Category Archives: Science

How to fake an OSX theme appearance in Linux Ubuntu MATE

I’ve recently been fiddling about and trying to fake an OSX-style GUI appearance in Linux Ubuntu MATE (15.04). This is partly because I prefer the OSX GUI (let’s be honest) and partly because most of my colleagues are also Mac users mainly (bioinformaticians…) and students in particular fear change! The Mac-style appearance seems to calm people down. A bit.

The specific OS I’m going for is 10.9 Mavericks, because it’s my current favourite and nice and clear. There are two main things to set up: the OS itself and the appearance. Let’s take them in turn.

1. The OS

I’ve picked Ubuntu (why on Earth wouldn’t you?!) and specifically the MATE distribution. This has a lot of nice features that make it fairly Mac-y, and in particular the windowing and package management seem smoother to me than the vanilla Ubuntu Unity system. Get it here: https://ubuntu-mate.org/vivid/.* The installation is pretty painless on Mac, PC or an existing Linux system. If in doubt you can use a USB as the boot volume without affecting existing files; with a large enough partition (the core OS is about 1GB) you can save settings – including the customisation we’re about to apply!

*We’re installing the 15.04 version, not the newest release, as 15.04 is an LTS (long-term stable) Ubuntu distribution. This means it’s supported officially for a good few years yet. [Edit: Arkash (see below) kindly pointed out that 14.04 is the most recent LTS, not 15.04. My only real reason for using 15.04 therefore is ‘I quite like it and most of the bugs have gone'(!)]

2. The appearance

The MATE windowing system is very slick, but the green-ness is a bit, well, icky. We’re going to download a few appearance mods (themes, in Ubuntu parlance) which will improve things a bit. You’ll need to download these to your boot/install USB:

Boot the OS

Now that we’ve got everything we need, let’s boot up the OS. Insert the USB stick into your Mac-envious victim of choice, power it up and enter the BIOS menu (F12 in most cases) before the existing OS loads. Select the USB drive as the boot volume and continue.

Once the Ubuntu MATE session loads, you’ll have the option of trialling the OS from the live USB, or permanently installing it to a hard drive. For this computer I won’t be installing to a hard drive (long story) but using the USB, so customising that. Pick either option, but beware that customisations to the live USB OS will be lost should you later choose to install to a hard drive.

When you’re logged in, it’s time to smarten this baby up! First we’ll play with the dock a bit. From the top menu bar, select “System > Preferences > MATE Tweak” to open the windowing management tool. In the ‘Interface’ menu, change Panel Layouts to ‘Eleven’ and Icon Size to ‘Small’. In the ‘Windows’ menu, we’ll change Buttons Layout to ‘Contemporary (Left)’. Close the MATE Tweak window to save. This is already looking more Mac-y, with a dock area at the bottom of the screen, although the colours and icons are off.

Now we’ll apply some theme magic to fix that. Select “System > Preferences > Look and Feel > Appearance”. Now we can customise the appearance. Firstly, we’ll load both the ‘Ultra-Flat Yosemite Light’ and ‘OSX-MATE’ themes, so they’re available to our hybrid theme. Click the ‘Install..’ icon at the bottom of the theme selector, you’ll be able to select and install the Ultra-Flat Yosemite Light theme we downloaded above. It should unpack from the .zip archive and appear in the themes panel. Installing the OXS-MATE theme is slightly trickier:

  • Unzip (as sudo) the OSX-MATE theme to /usr/share/themes
  • Rename it from OSX-MATE-master to OSX-MATE if you downloaded it from git as a whole repository (again, you’ll need to sudo)
  • Restart the appearances panel and it should now appear in the themes panel.

We’ll create a new custom theme with the best bits from both themes, so click ‘Custom’ theme, then ‘Customise..’ to make a new one. Before you go any further, save it under a new name! Now we’ll apply changes to this theme. There are five customisations we can apply: Controls, Colours, Window Border, Icons and Pointer:

  • ControlsUltra-Flat Yosemite Light
  • Colours: There are eight colours to set here. Click each colour box then in the ‘Colour name’ field, enter:
    • Windows (foreground): #F0EAE7 / (background): #0F0F0E
    • Input boxes (fg): #FFFFFF / (bg): #0F0F0E
    • Selected items (fg): #003BFF / (bg): #F9F9F9
    • Tooltips: (fg): #2D2D2D / (bg): #DEDEDE
  • Window borderOSX-MATE
  • IconsFog
  • PointerDMZ (Black)

Save the theme again, and we’re done! Exit Appearance Preferences.

