Should you buy a nanopore sequencer?

Saturday, February 18th, 2012

This morning twitter is awash with posts discussing the newly announced nanopore sequencers from Oxford Nanopore. Speculation has been rife for some time about the potential specifications of the first sequencers to be produced by the company, and it certainly appears that the company have fulfilled the expectations placed upon them.

I’m not going to go through the details of the two sequencers announced – others have done a good job of listing the specs here and here, but basically you have machines with either 512, or 2000 nanopores capable of sequencing fragments up to 40kb (but probably several kb routinely) and error rates of around 4%, mostly indels, and with promises of imminent improvements to bring this value down – all at a per-base cost similar to the best of the existing platforms.

Reading through the initial comments about this new platform my first reaction was that we have to get one (or more) of these, but after calming down and thinking about this for a bit I thought I’d have a stab at going through the use cases where this type of sequencer really makes sense.

De-dovo sequence assembly: Oh yes!

The one place where this new platform is a complete no-brainer is if you’re assembling de-novo genome sequence. Whilst Illumina sequencers can give you good coverage depth from paired end reads of around 100bp there are always regions of the genome whose repetitive nature mean that this will not provide enough context to allow a contiguous assembly. Currently you either need to start creating mate-pair libraries, which are notoriously difficult to produce, or you need to get your floor reinforced and stump up a huge amount of cash for a PacBio.  The prospect of generating reads of 10kb+ with a simple library prep should be music to your ears, and a 4% error rate with short indels should be easy to work around with a mixed assembly.

Metagenomics: Oh yes!

In the same vein as de-novo assembly the propect of longer reads should make metagenomic studies much easier.  Getting more context for your reads should allow you to distinguish between related species much more easily and assembly of mixed bacterial populations should be possible even with the slightly more limited throughput of these sequencers.

Genotyping: Possibly

I guess the main advantages of the nanopore platform for genotyping is the speed with which it can generate data.  Data collection begins almost immediately upon addition of the sample, and real-time monitoring of the data output means that you can immediately stop the run once you have observed all of the variants you are looking for.  The long read lengths should allow the illucidation of even the most complex genome re-arrangements.  The somewhat high error rates may be problematic, but if these really are mostly indels, then SNP calling might still be practical.  The per-base cost means that the current sequencers aren’t yet practical for real time diagnostic use, but future developments on this platform would seem to make this a possibility.

Epigentics: Possibly

One of the promises of nanopore sequencing was the ability to distinguish modified bases during the base calling process.  PacBio have shown that they are able to distinguish hydrox-methyl-cytosine from cytosine, and suggest that identification of methyl-cytosine is theoretically possible. In the reports I’ve seen so far Oxford Nanopore haven’t said anything concrete yet about the ability of their platform to call modified bases, but if this proves to be possible and reliable then this will become an essential bit of kit for labs working on epigentics.  The ability not only to directly read modifications directly, but to be able to put these in the context of a multi-kb fragment is truly exciting.  The addition of a hairpin structure at the end of a fragment would also allow these sequencers to read both strands of the same fragment, again providing contextual information which has so far been lacking.

It’s possible that the nanopore sequencers may still be of use to epigenetics even without the ability to read modifications directly.  Genome wide bisulphite sequencing is already being undertaken on Illumina sequencers, and should be possible on nanopore sequencers, however the bisulphite treatment itself is very harsh, and fragments the DNA sample as it modifies it, so the super-length reads able to be obtained from normal genomic DNA may be elusive once it has been modified.

 ChIP-Seq: Not really

The power of ChIP-Seq comes from the number of observations you make, not the length of those observations.  The nanopore sequencers seem to be best suited to sequencing fewer, longer fragments which would not be an advantage for ChIP-Seq.  There seems no obvious reason why short insert libraries couldn’t be sequenced on a nanopore platform, but at the moment we know very little about the overhead of starting a new sequence on the same nanopore so this may be feasible, but longer read lengths would simply reduce the resolution of the ChIP assay.  For some applications it might be interesting to monitor ChIP results in real time, and be able to halt a run once clear peaks had emerged, but in the short term I can’t see this being a good option for this type of experiment.

