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posted by takyon on Monday May 16 2016, @09:20PM   Printer-friendly
from the better-late-than-never dept.

You may recall that I did a TEDxWellington talk about two months ago. My talk was about sequencing on the Oxford Nanopore MinION. The video of this talk has now been edited and is available on the TEDx Youtube Channel.

Although I haven't explicitly said it in the talk, this is a live demonstration of DNA sequencing, and possibly the first such demonstration outside ones done by Oxford Nanopore Technologies. I spend the first half of the talk stalling for time while the initial QC finished, and then a bit of time after data analysis (i.e. a BLAST search) discussing where we could be heading.

To give a bit of an idea of the challenges involved in doing this, all my equipment for sequencing (excluding laptop) was brought to the venue the day before (for the dress rehearsal) in a 30cm polystyrene cube.

On arrival at the venue, I stored the ONT reagents in a freezer in the nearby kitchenette, and prepared the flow cell about half an hour before my talk. In my lab I had prepared two tubes with pre-mixed reagents (one with library + water + running buffer; one with fuel mix), so I was able to use a fine-nozzle pasteur pipette to do the final mixing and loading onto the flow cell.

I had a slightly flakey USB connection on the MinION, so couldn't start the run off-stage (it was very sensitive to bumping). Despite starting the run during the video prior to my talk, I still had a bit less run time than the 5 minutes I had planned for, so had to tweak my presentation a bit to fit the end of the QC step into my talk.

The sequencing run was carried out using a laptop I had purchased for $900 NZD and set up a couple of weeks prior to the conference. Sequencing was done from battery power only, using the WiFi connection because the wired connection was being used for conference live streaming -- this might be the reason why called sequences took a little longer than a couple of minutes to download onto the laptop. The dress rehearsal the day before was the first time I'd carried out a sequencing run on that particular laptop, and made me aware that the screen resolution was less than the recommended minimum requirements from ONT.

Despite everything that happened, I don't think any of the audience were aware that I had any problems with my run (apart from needing to use my dress-rehearsal backup sequences), which aligns very nicely with the themes of trust and secrecy for this year's conference.

For those interested in looking at the actual reads from that run, I've put the "pass" reads into a dropbox folder.

If you want a little low-hanging-fruit programming project to work on, then you can have a look at improving the recently published open source base callers:

  • DeepNano (Neural network basecaller): Deep Recurrent Neural Networks for Base Calling in MinION Nanopore Reads; Vladimír Boža et al., Comenius University
  • Nanocall (HMM basecaller): An Open Source Basecaller for Oxford Nanopore Sequencing Data; Matei David et al., Ontario Institute for Cancer Research and University of Toronto

And now, the answers to the Q&A:

takyon (881) asked:

The Nanopore website mentions connecting to a PC or laptop using a USB port. How about a smartphone? What software is used on the PC/laptop/phone to receive data?

Software called MinKNOW operates the MinION from the laptop or pc. It's not smartphone enabled yet but the company has previously expressed a desire to make it easier to take MinION in the field and this is one of the obvious ways to do that.

As well as MinKNOW, Oxford Nanopore is starting to offer other kinds of real time analysis solutions to people who might not have bioinformatics skills but want to perform their own experiment. These are available from Metrichor- one example is a workflow that allows people to identify species in their sample, in real time, against a reference dataset. [response from ONT based on public sources]

Does the MinION dump the raw data to a PC, which will then compress it?

When/where/how should the sequenced genome be compressed?

The electric current across 512 electrical sensing channels is sampled at [currently] 3kHz and transferred to the PC. By modifying the sequencing recipe source code, users have options in software to retain or discard that raw signal data. That signal is then processed into 'event' data which describes contiguous parts of the signal that fit into a similar range of current, generally attributed to the movement of a single base through the nanopore. These data are stored in an HDF5 file, which I believe has a bit of compression applied to it.

There's more information in the event data (and raw signal) than just the 4-base model of a DNA sequence, so I'm not convinced that it's a good idea to only store the final sequence information (and especially not now, given that the base caller needs a lot of improvement). Some time in the not-so-distant future we'll have to think of a better model than what we've got, and stop sweeping "out-of-model" genetic features under the carpet by calling them fancy names like epigenetics. Unfortunately, that also means that some changes need to be made with regards to reference genomes and associated compression schemes. I have no answers for how this might (or should) be done. Given that we're still surviving in the ultra-high-throughput phase of DNA sequencing with text files and gzip compression, I'm not yet convinced that any fancier compression is necessary.

Where do you acquire the graphene for nanopores (assuming that is what it used in current generations of the MinION), and have the costs fallen?

Graphene isn't yet used in the MinION, although this is in the pipeline. The current structure is a synthetic membrane into which biological nanopores are embedded. [response from ONT based on public sources]

Is Oxford Nanopore Technologies involved with the 100,000 Genomes Project or any other emerging population-scale sequencing efforts?

I expect not. ONT tends to do their own internal research in the lab and leave the users of the MinION to do the interesting stuff.