Finally we’ll install Solarized as the default terminal (command-line interface) theme, because I like it. In the MATE Terminal, Unzip the solarized-mate-terminal archive, as sudo. Enter the directory and simply run (as sudo) the install script using bash:


$ sudo unzip solarized-mate-terminal
$ cd solarized-mate-terminal
$ bash solarized-mate.sh

Close and restart the terminal. Hey presto! You should now be able to see the light/dark Solarized themes available, under ‘Edit > Profiles’. You’ll want to set one as the default when opening a new terminal.

Finally…

Later, I also installed Topmenu, a launchpad applet that gives an OSX-style top-anchored application menu to some linux programs. It’s a bit cranky and fiddly though, so you might want to give it a miss. But if you have time on your hands and really need that Cupertino flash, be my guest. I hope you’ve had a relatively easy install for the rest of this post, and if you’ve got any improvements, please let me know!

Happy Tweaking…

Messing about with the MinION

IMG_4543Molecular phylogenetics – uncovering the history of evolution using signals in organisms’ genetic sequences – is a powerful science, the latest expression of the human desire to understand our common origins. But for all its achievements, I’d always felt something was, well lacking from my science. This week we’ve been experimenting with the MinION for the first time, and now I know what that is: immediacy. I’ll return to this theme later to explain how exciting this realisation is, but first we better ask:

What is a MinION?

NGS nerds know about this already, of course, but for the rest of us: The MinION (minh-aye-on) is a USB-connected device marketed by a UK company, Oxford Nanopore, as ‘a portable real-time biological analyser’. Yes – that does sound a lot like a tricorder, and for good reason: just like the fictional device, it promises to make the instant identification of biological samples a reality. It does this using a radically different new way to ‘read’ DNA sequences from a liquid into a letters (‘A, C, G, T‘) on a computer screen. Essentially, individual strands of DNA are pulled through a hole (a ‘nanopore’) in an artificial membrane, like the membranes that surround every living cell. When an electric field is applied to the membrane, the individual DNA letters (actually, ‘hexamers’ – 6-letter chunks) passing through the nanopore can be directly detected as fluctuations in the field, much like a magnetic C-60 cassette tape is read by a magnetic tape head. The really important thing is that, whereas other existing DNA-reading (‘sequencing’) machines take days-to-weeks to read a sample, the MinION produces output in minutes or even seconds. It’s also (as you can see in the picture, above) a small, wait tiny device.

Real-time

This combination of fast results and small size makes the tricorder dream possible, but this is more than just a gadget. Really, having access to biological sequences at our fingertips will completely change biology and society in many ways, some of which Yaniv Erlich explored in a recent paper. In particular, molecular evolutionary biologists will soon be able to interact with their subject matter in ways that other scientists have long taken for granted. What I mean by this is that in many other empirical disciplines, researchers (and schoolkids!) are able to directly observe, or easily measure, the phenomena they study. Paleontologists dig bones. Seismologists feel earthquakes. Zoologists track lions. And so on. But until now, the world of genomic data hasn’t been directly observable. In fact reading DNA sequences is a slow, expensive pain in the arse. And in such a heavily empirical subject, I can’t help but feel this is a hindrance – we are burdened with a galaxy of baroque models to explain variations in the small number of observations we’ve made, and I’ve got a hunch more data would actually consign many of them to the dustbin.

Imagine formulating, testing, and validating or discarding genetic hypotheses as seamlessly as an ecologist might survey a new forest…

IMG_4531

Actual use

That’s the spiel, anyway. This week we actually used the MinION for the first time to sequence DNA (I’d been running some other technical tests for a couple of months, but this was our first attempt with real samples) and since it seems lots more colleagues want to ask about this device than have had a chance to use it, I thought I’d share our experiences. I say ‘we’ – work took place thanks to a Pilot Study Fund grant at the Royal Botanic Gardens, Kew, in collaboration with Dr. Alex Papadopulos (and really useful input from Drs. Andrew Helmstetter, Pepijn Kooij and Bryn Dentinger). It’s worth pointing out that although there’s a lot of hype about the device, it is still technically in a prototype/public-beta type phase, so things are changing all the time in terms of performance, etc.