RNA-Seq: It depends

As with ChIP-Seq, much of the power of RNA-Seq comes from the number of observations which have been made.  To make a reasonable measurement of low-expressed transcripts then very large numbers of sequences must be generated, and the existing short read platforms will likely have an advantage in this regard for some time, so for simple quantitation of transcripts the nanopore platform may not offer huge advantages.  Where the longer read lengths of the nanopore sequencers will  be of use will be in the elucidation and quantitation of splice variants.  Current RNA-Seq protocols provide coverage of a very small part of the transcript and often do not provide enough context to determine exactly which splice variant the reads came from.  Performing relative quantitation of the splice variants of a gene is therefore not a simple process.  Longer reads from a nanopore sequencer could cover the whole length of a transcript removing all doubt about exactly which variant it was.  Whether this proves to be a useful tool for expression quantitation will depend on whether the platform is able to generate an unbiased selection of reads (or a selection with a well understood bias) to allow accurate quantitation.

So…

So do I think we should get one of these sequencers?  Heck yes! For $900 a piece for the MinIon there’s absolutely no excuse for everyone not to get one and start playing with it to see what it can do. For much of the workload we currently have it may be that this platform isn’t going to revolutionise what we do, but if nothing else it will hopefully spur on the existing manufacturers to push forward the development of their existing platforms.  In any case the scientists win.  We live in exciting times…

Tags: hardware
Posted in Bioinformatics, Technology | No Comments »

Creating the ideal simple recording setup for live acousitc concerts

Sunday, February 6th, 2011

The Brief

I regularly perform with a number of groups ranging from small ensembles, to choirs and full orchestras.  For a long time now I’ve been wanting to have a simple recording setup which I could use to make high quality recordings of these events, mostly for personal use but also with an eye towards making CDs either just for members of the band, or maybe for commercial sale at a later date.

Although I wanted to be able to get good recording results I also needed to have a setup which was unobtrusive (no obvious microphones on stage), quick to set up and which wouldn’t require monitoring during the concert (because I have to play in it!).

The Setup

Having done a bit of research I quickly came to the conclusion that the ideal setup for this kind of recording would be a simple pair of condenser microphones which could be mounted on a high stand a few meters behind the conductor.  This avoids the need to have individual microphones for individual instruments or sections, but can hopefully get close enough to the players to avoid sounding like a purely ambient recording.  Since most of my concerts take place in churches it would be easy to set up in the central aisle.  Using this setup on a proper stage might be an issue, and would probably require the microphones to be suspended above the audience rather than put on a floor stand.

The recorder

I looked at several options when working out how I was actually going to record the sound.  Older technologies such as DAT tapes and minidiscs were dismissed, DAT is too big and fragile (and tape is a pain), and minidiscs are dying and lossy.  My original intention was to record straight onto a laptop.  However I wanted a setup which was unobtrusive, which means using a netbook.  Recoding this way would mean that I’d have to leave the computer open and running during the concert which would make it difficult to secure – most netbooks are also not completely silent which might be distracting for people sitting near to it.  Most importantly though, the sound hardware in every netbook I tried is terrible.  Even if you feed it a high quality signal then the internal hardware will add a significant amount of noise to the eventual recording.  You can get around this by using an external sound module which feeds into the laptop over USB, but this makes the whole setup more complicated and adds quite a lot to the cost.

In recent years there have been quite a few dedicated solid state recorders released.  These generally have inbuilt microphones, some of which can be pretty good, but may also have the option to record from an external sound source.  They generally record to an SD card so there are no moving parts and the units operate in complete silence.

Having seriously considered a couple of these units I found a couple of problems.  Firstly, the batteries of most of these units have a lifespan which wouldn’t be enough to comfortably record a rehearsal and concert in the same session without getting worried about them running out and the recharge time ran to several hours.  Secondly, although the internal mics on these units can be very good I wanted the option to use good professional microphones as an input.  The ideal microphones for this type of recording are small diaphragm condensers, and to work properly these require power (termed phantom power) to be sent to the microphones from the recorder.  Most of the recorders only have mini-jack connectors for the inputs and have no facility to provide phantom power.  To use these would therefore require a more complicated external power supply to power the mics.

My final solution therefore was the Tascam DR-100.  Although this is at the expensive end of this type of recorder it has the big advantage that in addition to the (really very good) directional microphones on the unit, it also has two XLR inputs on the bottom which can take a feed from an external microphone, and which can supply 48V phantom power to allow you to use condenser microphones without the need for additional hardware.