How are competing sequencing products better or worse than MinION, on factors other than portability/lack of portability?

I see sequencing as split into three different generations:

  1. Sequencing by Amplification -- copying entire sequences (e.g. sanger)
  2. Sequencing by Synthesis -- copying single bases (e.g. illumina)
  3. Sequencing by Observation -- no copying involved (e.g. nanopore)

As far as I'm aware, no other companies are trying to do sequencing without some form of synthesis, so it's hard to define a "competing" sequencing product. Some people will argue that PacBio is a direct competitor; here's my response, which I felt I needed to say in the previous discussion because I disagreed with the other answer that was given:

Both PacBio and ONT are producing sequencers that produce long reads off single molecules, but the technology and approach of sequencing is very different. In other words, while the output is [currently] similar, the way you get there is different.

The biggest difference, from my point of view, is that PacBio sequencers carry out sequencing by synthesis, while ONT sequencers (e.g. the MinION) carry out sequencing by observation. This has two fairly big advantages:

  1. Once samples are loaded onto the MinION device, no further reagent loading is necessary to get sequencing working.
  2. The MinION does something very close to model-free sequencing, by observing changes in electric current as the template passes through the pore.

We don't yet know how to properly use the output from the MinION, but a basecaller has been created by ONT to convert signal output into a base sequence. The assumed models are all in software (in the base-caller), and that can be updated and improved later in-silico.

Considering purely what is useful right now, Sequel is a bit cheaper per run ($700 vs $1200) for about five times the theoretical maximum yield (10 Gb vs 2 Gb), as long as you ignore the cost of purchasing (and maintaining) a Sequel.

However, that's not a particularly helpful answer. Most people who are using Sequencing By Synthesis (SBS) machines are probably not going to like (or change to) the MinION any time soon. People don't like change, and will cling onto whatever disadvantages they can find as an excuse to resist the change. These disadvantages for the MinION are fading away as the technology improves, but I doubt they'll ever disappear entirely:

  • The MinION doesn't generate enough data
  • The data it **does** generate is too low quality
  • The sequences it generates can't be processed in the same way as other sequence data

It's probably worth pointing out that the available MinION technology is (for the forseeable future) always going to be better than what can be seen in research papers, and the technology in development (by ONT) is always going to be better than what is available to users. The MinION technology is disruptive, and changes almost everything about how sequencing is carried out. Here's my initial attempt at generating a list of what's the **same** between the MinION and other sequencers:

  • In sample preparation, adapters are added to sequences to provide anchor points for the sequencing process to begin

And here's my list of MinION things that are different:

  • Sample preparation requires no amplification
  • Read length and quality out is associated with the quality of the sample preparation
  • A pipette is the only fluidics required for sequencing
  • Can sequence unknown DNA structures / variants
  • Can sequence hybrid DNA/RNA
  • Can sequence RNA directly
  • Fits into your pocket
  • Powered by USB2
  • Can sequence in real-time using a laptop running on a battery

I'm sure I've missed things off both of those lists; please feel free to add your own contributions.

What achievement is the company using to immediately promote the MinION? For example, are you or partners going out into the field and rapidly sequencing undiscovered bacteria, a certain taxonomical group of plants/animals/fungi, or an endangered population of big cats to preserve genetic diversity?

I need to once again clarify that I'm not an employee of ONT, I'm just a fanboy.

ONT prefers advertising the community achievements rather than what they've done internally in the lab, although they did produce a few posters on upcoming kits for their US meeting a few months ago. The nanopore sequencing community is fairly good at informing ONT about their publications, so the biggest effort on ONT's part in promotion is writing short stories about the community achievements.

The stories are many and various. I used two for my talk (tracking the Ebola virus through Africa, sequencing on the slopes of a volcano in the rainforests of Tanzania), and had to cut out another (about NASA sequencing in microgravity) due to time constraints. Most of the applications have been around things that could be done with other sequencers if samples were taken away and sequenced at another location, but I expect people will eventually get bored of that and explore the technology a bit more.

martyb (76) asked:

There has been much discussion on this site about the-powers-that-be vacuuming up all the information that they can. Not just from government entities (such as the NSA and GCHQ), but also corporate entities, as well (data brokers, insurance companies, Google, etc.)

I have a two-part question:

  1. What do you see as the greatest opportunities in the use of a tool such as MinION?
  2. How can we protect ourselves from having that information used against us?
  1. Making sequencing affordable, useful, and accessible for everyone. As a start, sequencers that are as cheap and as available as a high-end household appliance. Will you upgrade your phone this year, or buy a DNA sequencer?
  2. I don't know. DNA is everywhere. We certainly can't keep people from collecting our DNA, any more than we can stop them from recording our car license plate. With accessible sequencing, the same loss of control will extend to storing DNA sequences on private databases. This is not going to be a universal problem -- there are not many people who would be interested in keeping a record of every DNA sequence they come across -- but it is a problem that exists. Laws and acceptable ethical practise will prevent some bad uses, but not everything.

bitstream (6144) asked:

Is there any hindrance to provide software with the hardware that runs on the free Unixes like FreeBSD and Linux?