IMG_4547First up, the size. The pictures don’t really do it justice… compared with existing machines (fridge-sized, often) the MinION isn’t small, it’s tiny. The thing itself, plus box and cable, would easily fit into a side pocket on any laptop bag. So the ‘portable’ bit is certainly true, as far as the platform itself is concerned. But there’s a catch – to prepare a biological sample for sequencing on the MinION, you first have to go through a fairly complicated set of lab steps. Known collectively as ‘library preparation’ (a library here meaning a test tube containing a set of DNA molecules that have been specially treated to prepare them to be sequenced), the existing lab protocol took us several hours the first time. Partly that was due to the sheer number of curious onlookers, but partly because some of the requisite steps (bead cleanups etc) just need time and concentration to get right. None of the steps is particularly complicated (I’m crap at labwork and just about followed along) but there’s quite a few, and you have to follow the steps in the protocol carefully.

Performance

So how did the MinION do? We prepared two libraries; one from a control sample of bacteriophage lambda (a viral genome, used to check the lab steps are working) and another from Silene latifolia, a small flowering plant and one of Alex’s faves. The results were exciting. In fact they were nerve-wracking – initially we mixed up the two samples by mistake (incredible – what pros…) and were really worried when, after a few hours’ sequencing, not a single DNA read matching the lambda genome had appeared. After a lot of worrying, and restarting various analyses, including the MinION itself, we eventually realised our mistake, reloaded the MinION with the other sample and – hey presto! – lambda reads started to pour out of the software. In the end, we were able to get a 500x coverage really quickly (see reads mapped to the reference genome, below):

lambda

You can see from the top plot that we got good even coverage across the genome, while the bottom plot shows an even (ish) read length distribution, peaking around 4kb. We’d tried for a target size of 6kb (shearing using g-TUBEs), so it seems the actual output read size distribution is lower than the shearing target – you’d need to aim higher to compensate. Still, we got plenty of reads longer than 20, or even 30,000 base-pairs (bp) – from a 47kbp genome this is great, and much much much longer than typical Illumina paired-end reads of ~hundreds of bp.

Later on the next day, we decided we’d done enough to be sure the library preparation and sequencing were working well, so we switched from the lambda control to our experimental (S.latifolia) sample. Again, we got a good steady stream of reads, and some were really long, over 65,000bp. Crucially, we were able to BLAST these against NCBI and get hits against the NCBI public sequence database and get Silene hits straight back. We were also able to map them onto the Silene genome directly using BWA, even with no trimming or masking low-quality regions. Overall, we got nearly a full week’s sequencing from our flowcell, with ~24,000 reads at a mean length of ~4kb. Not bad, and we’d have got more if we hadn’t wasted the best part of the run sodding about while we troubleshot our library mix-up at the start (the number of active pores, and hence sequencing throughput, declines over time).

The bad: There is a price for these long, rapid reads. Firstly, the accuracy is definitely lower than Illumina – although the average lambda library sequencing accuracy in our first library was nearly 90%, on some of our mapped Silene reads it dropped much lower – 70% or so. This has been well documented, of course. Secondly, the *big* difference between the MinION in use and a MiSeq or HiSeq is the sheer level of involvement needed to get the best from the device – whereas both Illumina machines are essentially push-button operated, the MinION seems to respond well if you treat it kindly (reloading etc) and not if you don’t (introducing air bubbles while loading seemed to wreck some pores)

The very, very, good: Ultimately though, we came away convinced that these issues won’t matter at all in the long run. ONT have said that the library prep and accuracy are both boing improved, as are flowcell quality (prototype, remember). Most importantly the MinION isn’t really a direct competitor to the HiSeq. It’s a completely different instrument. Yes, they both read DNA sequences, but that’s where the similarity ends. The MinION is just so flexible, there’s almost an infinite variety of uses.

In particular, we were really struck that the length of the reads means simple algorithms like BLAST can get a really good match from any sample with just a few hundreds, or tens, of reads from a sample. You just don’t need millions of 150bp reads to match an unknown sample to a database with reads this long! Coupled with the fact that the software is real-time and flowcells can be stopped and started, and you have the bones of a really capable genetic identification system for all sorts of uses; disease outbreaks (in fact see this great work on Ebola using MinION); customs control of endangered species; agriculture; brewing – virtually anything, in fact, where finding out about living organisms’ identity or function is needed.