The audio hardware inside the DR-100 is excellent and every review I read commented how, even at high levels of gain (which you generally need to use) introduces virtually no noise.  It has the ability to record direct to MP3 if you want to be able to leave it running for hours on end, but can also record 24-bit wav files which are high enough quality for any application to which you might want to put your recording.  Half a concert recorded uncompressed at 24-bit easily fits on 2GB SD card, so you just need to swap cards in the interval.

Microphones

I should state up front that I am not a sound engineer or even an audiophile.  Having done some preliminary tests with the onboard mics on the DR-100 I was actually quite happy with the quality of sound I could record – although I could hear that it could be improved.  The biggest problem with using the onboard mics though is being able to position them correctly.  The DR-100 is quite small, but you can’t mount it on a high stand easily, and even if you could you can see or get to any of the controls.  I tried recording concerts from the front row of the audience, or from a point behind the orchestra, but found that the balance of the sound was too poor to use.

I therefore spent quite a long time researching options for good quality microphones.  I soon found out that:

1. There is no single correct answer.  Any microphone you care to mention will have both great and awful reviews depending on which site you look at.  I tended to go on weight of evidence, but don’t really have any way of judging how well my current setup is performing relative to what I would have got with a different choice of mics.
2. The sort of microphones you need for this kind of recording range in price from really expensive to really expensive.  In the end the microphones in my setup cost about as much as everything else put together.  There isn’t a way around this – if you want to do it you just need to bite the bullet and open your wallet.

My intention was to get two condenser microphones which would then be mounted in a crossed stereo pair, where the microphones are positioned at right angles to each other with the diaphragms positioned close together.

My choice in the end was a pair of Rode NT5 microphones.  Of all of the microphones I researched these were the ones which had the most consistently good reviews.  There are also a number of example recordings available made with these microphones which sounded excellent so these were what I ended up with.

I ordered a matched pair of mics which should be ideal for using for stereo recording.  However when using them I’ve found that they show very different input levels when recording onto the DR-100 (approx 1-2 stops on the gain selector), which doesn’t make them very matched – however I’ve not been systematically through all the pieces of the setup to confirm that it’s definitely the mics so I’m going to reserve judgement on this.

I have been very impressed by the quality of sound I can get from the NT5s.  I’ve used them both for full concert records, but also for close micing of individual instruments and have had consistently excellent results.  Getting the placement of the mics right has proved to be critical.  For close micing moving the mics a few centimetres can make a big difference to the sound produced, and for concert recording getting correct separation and angle of the microphones is crucial (but it doesn’t take too much playing around to find it).

Setup

Since I wanted to end up with a recording setup which was self-contained and could be left out during a concert without fear of having it damaged or disturbed I needed to add some more bits of kit.  Basic things I had to get were:

1. A pair of 5m XLR cables.  Nothing special about these.  I may get longer ones at some point to give me a bit more flexibility to position the recording gear exactly where I want.
2. A boom stand.  I got a relatively cheap boom stand from Tiger Stands however despite the cheap price this turned out to be a really solid piece of kit.  Fully extended this can raise the mics at least 3m up.  Ideally I’d like them a little higher, but that would require a more expensive stand, which would also have to have a larger footprint.  This stand doesn’t completely fill the aisle and even fully extended the stand seems to be pretty stable.
3. A stereo microphone mount.  I spent a while looking for this.  There are some ridiculously expensive mounts around for creating a stereo pair of microphones.  In the end I went for this simple mount from a site which mostly seems to see old records.  For less than £5 I have a perfectly good stereo mount.  The clips for the actual microphones were supplied with the NT5s.
4. A long power extension cord (10m) since often the nearest powerpoint is further away from the aisle than you’d like.  I’m thinking about carrying a second extension as I’ve already had a close shave where the 10m was only just long enough.  Some churches don’t have a wide selection of power points available!
5. A power adapter for the DR-100 so I can run it continuously without having to worry about the battery.  If anything happens to the power the unit will run without power for at least 30 mins, and this could be extended futher if I were to put in an external battery to the DR-100.
6. A pair of headphones.  I have a relatively cheap pair of Sennheiser HD-201 headphones which have proved to be fine for monitoring the recording setup.
7. A small study plastic box in which I can keep sundry items such as the remote control and USB cable for the DR-100, spare SD cards and that kind of stuff.

