The changing landscape of sequencing platforms that underpin genome assembly

From Flickr user itsrick208. CC BY-NC 2.0

From Flickr user itsrick208. CC BY-NC 2.0

In my last blog post I looked at the the amazing growth over the last two decades in publications that relate to genome assembly.

In this post, I try seeing whether Google Scholar can also shed any light on which sequencing technologies have been used to help understand, and improve, genome assembly.

Here is a rough overview of the major sequencing platforms that have underpinned genome assembly over the years. I’ve focused on time points when there were sequencing instruments that people were actually using rather than when the technology was first invented or described. This is why I start Sanger sequencing at 1995 with the AB310 sequencer rather than 1977.

Click to enlarge

Return to Google Scholar

So how can you find publications which concern genome assembly using these technologies? Well here are my Google Scholar searches that I used to try to identify relevant publications.

  1. Sanger — "genome assembly"|"de novo assembly" sanger -sanger.ac.uk — I had to exclude the Sanger’s website address as this was used in many papers that might not be talking about Sanger sequencing per se.
  2. Roche 454 — "genome assembly"|"de novo assembly" 454 (roche |pyrosequencing) — another tricky one as ‘454’ alone was not a suitable keyword for searching.
  3. Illumina — "genome assembly"|"de novo assembly" (illumina|solexa) — obviously need to include Solexa in this search as well.
  4. ABI SOLiD — "genome assembly"|"de novo assembly" “ABI solid”
  5. Ion Torrent — "genome assembly"|"de novo assembly" "ion torrent”
  6. PacBio — "genome assembly"|"de novo assembly" ("PacBio"|"Pacific Biosciences”)
  7. Oxford Nanopore Technologies — "genome assembly"|"de novo assembly" "Oxford Nanopore”

Now obviously, many of these searches are flawed and are going to miss publications or include false positives. This makes comparing the absolute numbers of publications between technologies potentially misleading. However, it should still be illuminating to look at the trends of how publications for each of these technologies have changed over time.

The results

As in my last graph, I plot the number of publications on a log scale.

Click to enlarge

Observations

  1. Publications about genome assembly that mention Sanger sequencing dominate the first decade of this graph before being overtaken by Illumina in 2009.
  2. The growth of publications for Sanger is starting to slow down
  3. Publications for Roche 454 peaked in 2015 and have started to decline
  4. Publications concerning Ion Torrent peaked a year later in 2016
  5. ABI SOLiD shows the clearest ‘rise and fall’ pattern with five years now of declining publications about genome assembly
  6. The rate of growth for PacBIo publications has been pretty solid but may have just slowed a little in 2017
  7. Oxford Nanopore, the newest kid on the block — in terms of commercially available products — has been on a solid period of exponential growth and looks set to overtake Ion Torrent (and maybe Roche 454) this year.

Are we about to reach ‘peak genome assembly’?

Sanger Peak. Image from Google Maps.

Sanger Peak. Image from Google Maps.

The ever-declining costs of DNA sequencing technologies — no, I’m not going to show that graph — has meant that the field of genome assembly has exploded over the last decade.

Plummeting costs are obviously not the only reason behind this. The evolving nature of sequencing technologies has meant that this year has pushed us into the brave new era of megabase pair read lengths!

Think of the poor budding yeast: the first eukaryotic species to have its (12 Mbp) genome sequenced. There was a time when the sequencing of individual yeast chromosomes would merit their own Nature publication! Now only chromosome IV remains as the last yeast chromosome whose length couldn’t be exceeded by a single Oxford Nanopore read (but probably not for much longer!). Update 2018-09-12: a 2.2 Mbp Nanopore read means that chromsome IV's length has now been eclipsed!

Looking for genome assembly publications

I turned to the font of all (academic) knowledge, Google Scholar, for answers. I wanted to know whether interest in genome assembly had reached a peak, and by ‘interest’ I mean publications or patents that specifically mention either ‘genome assembly’ or ‘de novo assembly’.