The future.

There’s absolutely no question at all: really, these devices are the future of biology. Maybe the MinION will take off (right now, the buzz couldn’t be hotter), or a competitor will find a way to do even more amazing things. It doesn’t matter how it comes about, though – the next generation of biologists really will be living in a world where, in 10, 15, 20 years, at most, the sheer ubiquity of sequencers like this will mean that most, if not all eukaryotes’ genomes will have been sequenced, and so can be easily matched against an unknown sample. If that sounds fantastic, consider that the cumulative sequencing output of the entire world in 1988 amounted to a little over 100,000 sequences, a yield equivalent to a single good MinION run. And while most people in 1988 thought that, since the human genome might take 30 or 40 years to sequence, there was little point in even starting, a few others looked around at the new technology, its potential, and drew a different conclusion. The rest is history…

Cheat on your exams

Had a heated discussion with a friend the other day. I went to a school, where ‘exam techniques’ were part of the standard toolkit given to students to get them the best possible grades at GCSE, A-levels, and beyond. She didn’t, and so hadn’t ever heard of a special ‘technique’ for exams until uni. She felt robbed – why should one group of students get an advantage over the others, because their school taught them how to cheat the system?

Well, it’s a fair point; but my reply was that exam techniques really aren’t that complicated. In fact, you can boil most of them down to three simple rules: Answer the QuestionPlan Your Time and Plan Your Answers. Look, I can even explain each one in 100 words or fewer 😉

Answer The Question

Every stack of exam scripts that’s ever been marked from Socrates* onwards contains at least one howling stink-bomb of a perfect answer. The student has a deep and broad understanding of their subject. The answer is comprehensive, incisive, and backed up by copious references. Unfortunately, because they’ve misread the question and gone off on a tangent, you sigh, marvelling at the intelligence that manages to completely grasp a difficult concept like mitochondrial introgression, but utterly failed to comprehend the text of the exam paper. Nil marks. Don’t let it be you: read the question carefully and give the examiner only what they’ve asked for.

*Socrates may not have actually sat a single GCSE, but you get the point.

Plan Your Time

This one is too obvious for words, but you must practice and be self-disciplined in the exam for it to work. Basically, most students subconsciously assume that the relationship between time spent on a question and marks collected is linear, something like this: 

Linear plot. Uses xkcd lib for matplotlib/numpy on python

(Edit: err, ignore the axes’ values… oops!)

Wrong. It’s minimally true that the longer you spend on a question, the more marks you’ll probably* get. But most exams have more than one question, so you must balance trying to get top marks on, say, Question 1, with getting at least some marks on Questions 2, 3 & 4! This is why it’s important to get a grip on the real relationship between effort and marks. It looks a lot more like this:

Sigmoid plot. Uses xkcd lib for matplotlib/numpy on python

Can you see what’s going on here? Most exam questions are deliberately structured so that getting a third-class (‘D’) grade is relatively easy, then marks are awarded more-or-less linearly up to the top of an upper-second (‘B). Finally, first-class (‘A’) grade answers usually require substantially more insight – and deliberately and rightly so, since these grades are supposed to mark out the very best exam scripts, usually around 5-10% of the class at most.

In other words, if you spend half of a 100-minute, four-question exam on Question 1, you might get an A for maybe 25% of the marks… but at the cost of achieving a C, maximum, on the other three questions. You’ll be lucky to average a C. Plan your time.

*’probably’ because after you’ve spent too long on a question there’s a temptation to start chucking in the kitchen sink as well, and after a while you’re at risk of saying something stupid or wrong which might actually cost you marks.

Plan Your Answer

When you turn over the exam paper and pick up your pen, your head is likely buzzing – with caffeine, formulae and references you’ve crammed in at the last minute, and possibly with the dull ache of worry that your Mum will kill you if you don’t pass this exam. You want to calmly and methodically, putting the first two exam techniques to devastating effect and dazzling your examiners. You’ll actually probably grab the first question you like the look of and start writing immediately.