I wanted to have a case which I could use to store the recording gear and which could also be used to house the gear during a concert.  Since this was likely to be too specialised to purchase I ended up making one.  The case is about 60cm x 60cm x 15cm.  It’s pretty sturdy – so much so that you can stand on it without problem.  The inside is split into two compartments, the rear compartment is left blank and is used for cables, the stereo mount and anything else which isn’t fragile.  The front compartment will be filled with foam containing cutouts for the recorder, headphones and microphones.

Flight case packed for transport

Flight case laid out in recording configuration

The case has cutouts at the back which allow power and XLR cables to pass into it.  The case can be padlocked shut, and can be left like that whilst recording so it can be relatively safely left unattended during a concert.  The security is only superficial, but it’s enough to stop people tampering with the equipment out of curiosity.  Finally the case has a handle on the side, so I can leave everything in place so I just need to grab the case and the boom stand and I’m good to go.

Whole recording setup in situ

There are a couple of additional items I want to add to the setup.

1. Some rubber cable covers to place over exposed cables to stop people tripping over them
2. Gaffer tape – because you never know what you need to stick down
3. Some kind of case for SD cards so I can keep track of which recordings are on which cards

Post-processing

After I’ve done on the on-site recording I am not trying to do any complicated mixing.  Post processing consists only of sorting out levels, adjusting the equalisation and splitting the recording into tracks.  For these simple processes I’m using Audacity on a Mac which seems to fit the bill very well.

Summary

In total I reckon the setup I’ve created has cost me around £900, which is a bit more than I set out to spend, but I now have a simple and yet versatile setup which is suitable to record a number of different types of event.  I shall make regular use of this so the investment will hopefully be worth it.  Also the quality of the recordings is such that I can make CDs from them, which will hopefully allow me to raise some money for the groups with which I play.

Tags: choral, concert, hardware, microphone, orchestra, recording
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The practical experience of owning a Kindle

Saturday, January 15th, 2011

I’ve now had my kindle for about two weeks, and during that time I’ve experienced the highs and lows of kindle ownership.

Overall I must say that I am very happy with my purchase.  Far from being a gimmick I have found the Kindle to be a genuinely useful piece of technology.  It’s been getting a lot of use at home and will definitely be coming with me on future trips.  Although it offers extended functionality (eg as a web browser), I’m only likely to use it as an e-book reader since the e-ink screen really isn’t suited for regular use in any kind of interactive application.

The Good Bits

I should start with the things I have really liked about the Kindle.  This seems only fair since my overall impression has been positive.

As a reading experience the Kindle has been excellent.  I’d not really seen an e-ink screen up close before buying the Kindle, and I was very impressed with the clarity of the screen.  I’ve also found that I have been able to read for long periods of time without becoming fatigued.  I can’t read on a computer screen for any significant period of time without getting eye strain, so the Kindle’s screen has been a welcome change.  I was also pleasantly surprised by the range of lighting conditions under which the screen was usable – everything from a small bedside light to full sunshine has provided an equally good experience.  That it can do this whilst consuming virtually no power is especially good – I’ve not charged mine since the day it arrived, and two weeks and four books later I still have over 75% battery remaining.

The form factor of the Kindle has also proved to be a bit hit.  It’s a very light device, but is quite rigid, and the soft feel of the case makes it very comfortable to hold in a variety of positions.  I’ve actually found it much easier to hold for long periods than a book.  Whereas with a book I found myself constantly changing position, the Kindle can be held naturally for extended periods without problem.  Some people have complained about the screen flash which occurs every time you turn the page, but this hasn’t bothered me at all (during the flash the new pages text is still visible on the screen, just inversed, so you can keep reading through the flash).  I have found that having the page turn buttons on the edge of the device makes it slightly uncomfortable to hold your thumb on the next page key.  I’d have personally preferred to be able to press slightly further into the bezel, but this is only a minor gripe about what  otherwise is a very ergonomic device.

