Index mis-assignment to Illumina's PhiX control

Multiplexing is the default option for most of the work being carried out in my lab, and it is one of the reasons Illumina has been so successful. Rather than the one-sample-per-lane we used to run when a GA1 generated only a few million reads per lane, we can now run a 24 sample RNA-seq experiment in one HiSeq 4000 lane and expect to get back 10-20M reads per sample. For almost anything other than genomes multiplexed sequencing is the norm.

But index sequencing can go wrong, and this can and does happen even before anything gets on the sequencer. We noticed that PhiX has been turning up in demultiplexed sample Fastq. PhiX does not carry a sample index index so something is going wrong! What's happening? Is this a problem for indexing and multiplexing in general on NGS platforms? These were the questions I have recently been digging into after our move from HiSeq 2500 to HiSeq 4000. In this post I'll describe what we've seen with mis-assignment of sample indexes to PhiX. And I'll review some of the literature that clearly pointed out the issue - in particular I'll refer to Jeff Hussmann's PhD thesis from 2015.

The problem of index mis-assignment to PhiX can be safely ignored, or easily fixed (so you could stop reading now). But understanding it has made me realise that index mis-assignment between samples is an issue we don not know enough about - and that the tools we're using may not be quote up to the job (but I'll not cover this in depth in this post).

The future of Illumina according to @chrissyfarr

In yesterdays Fast Company piece Christina Farr (on Twitter) gives a very nice write up of Illumina's history and where they are going with respect to bringing DNA sequencing into the clinic. I really liked the piece and wanted to share my thoughts after reading it with Core-Genomics readers.

Reporting on Fluidigm's single-cell user meeting at the Sanger Institute

The Genomics community is pushing ahead fast on single-cell analysis methods as these are revolutionising how we approach biological questions. Unfortunately my registration went in too late for the meeting running at the Sanger Institute this week (Follow #SCG16 on Twitter), but the Fluidigm pre-meeting was a great opportunity to hear what people are doing with their tech. And it should be a great opportunity to pick other users brains about their challenges with all single-cell methods.

Imaging mass-cytometry: the most exciting thing to happen in 'omics?

Mark Unger (Fluidigm VP of R&D) started the meeting off by asking the audience to consider the two axes of single-cell analysis: 1) Number of cells being analysed, 2) what questions can you ask of those cells (mRNA-seq is only one assay) - proteomics, epigenetics, SNPs, CNVs, etc.

Right now Fluidigm has the highest number of applications that can be run on single-cells with multiple Fluidigm and/or user developed protocols on the Fludigm Open App website; 10X Genomics only have single-cell 3' mRNA-seq right now, as do BioRad/Illumina and Drop-seq. But I am confident other providers will expand into non 3'mRNA assays...I'd go further and say that if they don't they'll find it hard to get traction as users are likely require a platform that can do more than one thing.

10X Genomics publications

Anyone that's been reading Core-Genomics will have seen my interest in the technology from 10X Genomics. I've been watching and waiting for publications to come out to get a better understanding of how people are using the technology and thought you might like my current list of articles: many of these are on the BioRxiv and should be available in a reputable journal if you're reading this in 2017 or later!

The number of 10X Genomics publications is going to grow rapidly; and this list will only be updated sporadically!

10X Genomics phasing explained

This post follows on from my previous one explaining the 10X Genomics single-cell mRNA-seq assay. This time round I'm really reviewing the method as described in a paper recently put up on the BioRxiv by 10X's Deanna Church and David Jaffe: Direct determination of diploid genome sequences. This follows on from the earlier Nat Methods paper which was the first 10X de novo assembly of NA12878, but on the GemCode system. While we are starting some phasing projects on our 10X Chromium box the more significant interest has been on the single cell applications. But if we can combine the two methods (or something else) to get single-cell CNV then 10X are onto a winner!