This is almost guaranteed to leave you sweating in a heap of confusion half-way through the exam, when you look up from polishing your first answer, realise time’s running out, and start flailing through the others. Without an answer plan you’ll find it hard to stick to time, and in the case of longer questions you’ll be more likely to stray off-piste as well.

Instead, invest some of the exam time into making a plan – with bullet points – for each answer. I always spend at least 5% of the time for each answer writing a plan. Do this on your answer script too, so that in the nightmare scenario that you run out of time the examiner can at least give you some marks. For instance:

Q2: Explain what is meant by Muller’s Ratchet, in the context of natural selection (30% of marks)

A: Outline answer:

  • Muller’s Ratchet (Muller, 1964) is accumulation of deleterious mutations in asexual populations
  • ‘Deleterious’ means lowering fitness of an organism
  • Asexual populations (or chromosomes) do not exchange genetic material in meiosis by recombination, unlike sexually-reproducing populations/chromosomes.
  • Mutations in genetic material occur over time at random
  • Most changes have little effect, or slightly deleterious
  • Some highly deleterious or advantageous
  • Natural selection filters randomly-occurring mutations. Under ‘neutral’ selection the effect of all mutations is negligible. Under ‘positive selection’ advantageous mutations’ benefit leads to their higher relative occurrence through evolutionary time. Under ‘negative selection’ deleterious mutations penalised highly
  • Muller’s Ratchet therefore implies that asexually-reproducing populations subject to negative selection will be disadvantaged compared to sexually-reproducing populations, as they cannot filter out deleterious mutations through recombination.

It probably took about 5 minutes to think about and write that plan – but it would likely get at least half-marks, on its own…

In many cases you will be able to get an idea of how many questions, and which topics, the exam will contain (using nefarious tricks like simply asking the course convenor). Armed with this, you can plan your overall exam strategy, with times. Something like this:

Plan for a 2 hour exam with 3 questions (choice of 7) starting at 10:30am:

  • 10:30 start exam.
  • 10:30-10:45 read exam and pick questions
  • 10:45 start first question
  • 11:15 start second question
  • 11:45 start second questions
  • 12:15-12:30 final proofreading

You can take your exam plan in with you (at least in your head) so you don’t waste valuable time trying to work out your timings in the real thing. You’ll also feel more confident and in control of your performance.

Bonus technique: Practice

The above three techniques will help you make the most of your hard-won knowledge (you did revise the content too, right?) but – trust me on this one, cos I’m an exam machine – you’ll be utterly unable to put them to effect without exam practice. This is probably the biggest difference between schools which actually spend precious teaching time on exam practice, and those which simply point students to blogs like this one as part of their revision.

Get a group of mates on the same course together, get a stack of past exam scripts, and practice in exam conditions. Compare, and mark each others’ scripts. Then repeat, again and again.

Last of all, don’t overdo the coffee. Good luck!

BaTS (and Befi-BaTS), SHiAT, and Genome Convergence Pipeline have moved!

Important – please take note!
Headline:

  • All my phylogenetics software is now on GitHub, not websites or Google Code
  • Please use the new FAQ pages and issue/bug tracker forms, rather than emailing me directly in the first instance

Until now, I’ve been hosting the open-sourced parts of my phylogenetics software on code.google.com. These include the BaTS (and Befi-BaTS) tools for phylogeny-trait association correlations; the alignment profilers SHiAT (and Genious Entropy plugin), and the Genome Convergence API for the Genome Convergence Pipeline and Phylogenomics Dataset Browser. However, Google announced that they are ending support for Google Code, and from August all projects will be read-only.

I’ve therefore migrated all my projects to GithubThis will eventually include FAQs, forums and issue/bug tracking for the most popular software, BaTS and Genome Convergence API.

The projects can now be found at:

 

I am also changing how I respond to questions and bug requests. In the past I dealt with questions as they came in, with the odd explanatory post and a manual or readme with each release. Predictably, this meant I spent a lot of time dealing with duplicates or missing bugs or feature requests. I am now in the process of compiling a list of FAQs for each project, as well as uploading the manuals in markdown format so that I can update them with each release. Please bear with me as I go through this process. In the meantime, if you have an issue with a piece of software or think you have found a bug, please:

  1. Make sure you have the most recent version of the software. In most cases this will be available as an executable .jarfile on the project github page.
  2. Check the ‘Issues’ tab on the project github page. Your issue may be a duplicate, or already fixed by a new release. If your bug isn’t listed, please open a new issue giving as much detail as possible.
  3. Check the manual and FAQs to see if anyone else has had the same problem – I may well have answered their question already.
  4. If you still need an answer please email me on joe+bioinformaticshelp@kitserve.org.uk

Thanks so much for your support and involvement,

Joe

Application note: ‘Befi-BaTS’ version 0.10.1 – Error rate and statistical power of distance-based measures of phylogeny-trait association.