Another good aspect has been the delivery of content to the device.  The Kindle came from Amazon (5 days after the predicted delivery date – but it was over Christmas) pre-registered to my Amazon account.  I like being able to browse for content on my PC since I don’t particularly like searching on the Kindle itself.  The purchasing process was very smooth (and even saved me when I mistakenly tried to buy the same book twice), and the whispernet delivery has worked flawlessly.  I’ve not tried the email delivery to the device yet, but this seems like it should be a simple way to get more content onto the device.  I’m not enamoured of the whole DRM system which wraps all of the purchased content – mostly because it means that I can’t purchase from other e-book stores, or borrow e-books from libraries at the moment, but hopefully as the market matures this will go the way of DRM on music downloads.

The Bad Bits

Within my overwhelmingly positive experience I have, unfortunately, encountered some problems.  Mostly these have revolved around the Kindle store, and I accept that some of these are beyond Amazon’s control, but nevertheless it’s very frustrating to see how much better the Kindle experience could be if some of these could be sorted out.

Let’s start with annoyances on the device.  The main thing which has bugged me has been the construction of the e-books.  In particular the one thing which you can’t do on all of the books I’ve read is to see how close you are to the end of the current chapter.  This is really frustrating since the device itself supports this functionality in the form of way markers on the progress bar, it’s just that this information isn’t annotated in the e-book itself so the bar often appear empty.  The speed of screen refresh means it’s not feasible to flick forwards and back quickly to find the next chapter.  It also means that as the chapters don’t appear in the table of contents you can’t easily jump to the place you want in the book unless you happen to know a text search which will take you there.  This is just laziness on the part of whoever created the e-book, and a small amount of addition effort would make the books much more usable.

The other thing which lots of people have commented on is the poor quality of the OCR in some books.  It’s surprising how suddenly coming to a mis-OCRd word in the middle of a paragraph can take you out of the book as you have to figure out that it’s the wrong word and figure out the correct meaning of the sentence.  This is worse than coming across a misspelled word in a book (which you can often read through without spotting), since many times you will have a correct word which is in completely the wrong context.  I realised that with the volume of OCR going on that mistakes are inevitable, but how about putting in place a way to report back mistakes.  You can already highlight words and add notes, so having a system to report errors can’t be that hard – that way we can gradually improve the quality of the books available.

The Kindle Store

Since most of the bad experience I’ve had has been in the Kindle store it gets its own section.  Since online retail is the stock in trade of Amazon I was surprised that so much about the Kindle store was so poor.  The main things which have bugged me are:

1. The range of e-books available is very poor.  Whether this is due to publishers refusing to release e-book rights, a backlog of books to be OCR’d or just the economics not adding up I was surprised to see how many very well known books are not available in e-book form.  As a quick example take a look at this excellent list of science fiction classics and note how many are available as e-books. Not many.
2. Following on from point 1, the presentation of e-books on the store is very poor.  I’ve had a number of occasions where I’ve searched for something and got back a set of results and found it very hard to tell whether that was what I wanted or not.  How about a book with the title of another book in brackets or a collection of stories where the description doesn’t even tell you which stories are in the collection?
3. The final big annoyance with the Kindle store is the pricing.  Maybe I’m still paying for being an early adopter (although I’m not sure that I count as that any more) but the prices being charged for many e-books are verging on the comical.  I fail to see the justification for charging a mark up on the price of the equivalent new paperback to get an e-book with no printing or distribution costs.  As a point of principle I will not buy e-books which are priced higher than the cheapest new paperback, and frankly they should be considerably cheaper.  Maybe increased adoption and competition will bring prices down eventually, but at the moment I’ll be curtailing my purchases simply due to the prices.

Having had a Kindle for a relatively short time I’m thoroughly convinced of its utility and I can only see the market for these devices increasing – probably to the point of replacing most conventional publishing.  Hopefully the problems which are currently present in the e-book marketplace particularly will be sorted out over time and there will then be even more to recommend these excellent devices.

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First impressions of iOS4 on new iPod touch

Sunday, July 4th, 2010

My original first gen iPod touch has been, without doubt, the best gadget I’ve ever purchased.  It’s been used daily for almost 3 years now as an MP3 player, email client, TV, photo album, and PDA.  However the headphone socket has only played out of one ear for about 6 months, and about a month ago I lost a whole swathe of pixels at the bottom of the screen, so today I went out a bought a new one.

Initially I though this was going to work out well.  I synced the new iPod with iTunes and it said it was going to upgrade to iOS 4 and restore the settings from my old iPod – great.  However I’m now collecting a list of problems which I thought I’d enumerate here.

