Thursday, 28 July 2016

10X Genomics single-cell 3'mRNA-seq explained

10X Genomics have been very successful in developing their gel-bead droplet technology for phased genome sequencing and more recently, single-cell 3'mRNA-seq. I've posted about their technology before (at AGBT2016, and March and November 2015) and based most of what I've written on discussion with 10X or from presentations by early access users. Now 10X have a paper up on the BioRxiv: Massively parallel digital transcriptional profiling of single cells. This describes their approach to single-cell 3'mRNA-seq in some detail and describes how you might use their technology in trying to better understand biology and complex tissues.

Monday, 25 July 2016

RNA-seq advice from Illumina

This article was commissioned by Illumina Inc.

The most common NGS method we discuss in our weekly experimental design meeting is RNA-seq. Nearly all projects will use it at some point to delve deeply into hypothesis driven questions, or simply as a tool to go fishing for new biological insights. It is amazing how far a project can progress in just 30 minutes of discussion, methodology, replication, controls, analysis, and all sorts of bias get covered as we try to come up with an optimal design. However many users don't have the luxury of in-house Bioinformatics and/or Genomics core facilities so they have to work out the right sort of experiment to do for themselves. Fortunately people have been hard at work creating resources that can really help and most recently Illumina released an RNA-seq "Buyer’s Guide" with lots of helpful information....including how to keep costs down.

Thursday, 21 July 2016

Core Genomics is going cor-porate (sort of)

I've just had my five year anniversary of starting the Core Genomics blog! Those five years have whizzed by and NGS technologies have surpassed almost anything I dreamed would have been possible when I started using them in 2007. My blog has also grown beyond anything I dreamed possible and the feedback I've had has been a real motivating factor in keeping up with the writing. It also stimulated my move onto Twitter and I now have multiple accounts: @CIGenomics (me), @CRUKgenomecore (my lab) and @RNA_seq, @Exome_seq (PubMed Twitter bots).

The blog is still running on the Google Blogger site I set up back in 2011 and I feel ready for a change. This will allow me to do a few things I've wanted to do for a while and over the next few months I'll be migrating core-genomics to a new WordPress site:

Saturday, 16 July 2016

Whole genome amplification improved

A new genome amplification technology from Expedeon/Sygnis: TruePrime looks like it might work great for single-cell and low-input anlyses - particularly copy number. TruePrimer is a primer-free multiple displacement amplification technology. It uses the well established phi29 DNA polymerase and a new TthPrimPol primase, which eliminates the need to use random primers and therefore avoids their inherent amplification bias. The senior author on the TthPrimPol primase paper, Prof Luis Blanco, is leading the TruePrime research team.

Thursday, 14 July 2016

How much time is lost formatting references?

I just completed a grant application and one of the steps required me to list my recent papers in a specific format. This was an electronic submission and I’m sure it could be made much simpler, possibly by working off the DOI or PubMed ID? But this got me thinking about the pain of reformatting references and the reasons we have so many formats in the first place. It took me ten minutes to get references in the required format, and I've spent much longer in the past - all wasted time in a day that is already too full!

Saturday, 2 July 2016

Comparison of DNA library prep kits by the Sanger Institute

A recent paper from Mike Quail's group at the Sanger Institute compares 9 different library prep kits for WGS. In Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing, the authors used a digital PCR (ddPCR) assay to look at the efficiency of ligation and post-ligation steps. They show that even though final library yield can be high, this can mask poor adapter ligation efficiency - ultimately leading to lower diversity libraries.

In the paper they state that PCR-free protocols offer obvious benefits in not introducing amplification biases or PCR errors that are impossible to distinguish from true SNVs. They also discuss how the emergence of greatly simplified protocols that merge library prep steps can significantly improve the workflow as well as the chemical efficiency of those merged steps. As a satisfied user of the Rubicon Genomics library prep technology (e.g. for ctDNA exomes) I'd like to have seen this included in the comparison*. In a 2014 post I listed almost 30 different providers.

Hidden ligation inefficiency: The analysis of ligation efficiency by the authors sheds light on an issue that has been discussed by many NGS users - that of whether library yield is an important QC or not? Essentially yield is a measure of how much library a kit can generate from a particular sample, but it is not a measure of how "good" that library is. Only analysis of final library diversity can really act as a sensible QC.

The authors saw that kits with high adapter ligation efficiency gave similar yields when compared to kits with low adapter ligation efficiency (fig 4 reproduced above). They determined that the most likely cause was that the relatively high amount of adapter-ligated DNA going into PCR inhibits the PCR amplification reaction leading to lower than expected yields. For libraries with low adapter ligation efficiency a much lower amount of adapter-ligated DNA would make it into PCR, but because there is no inhibition the PCR amplification reaction leads to higher than expected yields. The best performing kits were Illumina Truseq Nano and PCR free, and KAPA Hyper kit with ligation yields above 30%; and the KAPA HyperPlus was fully efficient.

