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.

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.

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: Enseqlopedia.com. 

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.

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!

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.