Nuclear sharks live for 400 years

A wonderful paper in a recent edition of Science uses radiocarbon dating to show that the Greenland shark can live for up to 400 years - making it the longest lived vertebrate known. See: Eye lens radiocarbon reveals centuries of longevity in the Greenland shark (Somniosus microcephalus).

Sequencing base modifications: going beyond mC and 5hmC

A great new resource was recently brought to my attention on Twitter and there is a paper describing it on the BioRxiv: DNAmod: the DNA modification database. Nearly all of the modified nucleotide sequencing we hear and read about is modifications to Cytosine mostly methyl cytosine and hydroxymethyl cytosine; you may also have heard about 8-oxoG if you are interested in FFPE analysis. All sorts of modified nucleotides occur in nature and may be important in biological processes where they can vary across tissue of an organism, or may just be chemical noise. The modifications are most important when they change the properties of the DNA strand, how is is read, and what might or might not bind to it e.g mC.

Celebrating 10 years at the CRUK-Cambridge Institute today

Today I have been working for Cancer Research UK for ten years! September 1st 2006 seems like such a short time ago but a huge amount has changed in that time in the world of Genomics. NGS has changed the way we do biology, and is changing the way we do medicine. The original Solexa SBS has been pushed hard by Illumina to give us the $1000 genome, and perhaps just as exciting are the results coming out of Oxford Nanopore's MAP community - this maybe the technology to displace Illumina? What the next ten years will hold is difficult to predict, but today I wanted to focus on the highlights of the last ten years at CRK for me.

CRUK-Cambridge Institue circa early 2006

Optalysys eco-friendly genomics analysis

The amount of power used in a genome analysis is not something I'd ever thought of until I heard about Optalysys, a company developing optical computing that has the potential to be 90% more energy-efficient and 20X faster than than standard (electronic) compute infrastructure. Read on if you are interested in finding out more, and watch the video below - featuring Prof Heinz Wolff!

Optalysys was originally spun out from the University of Cambridge and the technology needs a lot more explanation that I'll give: briefly they split laser light across liquid crystal grids where each "pixel" can be modulated to encode analogue numerical data in the laser beam, this diffracts forming an interference pattern and a mathematical calculation is performed - all at the speed of light. The beam can be split across many liquid crystals to increase the multiplicity and complexity of mathematical operations performed.

Optalysys and the Earlham Institute in Norwich are collaborating on a project to build hardware/software that will be used for metagenomic analysis. This is a long way from comparing 500 matched tumour and normal genomes in an ICGC project; but if Optalysys can build systems to handle this scale then the huge compute processing tasks might be carried out at a fraction of the current costs and whilst running from a standard mains power supply.

PS: do you remember the Great Egg race as fondly as I do?

Upcoming Genomics conferences in the UK

It is almost time for the kick off at Genome Science, probably the best organised academic conference in the UK. It runs from August 30th to September 1st next week and sadly I can't be there (just returned from holidays and too much going on). You can hear from a wide range of speakers in a jam packed agenda. This year it is hosted by the University of Liverpool, and the evening entertainment comes from Beatles Tribute Band “The Cheatles”!

What other conferences are available for Genomics in the UK, and which one should you attend if you too can't make it over to Liverpool? The Wellcome Trust Genome Campus is holding their first Single Cell Genomics conference from September 9th (sold-out I'm afraid). Personally I thought that the London Festival of Genomics was excellent and I've high hopes for the January 2017 meeting. 

Often it is word of mouth that brings a conference to my attention, but there are a couple of resources out there to help.

• AllSeq maintain a list of conferences.
• GenomeWeb has a similar list, but it seems less focused than AllSeq.
• NextGenSeek has a list for 2016, but nothing on the cards for 2017 yet.
• Nature has an events page (searchable) that lists 50 upcoming NGS conferences.

PS: please do let me know if you've particular recommendations on conferences to attend. And do get in touch with the groups above to list your conference on their sites.

PPS: If you can justify it then the HVP/HUGO Variant Detection Training Course - "Variant Effect Prediction" running from 31st October 2016 is in Heraklion, Crete - a beautiful place to learn!