Some obvious caveats:

  1. Google Scholar is not a perfect source of publications: some papers are missing, some appear multiple times, and occasionally some are associated with the wrong year.
  2. Publications are increasing in many fields due to more scientists being around and the inexorable rise of if-you-pay-us-money-and-randomly-hit-keys-on-your-keyboard-we-will-publish-it publishing. So a rise in publications in topic 'X' does not necessarily reflect more interest in that topic.
  3. Not all publications concerning genome assembly will contain the phrases ‘genome assembly’ or ‘de novo assembly’.

Caveats aside, let’s see what Google thinks about the state of genome assembly:

Click to enlarge

Does this tell us anything?

So there’s clearly been a pretty explosive growth in publications concerning genome assembly over the last couple of decades. Interestingly, the data from 2017 suggest that the period of exponential growth is starting to slow just a little bit. However, it would seem that we have not reached ‘peak genome assembly’ just yet.

There are, no doubt, countless hundreds (thousands?) of publications that concern technical aspects of genome assembly which have reached dead ends or which have become obsolete (pipelines for your ABI SOLiD data?).

Maybe we are starting to reach an era where the trio of leading technologies (Illumina, Pacific Biosciences, and Oxford Nanopore) are good enough to facilitate — alone, or in combination — easier (or maybe less troublesome) genome assemblies. I’ve previously pointed out how there are more ‘improved’ assemblies being published than ever before.

Maybe the field has finally moved the focus away from ‘how do we do get this to work properly?’ to ‘what shall we assemble next?’. In a follow-up post, I’ll be looking at the rise and fall of different sequencing technologies throughout this era.

Update 2018-08-13: Thanks to Neil Saunders for crunching the numbers in a more rigourous manner and applying a correction for total number of publications published per year. The results are, as he notes, broadly similar.

How and why the Institute of Cancer Research are using their new Illumina NovaSeq →

Image credit: The Institute of Cancer Research, London

Image credit: The Institute of Cancer Research, London

Yesterday, the The Institute of Cancer Research — disclaimer: that's where I work — published a new blog post where they spoke to Nik Matthews, Genomics Manager in the ICR’s Tumour Profiling Unit, about the Illumina NovaSeq sequencing platform.

It's a little more technical than some of the ICR 'Science Talk' blog posts that we usually publish which is why I thought I'd link to it here.

As someone who was started their PhD around the time the yeast genome was being finished I still am shocked by how far the world of DNA sequencing has come. This is something Nik refers to in his opening answer:

We can now produce data equivalent to the size of the original human genome project every six minutes, which is astonishing.

By comparison, the yeast genome project — an international collaboration involving many different labs — took over five years to sequence its genome…all 12 Mbp of it! Read the full blog post to find out more about how, and why, the ICR adopted the NovaSeq platform:

Illumina's new NovaSeq platform unveiled at The Institute of Cancer Research, London →

Dr Nik Matthews, Genomics Manager in the ICR's Tumour Profiling Unit. Credit: ICR

Dr Nik Matthews, Genomics Manager in the ICR's Tumour Profiling Unit. Credit: ICR

It feels a bit strange to be using this blog to link to a news post at my current employer, but I'm happy to share the news that the ICR has become the first organisation in the UK to deploy Illumina's NovaSeq platform.

The ICR's Dr Chris Lord, Deputy Director of the Breast Cancer Now Research Centre, had this to say:

One key area we are keen to use the NovaSeq sequencer for is to discover new ways to select the best available treatment for each individual cancer patient’s specific disease.

If we can do this, we should be able to improve how a significant number of patients are treated. With the NovaSeq system, this kind of work is now feasible – this will be a real game-changer for a lot of the work across the ICR.

Read more in the full news article on the ICR website:

Chromosome-Scale Scaffolds And The State of Genome Assembly →

Keith Robison has written another fantastic post on his Omics! Omics! blog which is a great read for two reasons.