1. Couldn’t connect to my home wifi network.  The new iPod picked up the stored password from my settings, but couldn’t connect to my network.  All other machines (and my old iPod) worked fine.  I had a very similar problem where my Macbook Pro wouldn’t connect to my parents network after upgrading to snow leopard.  Research for that problem let me to several posts which linked the problem to the use of WEP on the connection.  I therefore changed the encryption to WPA and the new iPod now connects.  Is this a backdoor route through which Apple hope to increase the security of home networks?
2. Couldn’t connect to my email.  Although my email settings were carried over, the password wasn’t.  Oddly I wasn’t prompted for it but rather told to go into the mail settings to find the appropriate box in which to add it.  Once I’d added the password I could connect.
3. But – I’ve had some oddities with my email.  I uses an SSL encrypted IMAP account and I’ve had messages which wouldn’t move to unread status no matter how many times I viewed them.  I also had a ‘ghost message’ with no title and no content – but it was apparently unread and couldn’t be read or deleted.  It took a reboot to get rid of that, but it’s reoccurred after some more use.  It seems that I’m not alone but the immediate fix (use the multitaking switcher to kill mail) doesn’t work for me because this iPod doesn’t support multitasking.
4. Although all my media seems to have synced over OK, one of my podcasts (This Week in Tech as it happens) is duplicated on my iPod.  The same episodes appear in both copies.  I’ve removed and readded the podcast from iTunes, but it just duplicates it all over again.  No idea what else to do with this.

This experience has further reinforced my love hate relationship with Apple.  When their stuff works it’s great.  Well thought out, nicely implemented and consistently integrated.  Everything you could hope for really.  The problem comes when things don’t work out properly.  The ecosystem within which Apple operates is so closed that they won’t acknowledge when problems exist and you’re left with an unknown wait until a future update magically fixes things.  People contacting apple support about the IMAP problems have been talked through resetting their mail accounts and a few other fixes which Apple must know don’t work.  Contrast this with open source solutions where all bug tracking is out in the open so you can see not only that the problem exists and has been identified, but also get an official response for the best work around and can track the progress on a permanent fix.  Would adopting this kind of system really cause harm to Apple?

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iPlate lives up to the hype

Sunday, October 12th, 2008

Our house is somewhat isolated, and until quite recently was too far from our nearest exchange to be able to get ADSL broadband.

Since we did get broadband our connection speed has been somewhat dissapointing varing from 300-600kb/s, and peppered with frequent (though usually short) outages.  Similar problems have plagued our neighbours but we’re grateful for what we can get!

I’ve already tried a few different modems to try to improve the reliability or stability of our connection, and although there was a definite variation between different models the connection only varied between slightly dodgy to downright awful.

I was intrigued therefore to hear that BT wholesale had released a new device called an iPlate.  This is a small hardware device which attaches to your master socket and is supposed to improve the reliability and speed of your connection.  When I heard this I was (as I usually am) sceptical.  From my understanding the limitations on our connection were due to the distance our signal had to travel, and it seemed unlikely that there would be a simple and cheap way to improve this – otherwise why wouldn’t they have been doing this before.  However – the initial reports I read about people who had tried the iPlate were universally positive.  People were reporting anything from a 20% to 50% increase in connection speed, which in our case would be a big improvement.

I therefore decided to take a punt on this being real and bought an iPlate from Tamar Communications (no affiliation), which cost a grand total of £12.33 (including postage and VAT).  It duly turned up the next day.

Fitting the iPlate is dead simple.  The i stands for ‘interstitial’ because the way you fit it is to unscrew the front plate from your master socket (leaving the wires connected!), you plug the iPlate into the backplate of the socket, and then plug the original front plate into the iPlate.  The whole thing took about 2 minutes.

Upon reconnecting my modem it reconnected almost instantly (previously establishing a connection took around 2 mins).  Looking at the stats page my connection speed was 2Mb/s (a 6-fold increase on our typical connection speed!), and it’s stayed there ever since.  To my mind this is one of the best paybacks I’ve ever had for a piece of tech.

I suspect that we’re the ideal candidates for the iPlate, those with weak or noisy connections are likely to see the best increases – if your connection speed is already good then the difference may be negligible, but for houses like ours it’s been a godsend.

Tags: adsl, broadband, iplate
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