illumina) sequencers. The low-cost is going to be attractive, but only to a point. If Ion can deliver a truly end-to-end solution then they may be onto a winner.

AllSeq got their first with their digging around on Google, and Keith at Omics Omics got a sneak peek, "Ion have identified a profitable market segment...fast, targeted sequencing -- and is going full bore in this area". Keith points out that the new chips are not compatible with PGM or Proton, "the concept that all upgrades are encased in the consumable is long dead". He goes into some depth about the competition for these new instruments; MiSeq (takes four hours to deliver 1x36bp); GnuBio, Genia/Roche and QIAGEN have gone all but silent (expect more from Qiagen very soon), and ONT's MinION (possibly).

See it yourself on the Ion Torrent world tour. The UK Genome Sciences meeeting does not feature on the list of upcoming conferences where Ion will present data - I am sure there's still room. You can catch them in London on the 10th.

I'll be interested to start talking with users at AGBT in February. Whether the S series will take off where PGM and Proton did not may partly depend on whether PGM and Proton users get a good trade-in.
Sign up for Ion World Tour location near you >
Sign up for Ion World Tour location near you >
Sign up for Ion World Tour location near you >

PS: If I wanted to trade-in a HiSeq 2000 for amplicon work what would Ion offer me?