First he looks at the issues regarding chromosome-size scaffolds that can now be produced with Hi-C sequencing approches. He then goes on to provide a brilliant overview of what the latest sequencing and mapping technologies mean for the field of genome assembly:

For high quality de novo genomes, the technology options appear to be converging for the moment on five basic technologies which can be mixed-and-matched.

  • Hi-C (in vitro or in vivo)
  • Rapid Physical Maps (BioNano Genomics)
  • Linked Reads (10X, iGenomX)
  • Oxford Nanopore
  • Pacific Biosciences
  • vanilla Illumina paired end

This second section should be required reading for anyone interested in genome assembly, particularly if you've been away for the field for a while.

Read the post: Chromosome-Scale Scaffolds And The State of Genome Assembly

Great Scott! Five fun facts about DNA sequencing from 1985

As everyone is celebrating a certain 2015–themed calendar event today, I thought we could instead go back to the future past of DNA sequencing.

 

1.

Thirty years ago there were no automated sequencing machines. However, Sanger sequencing technology could still provide longer reads than most of Illumina's machines today, e.g. from this paper (A rapid procedure for DNA sequencing using transposon-promoted deletions in Escherichia coli):

The length of the sequence that could be read from each gel in a single run varied from 175 to 200 nt.

 

2.

The idea of sequencing nuclear genomes was still largely a pipe dream, but smaller genomes were tractable. 1985 saw the addition of the Xenopus laevis mitochondrial genome to the tiny collection of organelle genome sequences. Figure 3 of this paper displayed the full sequence, spread over six pages that looked like this:

Including long DNA sequences in journal articles was a surprisingly common practice at this time.

 

3.

There were two releases of GenBank in 1985. The second release saw the database grow to an astounding set of 5,700 sequences, totalling 5,204,420 bp. For comparison, this year also saw the release of the Commodore 128 home computer which came with 128 KB of RAM. The first 3.5" hard drives were only a couple of years old, and could store 10 MB (so capable of storing the DNA sequences in GenBank, but possibly not the associated annotation).

 

4.

The SEQ-ED program was published, allowing the handling of 'long DNA sequences' that were 'up to 200 Kbp'.

 

5.

Somewhat amazingly, people were writing bioinformatics software for Apple computers. The journal CABIOS included this paper:

But how did people distribute software in the days when there was no GitHub, SourceForge, or indeed…no world wide web?

For both code and source of PEGASE, please send two blank 5" diskettes and indicate precisely your system configuration (there is a slight difference between the Apple II+ and the Apple lIe version which depends on the availability of lower case characters).

What a difference a day makes: markets react to PacBio's new sequencing platform

Daily change in share price at close of trade on October 1st, 2015:

Figures from Google Finance

Update: Earlier version of this figure incorrectly cited a 19% drop in Illumina's share price. My bad: -18.6 was the price change in dollars, not the percentage change.

 

Financial disclaimer: I do not own shares in any biotechnology company. 

Who is saying what about the new PacBio Sequel system?

The big news from the world of DNA sequencing this week was that Pacific Biosciences has launched a new sequencing platform. The successor to their RS II platform has been named The Sequel System and it will be on display at the upcoming American Society of Human Genetics meeting. The new system promises a cost of sequencing a human genome (at 10x coverage) for $3,000.

The early buzz already seems pretty positive, and hopefully this sequel will turn out to be more like The Empire Strike Back than, say, Highlander II. What follows is a fairly comprehensive roundup of what people have been saying about this new platform — note that this story has been updated several times since I first wrote it (details of these updates are included at the end of this post):

From PacBio

From science news websites

'Traditional' news outlets

From blogs

From discussion forums

From the world of finance

I guess the question that everyone is asking now concerns the possibility of someone making a genome assembly from sequence data using this platform, and then using this tool to produce a better version of the assembly. In this case, would it be a sequel Sequel SEQuel genome assembly?