The software we're using for interfacing with the hardware is mostly python, and a Mac client is in the works, so I don't think there are any technical issues. ONT seems to be spread far too thinly on the software development front, and aren't particularly keen on releasing specifications for interfacing with their devices using free and open source software. The company appears to be interested in making money out of their software ideas as well as their hardware, and that worries me slightly.

VLM (445) asked:

How do you handle the HIPPA problems of the volume of data?

I guess to expand on what I'm talking about, its one thing to "lock down" and "keep secret" my O+ blood type (err, I think thats what it is, anyway). How do you handle "large amounts" of genetic data?

Sorry, I'm not familiar enough with HIPPA to make informed comments on that. Nanopore data is unlikely to overtake Illumina data in the next few years in terms of the amount of data produced, but the locking up of genetic data for privacy reasons is basically a moot point. Who needs DNA sequences to be stored, when you can just pick more DNA off the street, or out of a rubbish bin? I can store genetic data on a hard drive and keep it in a locked cabinet (or embedded in micro flash storage under my skin somewhere), but there's not really anything I can do to stop my body from discarding dead skin cells, or to stop one of my relatives agreeing to having their DNA sequenced instead. We need to think about a world where the information we can store is no different from the physical things that we have access to.

sbgen (1302) asked:

Is this gadget useful for testing of the food supply? I'm not talking about paranoid GMO stuff, but is it reasonably sensitive such that you could grind up wheat into flour, mix thoroughly, and test for bug DNA to verify bug contamination of the original wheat? Most of the stuff in the flour would be gluten protein and "wheat parts" but could you search for bug DNA specifically instead?

Yes, it is. And the more it is used for that purpose, the more useful it will become because the public databases of discovered sequences will increase in diversity. For my TEDx demonstration I did a live sequencing of a tomato source: tomato that my wife bought at the local market, which I extracted DNA from at home using a mortar and pestle, salt, detergent, a sieve, and some meths. I needed to do a bit of purification and sample preparation in the lab prior to sequencing [ONT is working on fixing that issue with something they call VolTRAX], but about 15 minutes after it was loaded onto the MinION I had sequence that could be BLASTed to a public database of sequences.

I did previously try bread and butter, but my lab skills aren't good enough for the high DNA concentration that was required (1.5μg in 50μl). There's plenty of DNA there, but it was mixed in with a lot of liquid as well. I think given a bit more time and money, I could probably work out a reasonable protocol for bread and butter.

If you just want to look for bacterial sequences in your food, that can be done as well. However, it tends to be the case that 99% of the sample DNA is host sequence and a waste of sequenced reads, so it can take a bit longer to get enough sequence to properly establish the microbial fraction of your sample. I don't think I could achieve that in a 15-minute demonstration.

Anonymous Coward asked:

What are your feelings on the current unreliability of polymorphisms at producing meaningful health outcomes? Besides obvious hereditary diseases and tumor profiling, there doesn't really seem to be much predictive power of having a genome sequence for a patient.

On the subject of polymorphisms, I think we put too much trust in our four-base model of DNA, and also don't put enough consideration into local genomic structure. I spent a couple of years doing research on haplotypes (i.e. combining multiple adjacent genetic variants), and was able to show that results and associations could change depending on whether or not haplotypes were taken into consideration. I also talk frequently with someone who's discovering interesting things about methylation, and have attended talks that discuss how the 3-dimensional structure of DNA can influence gene expression.

With regards to genome sequencing, it's very effective for conditions that have an obvious genetic basis, but also very expensive. Unfortunately there are a whole bunch of unique disease-causing variants floating around (especially in cancer), so it's frequently the case that finding one cause for one person doesn't translate to finding the ultimate cause for everyone.

devlux (6151) asked:

I worked in a lab for years. Paid for part of my college that way. Contamination and cross contamination of samples is always A HUGE problem. I see nothing here that addresses demunging of results or even that there is an attempt.

Consider a sample taken in a hotel room. Even after cleaning, there are literally thousands of people's skin & hair flakes laying about all over the room. There is no way to distinguish one from another and it's unlikely that normal room swabbing would be able to distinguish one person from another.

So how does this system purport to differentiate or at least make some control against contamination?

The biggest contamination issue with the way sequencing is done at the moment is that it requires an amplification step prior to the actual sequencing. This means that any contaminants are also amplified, and possibly preferentially amplified. This is such a big problem that labs are frequently split into pre-amplification (or pre-PCR) areas and post-amplification areas. The MinION has no requirement for amplification, so the reads that come out of the machine are a closer representation of the samples that go in.

Longer reads also help in this regard. The longer the read is, the more chance you have that a given sequence can be distinguished if it came from a different sample.