Control amplicon bias: the PhiX control used had three separate PCR amplicons amplified to assess bias. The kits with the lowest bias at less than 25 % for each fragment size were KAPA HyperPlus and NEBNext. The Illumina TruSeq Nano kit showed different biases when using the "Sanger adaptors" rather than "Illumina adaptors", which the authors suggest highlights that both adapter and fragment sequence play a role in the cause of this bias.

Which kit to choose: The authors took the same decision as most kit comparison papers and shied away from making overt claims about which kit was "best". The did discuss fragmentation and PCR-free as important points to consider.
  • If you have lots of DNA then aim for PCR-free to remove any amplification errors and/or bias.
  • If you don't have a Covaris then newest enzymatic shearing methods e.g. KAPA fragmentase have significantly less bias than previous chemical fragmentation methods.
Ultimately practicability, the overall time and number of steps required to complete a protocol, will be uppermost in many users minds. The fastest protocols were NEBNext Ultra kit, KAPA HyperPlus, and Illumina Truseq DNA PCR-free.

*Disclosure: I am a paid member of Rubicon Genomics' SAB.

Wednesday, 29 June 2016

Measuring translation by ribosomal footprinting on MinION?

Oxford Nanopore should have kits for direct RNA sequencing available later this year, and have several examples of how these might be used on their website. The method presented at London Calling (see OmicsOmics coverage), is primarily for mRNA, but it likely to be adapted for other RNA species in due course.

One of the ideas I've briefly thought about is using the MinION to perform ribosome profiling - a basic method would involve ligating adapters to RNA after cell lysis so Ribosomes  are fixed to mRNAs with cyclohexamide treatment. Fast mode sequencing would identify the 3' end and the transcript, then sequencing speed would be massively ramped up to zip the mRNA through the pore; the bound ribosomes should cause sequencing to stall allowing counting of stall events and therefore the number of ribosomes attached to an mRNA.

Friday, 24 June 2016

I don't want to leave Europe

Brexit sucks...probably. The issue is we don't really know what the vote really means, or even if we'll actually leave the European Union in the next couple of years at all. However one thing cannot be ignored and that is the two-fingered salute to our European colleagues from 52% of the UK voting population that got out of bed on a rainy Thursday.

I am privileged to work in one of the UK's top cancer institutes at the top UK University: the Cancer Research UK Cambridge Institute, a department of the University of Cambridge. The institute, and the University, is an international one with people from all across the globe, many of the staff in my lab have come from outside the UK and they are all great people to work with. I dislike the idea that these people feel insecure about their future because our politicians have done such a crap job on governing the country.

I'd like to keep the international feel so if you're still thinking that working in the UK would be good for you (and your a genomics whiz) then why not check out the job ad for a new Genomics Core Deputy ManagerWe're expanding the lab and putting lots of effort into single-cell genomics service (10X Genomics and Fluidigm C1 right now). I'm looking for a senior scientist of any nationality to help lead the team, with NGS experience, and ideas about single-cell genomics.

You can get more information about the lab on our websiteYou can get more information about the role, and apply on the University of Cambridge website.

Friday, 17 June 2016

Come and work in my lab...

I've just readvertised for someone to my lab as the new Genomics Core Deputy Manager. We're expanding the Genomics core and building single-cell genomics capabilities (we currently have both 10X Genomics and Fluidigm C1). I'm looking for a senior scientist who can help lead the team, who has significant experience of NGS methods and applications; and ideally has an understanding of the challenges single-cell genomics presents. You'll be hands-on helping to define the single-cell genomics services we offer, and build these over the next 12-18 months.

You'll have a real opportunity to make a contribution to the science in our institute and drive single-cell genomics research. The Cancer Research UK Cambridge Institute is a great place to work. It's a department of the University of Cambridge, is one of Europe's top cancer research institutes. We are situated on the Addenbrooke's Biomedical Campus, and are part of both the University of Cambridge School of Clinical Medicine, and the Cambridge Cancer Centre. The Institute focus is high-quality basic and translational cancer research and we have an excellent track record in cancer genomics 123. The majority of data generated by the Genomics Core facility is Next Generation Sequencing, and we support researchers at the Cambridge Institute, as well as nine other University Institutes and Departments within our NGS collaboration.

You can get more information about the lab on our websiteYou can get more information about the role, and apply on the University of Cambridge website.

Tuesday, 14 June 2016

SPRI alternatives for NGS: survey results

Everyone loves bead cleanups, and it appears that almost everyone (85%) who read my recent post about SPRI alternatives loves Agencourt AMPureXP. I'd asked readers to take a survey asking if they used AMPure XP, a commercial alternative, or a home-brew version - the results are below.
Take the survey:

I was surprised to see more home-brew responses than commercial alternatives, but this could simply reflect the attitudes of people reading CoreGenomics.