 

Questions from the conference call

There were a lot of questions asked in the hour long conference call. I've transcribed some of them and indicated the time point where you can jump to if you are interested in hearing PacBio's answers to specific questions:

  • 7:40:"Can you give us some thoughts on turnaround time and cost per genome?"
  • 11:20:"Can you talk about the use case beyond your current customer base? How this expands the number of applications?"
  • 15:17:"Can you help us think about some of the major changes that went into the system? Is there still a manifold that moves in three dimensions?"
  • 19:20:"From a user standpoint, are there any changes to site preparations that you would have to make from Sequel vs RS II; any limitations on things like putting it on 2nd/3rd/4th floor?"
  • 22:25:"You've introduced a number of kits with various applications for the RS II, will the Sequel be able to run all of the applications from the beginning, or will it take time to introduce certain applications to the system?"
  • 24:34:"Are there specific customer types that you think are positioned to be more on the earlier side of adoption, such as human sequencing, or microbiology, plant, animal etc.?"
  • 33:20:"Can you give a perspective on what the scalability of this platform looks like comparatively (to the RS II)?"
  • 35:08:"In terms of the metrics you gave around price per human genome, can you help us think about that relative to Illumina? If you take a 30x coverage genome on Illumina, what is the equivalent coverage you would need on the Sequel to get something similar…and how long would that take you to do?"
  • 38:29:"Recognising a lot has been achieved with this launch: different computer architecture, different form factor, new optical systems, higher density, with a smaller footprint. I just want to make sure, there's no compromise in raw accuracy expected relative to the RS II?"
  • 47:46:"Could you describe in layman's terms the benefits of methylation detection for your system?"
  • 50:50:"With your technology relative to other platforms, can you help us understand — if you have these larger pieces of the puzzle if you will — how advantageous that could be after you're done generating data, when you get down to assembling the genome?"
  • 53:16:"I'm curious what percentage of potential customers that looked at the RS II passed given the high price tag? What is the incremental buyer opportunity at the price point of $350,000?"
  • 57:35:"Still trying to understand what percentage of competitive platforms you think you can swap out with the Sequel?"
 

Updates

2015-10-01 13.46: Added some more sources of news, including questions asked in conference call
2015-10-01 20.04: Added in more conference call details, with time points of different questions.
2015-10-01 20.39: Added Keith Robison's blog post
2015-10-02 06:34: Changed link for Bio-IT World's piece
2015-10-02 09.08: Added more links about PacBio's presentation at ASHG 2015
2015-10-02 09.41: Added link to CoreGenomics post and added disclaimer
2015-10-02 11.54: Added links to Sequel-related discussions on SEQanswers and reddit
2015-10-02 13.28: Added Biomusings and Checkmate Scientist blog posts, and split main part of article into different sections
2015-10-12 09.52: Addition of NBC Bay Area News piece
2015-10-14 16.57: Addition of 2nd GenomeWeb story
2015-10-23 20.02: Addition of 3rd GenomeWeb story

 

Financial disclaimer: I do not own shares in any biotechnology company.

An 18 Kbp read from a MinION sequencer!

The UC Davis Genome Center was fortunate to receive a few MinIONs from Oxford Nanopore the other week:

One of the things that we have been trying to do with these wondrous machines is to study variation in a mixed pan-European population. For this study, we simply combined saliva samples from individuals that represent 32 distinct European ethnicities (but no Belgiums, obviously), and the combined sample was applied directly to the MinION using the WF10 setting (WF = warp factor).

The preliminary results look very promising with an N50 read length of 12.2 Kbp (and this was before applying N50 Booster!!!). Here is the very first read from the device...18,731 bp of pan-European goodness (though note that there was a problem with base quality at the end of the read...contamination with Belgium DNA maybe?).

>PanEuroMix_read00001 

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MinIONs...do my bidding!

Oh the fun you can have with new sequencing technologies...