But at the end of the day, what you get out is only as good as what you put in. If you're putting the contents of someone's garbage into the MinION, don't be surprised if garbage comes out of it.

Anonymous Coward asked:

Is there any relationship between trust/secrecy and the talk topic?

The TEDx speakers for this year weren't **required** to involve trust in their talk, but I'd say that every speaker/performer did have the theme of trust running through their talk at some level.

There were a few trust themes in my talk:

  • I talked in jeans and a T-shirt; not the expected attire from a professional who works on genes
  • False trust in models vs observing the world
  • DNA extraction in front of my kids
  • Anxiety over the capabilities of the device (prerecorded video of a sequencing run)
  • Waiting for a sound clip to play
  • Trust needed prior to technology development
  • Trust needed prior to funding for research

But for me, trust was all over the place. Perhaps it wasn't obvious in the talk as presented, but it was definitely there:

  • My work didn't know in advance about my talk
  • It was my second conference as a speaker
  • I was talking to an audience of ~120, but livestreaming to an audience of thousands
  • I did a live technology demonstration
  • As far as I know it was the first MinION sequencing demonstration carried out by a non-ONT person
  • The device I was using had a flakey USB connection that disconnected when the laptop was moved
  • I didn't know in advance how much time I would get before my talk to start the sequencing run -- I had planned for 5 minutes, but only ended up with about 2 minutes. This meant I had to shift my talk around and fill in time until the run was ready to produce DNA sequences
  • I loaded the samples onto the sequencer using a pasteur pipette
  • I needed to use my backup sequences for demonstrating BLAST searches, because the ONT servers were a bit slower than they were in the dress rehearsal

So, yeah. There was a bit of a relationship between trust and my talk idea.


Original Submission

Related Stories

TEDxWellington Talk: Sequencing that Stimulates the Sensors; Author Q&A 29 comments

[Ed note: We are experimenting with something new with this story on SoylentNews. The submitter has agreed to answer some of your questions. Please submit one question per comment. The most highly-rated comments will be given to the submitter. We'll post another story later with the replies.

Previous stories on MinION:

The MinION - Genome Sequencing in a Handheld Device
A MARC in the Silicon: Sequencing E. coli with the MinION]

I was invited to be a speaker at the TEDxWellington 2016 conference on 6th March. The theme of the conference was trust. Along with all other speakers and crew, I was trusted not to tell anyone about my role in it until I had finished speaking. Attendees were transported from a meeting place at the National War Memorial to the venue in buses that had been blacked out The conference was the first public event that was held inside Peter Jackson's private cinema at Park Road Post Production in Miramar, Wellington, New Zealand.

Due to an enclosed fabric tunnel, the cinema inside the venue was the first thing that attendees saw after getting into the buses. Only a few people knew the speaker lineup, and speakers for the second and third sessions were distributed throughout the audience, so that attendees couldn't tell who else would be talking.

The talks, prior to post-production and editing are available on a live stream feed. My talk was about DNA sequencing using the Oxford Nanopore MinION and starts at timecode 01:29:00 on the recorded video. A video of my presentation will be edited and uploaded to the TEDx YouTube channel in a few weeks.

Here's a brief point summary of my talk:

[Continues.]

Weird Science Heading to the ISS 5 comments

Writing at Space.com, Sarah Lewin describes some of the scientific cargo that tomorrow's Dragon9 launch will carry to the International Space Station:

SpaceX's ninth commercial cargo mission, launching early Monday (July 18), is lugging a selection of strange science to the International Space Station -- living, beating heart cells, microbes from a nuclear disaster, a tiny DNA sequencer and more.

[...] One experiment won't take up a lot of space, but it has the potential to be a huge research boon to the orbiting lab -- the space station's first DNA sequencer, which is about the size of a "fun-size Snickers bar," said Sarah Wallace, a microbiologist at Johnson Space Center (JSC) in Houston.

[...] The astronauts will also be bringing aboard live heart cells, which they will cultivate for one month to test for changes in their sizes, shapes and beating patterns.

[...] Another experiment that investigates bone loss in space will test some technology that could potentially save researchers from sending similar experiments aloft: It will compare changes in bone cells that have been flown to space with ones that got the zero-g experience on Earth, levitated magnetically in a microgravity simulator.

[...] In addition to sending up those heart and bone cells, plus some tomato seeds that will be grown in schools once they return to Earth, the spacecraft is bringing another strange visitor: microbes that emerged after the Chernobyl nuclear reactor, located in Ukraine, melted down catastrophically in 1986.

[...] Researchers are also sending up a demo of a phase-change material heat exchanger, which will test wax-based and water-based substances that can melt and freeze as the temperature outside a spacecraft changes, absorbing excess heat and re-releasing it to keep the craft warm as it orbits in and out of Earth's shadow, going from blazing heat to chilling cold. They're also testing a receiver that will track ships on the ocean, computer processors to stick outside the station and check for radiation-related errors, and a more efficient solar cell.

The photo of the DNA sequencer shows that it really does resemble a "fun-size Snickers bar", though with two layers!