In prep.

SUMMARY

Building on work presented previously (Parker et al., 2008), we study a number of more complex measures of phylogeny-trait association (implemented in the program Befi-BaTS / BaTS v0.10.1) which take into account the branch lengths of a phylogenetic tree in addition to the topographical relationship between taxa. Extensive simulation is performed to measure the Type II error rate (statistical power) of these statistics including those introduced in Parker et al. (2008), as well as the relationship between power and tree shape. The technique is applied to an empirical hepatitis C virus data set presented by Sobesky et al. (2007); their original conclusion that compartmentalization exists between viruses sampled from tumorous and non-tumorous cirrhotic nodules and the plasma is upheld. The association index (AI), migration (PS), phylodynamic diversity (PD) and unique fraction (UF) statistics offer the best combination of Type I error and statistical power to investigate phylogeny-trait association in RNA virus data, while the maximum monophyletic clade size (MC) and nearest taxon (NT) statistics suffer from reduced power in some regions of tree space.

Keywords: BaTS, hepatitis C virus, Markov-chain Monte Carlo, Phylogeny-trait association, Phylogenetic uncertainty, simulation.

Manuscripts in progress (all rights reserved – you may not copy or distribute these files; content and conclusions subject to change; strictly embargoed until publication in a peer-reviewed journal/book):

  • v1: (): .doc
  • v2 (01/01/2014): .docx
  • v3 (16/06/2017): .pdf
  • View this project on GitHub

 

Application note: CONTEXT, a Phylogenomic Dataset Browser

In prep. (v3 – 14 Jun 2017)

Summary. The CONTEXT (COmparative Nucleotides and Trees Exploration Tool) is a phylogenomics dataset browser that consists of a Java API and an executable binary jarfile with graphical user interface (GUI) for the high-throughput analysis of phylogenomic datasets to detect convergent molecular evolution.

Motivation. Comparative genomics studies have become increasingly common, but these analyses are sensitive to the quality and heterogeneity of input datasets (multiple sequence analyses and phylogenies). Currently few tools exist to readily compute descriptive statistics, or to visualise large numbers of input datasets. CONTEXT facilitates these analyses in a lightweight application which allows any user to rapidly visualise, inspect, score, and sort input datasets to identify outlying datasets which may need additional processing or filtering.

Results. The application has been successfully implemented on a variety of infrastructures. A variety of common input data formats including FASTA, Phylip/PAML, Nexus, and Newick conventions are automatically read and parsed.

 

Manuscripts in progress (all rights reserved – you may not copy or distribute these files; content and conclusions subject to change; strictly embargoed until publication in a peer-reviewed journal/book):

 

  • v3 (14/07/2017): .pdf
  • v2 (03/04/2017): .pdf
  • v1 (24/02/2015): .doc
  • View this project on GitHub

Detection of molecular convergence – literature review

In prep. (v2 – 21 April 2015)

Abstract

Convergent evolution is a process by which neutral evolutionary processes and adaptive natural selection in response to niche specialisation lead to similar forms arising in unrelated taxa. Phenotypic convergence has been appreciated for well over a century (recognised as a confounding factor in morphological cladistics). Recently several studies have demonstrated that convergent-type signals exist in some molecular datasets. Extending these studies to genome scale data presents substantial challenges and opportunities. This chapter reviews the definition of convergence (compared to parallelism), and the biological interpretation of apparently convergent molecular data. Recent methodological developments and applications are examined and future problems outlined. These include suitable null and alternative models, and the role of multiple test phylogenies in convergence detection by the congruence / phylogeny support method.

 

Manuscripts in progress (all rights reserved – you may not copy or distribute these files; content and conclusions subject to change; strictly embargoed until publication in a peer-reviewed journal/book):

 

  • v1 (10/04/2015): .doc
  • v2 (21/04/2015): .doc