The "DNA sequencer" is made by Oxford Nanopore Technologies and is called MinION about which we have had a few stories posted here.

Related:
SpaceX Set to Launch 9th Dragon CRS Mission 12:45am EDT (04:45am UTC) on July 18


Original Submission

Human Genome Sequenced With MinION Nanopore Sequencer 3 comments

Pocket-Size Nanopore Device Sequences Entire Human Genome

Researchers have assembled the entire human genome using a nanopore sequencer, according to a study published today (January 29) in Nature Biotechnology [open, DOI: 10.1038/nbt.4060] [DX]. Using a pocket-size device, dubbed MinION, the team was able to fill 12 gaps in the sequenced human genome by achieving reads of DNA sequences nearly one million bases in length—the longest to date.

Also at BBC.

Nanopore sequencing and assembly of a human genome with ultra-long reads (linked above)

We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ∼30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ∼3 Mb). We developed a protocol to generate ultra-long reads (N50 > 100 kb, read lengths up to 882 kb). Incorporating an additional 5× coverage of these ultra-long reads more than doubled the assembly contiguity (NG50 ∼6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4-Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length, and closure of gaps in the reference human genome assembly GRCh38.

Previously: The MinION - Genome Sequencing in a Handheld Device
A MARC in the Silicon: Sequencing E. coli with the MinION
Update: Sequencing That Stimulates the Sensors, and MinION Q&A Responses

Related: 3D Genome Assembly Could Create a Human Reference Genome for Under $10,000


Original Submission

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  • (Score: 0) by Anonymous Coward on Monday May 16 2016, @09:33PM

    by Anonymous Coward on Monday May 16 2016, @09:33PM (#347046)

    WTF did I just scroll through... pages and pages and pages and pages... Did that guy from over at /. get to be an editor here on SN? I though we fled from that guy... (and beta, buck feta!)

    • (Score: 2) by takyon on Monday May 16 2016, @09:57PM

      by takyon (881) <reversethis-{gro ... s} {ta} {noykat}> on Monday May 16 2016, @09:57PM (#347054) Journal

      I think you will recover 👌👌👌

      --
      [SIG] 10/28/2017: Soylent Upgrade v14 [soylentnews.org]
      • (Score: 0) by Anonymous Coward on Monday May 16 2016, @10:20PM

        by Anonymous Coward on Monday May 16 2016, @10:20PM (#347060)

        My scrolling finger is sore. ✘

    • (Score: 2) by gringer on Tuesday May 17 2016, @12:35AM

      by gringer (962) on Tuesday May 17 2016, @12:35AM (#347082)

      Sorry about that. There were so few questions in the last post that I decided to answer all of them, and I'm not so great at giving short answers.

      --
      Ask me about Sequencing DNA in front of Linus Torvalds [youtube.com]
      • (Score: 2) by opinionated_science on Tuesday May 17 2016, @01:41AM

        by opinionated_science (4031) on Tuesday May 17 2016, @01:41AM (#347095)

        so how long until we get the $1000 genome?
        I understand the minion cannot be reused. Is there a technical reason that could maybe overcome?

        • (Score: 2) by gringer on Tuesday May 17 2016, @03:45AM

          by gringer (962) on Tuesday May 17 2016, @03:45AM (#347125)

          so how long until we get the $1000 genome?

          A $1000 genome is already here, depending on your definitions. For example, Genohub [genohub.com] tells me that I can get sequencing for 24 individuals done for less than $1000 per sample from a place in Australia (although library prep is another $2,500 on top of that). If you want a single bacterial (or yeast) genome, that can also be done for less than $1000.

          I just watched a video from Jens Gundlach [youtube.com] (one of the key people who helped work out nanopore sequencing technology) where he talks about what could come in the near future with nanopore sequencing in terms of sequencing cost (his graph suggests ~10 years):

          • $100 per sequence run: every person gets their genome sequenced
          • $10 per sequence run: doctors prescribe genomic DNA sequencing as a routine test
          • $1 per sequence run: doctors prescribe metagenomic DNA sequencing as a routine test

          I understand the minion cannot be reused. Is there a technical reason that could maybe overcome?

          The MinION can be reused, but at the moment it is just a relatively dumb interface between the flow cell and the computer. The flow cell is where all the complicated circuitry is, and that's something that currently needs to be returned to ONT to get recharged. ONT are in the process of developing the technology and shift the complex circuitry into the MinION, leaving just plastic and gel as a consumable part. I expect that approach will bring the consumable cost for a full run under $200, with partial runs possible for a smaller portion of that cost.

          I'll be attending the London Calling conference in a couple of weeks (May 26/27th), so should be able to give a few more concrete answers then about where the technology is heading. Answering "when" is a problem even for Oxford Nanopore; I'm starting to realise it's best to just talk about the things that I have access to and can vouch for myself.

          --
          Ask me about Sequencing DNA in front of Linus Torvalds [youtube.com]
        • (Score: 1) by sbgen on Tuesday May 17 2016, @03:20PM

          by sbgen (1302) on Tuesday May 17 2016, @03:20PM (#347333)

          I came here to repeat what gringer explained well: the cost of genome at the moment is less than $1000 depending upon your definition of it. I also came here to say that the real cost now is in preparing the sample ('library prep') and more importantly in analyzing the data. The computational needs for the latter are a cost center too.

          --
          Warning: Not a computer expert, but got to use it. Yes, my kind does exist.
          • (Score: 2) by gringer on Wednesday May 18 2016, @11:24AM

            by gringer (962) on Wednesday May 18 2016, @11:24AM (#347753)

            The computational needs for the latter are a cost center too.

            A recent Linux computer provides sufficient resources to do the majority of genetic research that's done these days. I built my current computer about 4 years ago from component parts for about $2500 NZD (6-core computer with 12 processing threads, 64GB memory, SSD drive), and at that time it pretty much only struggled with de-novo genome assembly. With computational advances, sequence length improvements, and better software it's actually become easier for me to do work on that same computer, and I'm starting to get into doing assembly on it as well.

            I've needed access to a HPC cluster for whole-genome mapping of 104 human samples in a timely fashion (~100GB genetic data per person; mapping and calling took about 2 weeks), but now that the mapping has been done and genetic variants obtained, that project is back to single-computer stuff. Probably the biggest issue I'm having at the moment is data storage, and that's not really that big of a problem. I buy another multi-terabyte hard drive every year or so for my own work, make sure I have my scripts backed up externally, and encourage my clients to do something similar. Data volume in genetics is also likely to reduce with single-molecule sequencing, as less genetic data will be needed to cover a greater proportion of the genome. If storage ever becomes a bigger problem for me, then I'll probably invest in something like the storinator [45drives.com].

            --
            Ask me about Sequencing DNA in front of Linus Torvalds [youtube.com]
      • (Score: 2) by bitstream on Tuesday May 17 2016, @07:13AM

        by bitstream (6144) on Tuesday May 17 2016, @07:13AM (#347175) Journal

        * Is there any means to read the phenotype of cells?

        * Even if the software is closed they could still release a client for BSD and Linux?

        * If more than one DNA is present in a sample, how does one separate them on the computer?

        for about five times the theoretical maximum yield (10 Gb vs 2 Gb),

        Does this mean it won't decode the full DNA sequence?

        The device I was using had a flakey USB connection that disconnected when the laptop was moved

        Add a capacitor across the +5V and ground on the USB cable with a Schottky diode towards the computer. That would keep the voltage up even when intermittently disconnected. And the diode would prevent flow of current back to the computer.

        • (Score: 2) by gringer on Tuesday May 17 2016, @10:06AM

          by gringer (962) on Tuesday May 17 2016, @10:06AM (#347220)

          Is there any means to read the phenotype of cells?

          Flow cytometry [wikipedia.org] is a common way to do that at the moment.

          Even if the software is closed they could still release a client for BSD and Linux?

          Yes, they could. Clients exist for OSX (and existed a year ago), but they're not yet released for public use. I suspect that ONT has a lack of skilled programmers, because there are a lot of front-end issues that have remained broken for over a year.

          If more than one DNA is present in a sample, how does one separate them on the computer?

          The DNA sequences are easily separated by the laptop during signal acquisition, because the pore is in a high current state unless there is DNA blocking it; separate sequences are discovered by looking for big jumps in the signal.

          If different genomic regions are sequenced at the same time, it's possible to differentiate them by mapping to a reference genome. If there is no reference genome, then you can cluster sequences based on their similarity.

          However, some times you can get sequences from completely different genomic regions (or different organisms), but they are similar enough that they will cluster together. There are gene duplications that happen, as well as regions of low complexity (i.e. highly compressible if run through a gzip algorithm), and trying to distinguish these almost-similar sequences is a hard computational problem, made a bit harder due to sequencing error.

          for about five times the theoretical maximum yield (10 Gb vs 2 Gb),

          Does this mean it won't decode the full DNA sequence?

          The current ball-park target for genome assembly from long reads is about 5 times coverage, which is expected to capture most of the genome by random sampling. For shorter reads, it's about 20-30 times. With a human genome size of 3.2 Gbp, that's only about 3 times coverage, so there would probably be a few large gaps in any such sequencing. However, it's not typical to do de-novo assembly of human genomes, and even an incomplete coverage (maybe as low as 5%) can be useful for genetic fishing expeditions to discover unexpected variants.

          Add a capacitor across the +5V and ground on the USB cable with a Schottky diode towards the computer. That would keep the voltage up even when intermittently disconnected. And the diode would prevent flow of current back to the computer.

          Thanks for the advice. I'm not sure how ONT would feel about modifying USB cables to work around a hardware issue on their device. The problem was a mechanical connection issue within the MinION device, rather than the USB cable; the cable connector needed to have pressure on the bottom of it at an angle to get the device to connect. I realised that this was the problem only a few weeks before my TEDx talk, and contacted ONT for a replacement. I got that replacement about a month later, but it didn't quite arrive in time for the TEDx conference.

          --
          Ask me about Sequencing DNA in front of Linus Torvalds [youtube.com]
  • (Score: 0) by Anonymous Coward on Monday May 16 2016, @10:11PM

    by Anonymous Coward on Monday May 16 2016, @10:11PM (#347058)

    The link is dead for me. How much does the sequencer cost?

  • (Score: 2) by gringer on Tuesday May 17 2016, @12:37AM

    by gringer (962) on Tuesday May 17 2016, @12:37AM (#347084)

    > about two months ago[s].

    --
    Ask me about Sequencing DNA in front of Linus Torvalds [youtube.com]
  • (Score: 0) by Anonymous Coward on Tuesday May 17 2016, @01:37AM

    by Anonymous Coward on Tuesday May 17 2016, @01:37AM (#347093)

    Don't do TED anything if you want to be taken seriously.

    • (Score: 2) by takyon on Tuesday May 17 2016, @02:03AM

      by takyon (881) <reversethis-{gro ... s} {ta} {noykat}> on Tuesday May 17 2016, @02:03AM (#347100) Journal

      It's just a platform to speak on. Much like anonymous postings.

      I would never say "Don't take anonymous posts seriously", no matter how many of them are worthless.

      --
      [SIG] 10/28/2017: Soylent Upgrade v14 [soylentnews.org]
      • (Score: 0) by Anonymous Coward on Tuesday May 17 2016, @03:17AM

        by Anonymous Coward on Tuesday May 17 2016, @03:17AM (#347118)

        That's because some of us write something useful without pretension every now and then, unlike you and your beloved TED whatsit.

    • (Score: 2) by gringer on Tuesday May 17 2016, @03:54AM

      by gringer (962) on Tuesday May 17 2016, @03:54AM (#347134)

      The TEDx speaking position hit me a bit by surprise. Through a misunderstanding of the application form, I originally expected that someone else would be talking about my idea.

      I don't mind if people don't take me seriously. I tried to throw a few jokes into my talk, carried out a sequencing run that wasn't associated with any research project, and turned DNA into sound. As long as people realise that DNA sequencing is much more accessible than it was a few years ago, I'll be happy.

      --
      Ask me about Sequencing DNA in front of Linus Torvalds [youtube.com]
    • (Score: 2) by bitstream on Tuesday May 17 2016, @07:02AM

      by bitstream (6144) on Tuesday May 17 2016, @07:02AM (#347172) Journal

      What's the problem with TED?

      • (Score: 2) by gringer on Tuesday May 17 2016, @10:35AM

        by gringer (962) on Tuesday May 17 2016, @10:35AM (#347231)

        For a start, it's easily confused with TEDx (and TED doesn't like it when people do that). TED (and TEDx) talks have a degree of sameness to them where someone tries to sell a complicated idea to a lay audience, usually by trying to be exciting and vague at the same time.

        TEDx events are independently organised with a smaller budget, cheaper tickets, and a lower bar for entry. While the training / coaching process for talks is similar to TED, TEDx suffers from the usual problems of crowd sourcing talent: you need a good filtering system in order to find a good TEDx talk.

        --
        Ask me about Sequencing DNA in front of Linus Torvalds [youtube.com]
        • (Score: 2) by bitstream on Wednesday May 18 2016, @12:53AM

          by bitstream (6144) on Wednesday May 18 2016, @12:53AM (#347587) Journal

          So TED has a good filtering while at TEDx the filtering is yourself?
          And that is the difference?

          I think lecturers should make things as simple as possible but never so simple as to distort any information. It's up to the audience to make sure they know enough to make sense of what is said. Ie don't go to a electrodynamics lecture until at least high school physics is understood. Thus the lecturer should be clear and go after that informative and relevant facts. Not let excitement kicks distort the core mission.

          • (Score: 2) by gringer on Wednesday May 18 2016, @01:16AM

            by gringer (962) on Wednesday May 18 2016, @01:16AM (#347595)

            So TED has a good filtering while at TEDx the filtering is yourself?

            TED has better filtering, and more money. Whether or not that's considered good enough is up to the viewer — you can find out by comparing the videos on ted.com [ted.com] (which includes some TEDx videos that have been noticed and put on a pedestal by TED) and the TEDx youtube channel [youtube.com].

            --
            Ask me about Sequencing DNA in front of Linus Torvalds [youtube.com]
            • (Score: 2) by bitstream on Wednesday May 18 2016, @01:33AM

              by bitstream (6144) on Wednesday May 18 2016, @01:33AM (#347605) Journal

              I read reports that TED is going in the direction of being snobby and only allowing "the right" people in etc. Such that they lost the openness for all disruptive knowledge.

  • (Score: 1) by anubi on Tuesday May 17 2016, @06:15AM

    by anubi (2828) on Tuesday May 17 2016, @06:15AM (#347161) Journal

    I am simply blown away by what you guys are doing.

    Understanding how to code in DNA to build life itself.... unbelieveable!

    --
    "Prove all things; hold fast that which is good." [KJV: I Thessalonians 5:21]
    • (Score: 2) by bitstream on Tuesday May 17 2016, @07:04AM

      by bitstream (6144) on Tuesday May 17 2016, @07:04AM (#347173) Journal

      Actually asfaik. There is no substantial understanding of the DNA code. It's more like an firmware dump where people find some ASCII strings and experiment with blanking parts or moving them around. There are results but not much understanding in the sense that people can actually read the sequence and understand it's meaning.

      • (Score: 4, Interesting) by gringer on Tuesday May 17 2016, @10:23AM

        by gringer (962) on Tuesday May 17 2016, @10:23AM (#347225)

        We understand a lot about the DNA code, but there's still a lot to learn. We understand that DNA is converted to RNA, which is converted to amino acid sequences (proteins, with other modifications). We know how to use and modify DNA to modify pre-existing coded protein sequences (which was an essential part of creating the nanopore that DNA moves through on the MinION sequencing device), and know how to generate novel protein subsequences from DNA.

        We also know that there's a particular set of genes [nature.com] that creates and maintains a living organism and can be created from scratch, even though a substantial portion of those genes have unknown function.

        We understand a little bit about how DNA packs together, but there's a lot more to learn about DNA modifications even at the linear sequence level that can't be discovered using current sequencing-by-synthesis methods (e.g. methylation, abasic regions, non-standard bases). We don't know all that much about long-range DNA effects (e.g. why does a particular sequence 1 Mbp away affect the expression of a gene). We have a [mostly] one-dimensional view of DNA that is a side-effect of the sequencing that we've been doing up till now. Hopefully by the development of additional observational sequencing technologies, our understanding of how DNA works will expand into the other dimensions.

        --
        Ask me about Sequencing DNA in front of Linus Torvalds [youtube.com]
        • (Score: 2) by bitstream on Wednesday May 18 2016, @12:46AM

          by bitstream (6144) on Wednesday May 18 2016, @12:46AM (#347580) Journal

          But do the research community understand what specific protein structure a specific DNA sequence will result in? 3 bases will result in one aminoacid asfair. The question then becomes how do one know which such ones that belong together?
          (bonus points for figuring out what all the proteins actually do..)

          And can one decode a protein into DNA sequence and thus find the spots which codes for it?

          • (Score: 3, Informative) by gringer on Wednesday May 18 2016, @01:51AM

            by gringer (962) on Wednesday May 18 2016, @01:51AM (#347612)

            But do the research community understand what specific protein structure a specific DNA sequence will result in?

            Not the 3D Structure. The amino acid sequence is well defined (i.e. the one-dimensional structure), but the 3D structure of proteins depends on a lot of environmental things that are not encoded in the DNA (e.g. pH, solvent concentration, post-translational modification, folding proteins). There are a few patterns of amino acids that tend to generate particular three-dimensional structures, but it's not possible to take a given sequence and know precisely what structure it will form in all conditions.

            And can one decode a protein into DNA sequence and thus find the spots which codes for it?

            This is a little harder to do due to redundancy in the translation. It's much easier to work at this in reverse: carry out six different translations of the genome into amino acid sequences and match the amino acid sequences to the protein. The NCBI tool tBLASTn [nih.gov] will do such a reverse search.

            --
            Ask me about Sequencing DNA in front of Linus Torvalds [youtube.com]
            • (Score: 2) by bitstream on Wednesday May 18 2016, @02:00AM

              by bitstream (6144) on Wednesday May 18 2016, @02:00AM (#347616) Journal

              Not the 3D Structure. The amino acid sequence is well defined (i.e. the one-dimensional structure), but the 3D structure of proteins depends on a lot of environmental things that are not encoded in the DNA (e.g. pH, solvent concentration, post-translational modification, folding proteins). There are a few patterns of amino acids that tend to generate particular three-dimensional structures, but it's not possible to take a given sequence and know precisely what structure it will form in all conditions.

              But even these environmental factors are a consequence of other codings in the DNA? at least internally.

              • (Score: 2) by gringer on Wednesday May 18 2016, @08:46AM

                by gringer (962) on Wednesday May 18 2016, @08:46AM (#347727)

                But even these environmental factors are a consequence of other codings in the DNA? at least internally.

                That depends on how many turtles you want to count. Protein construction processes can be affected by chemical signals sent through the blood, and also by metal ions moving around in the cells. There are steroids that can be applied with a topical cream, and burrow down through the cell membrane and nuclear membrane, and then bind to receptors attached to DNA [oxfordjournals.org]. If a cell is heated up at the focal point of a lens in direct sunlight, any created proteins will be denatured ("cooked"), and form a structure that is usually more predictable, but quite far from it's native structure in normal physiological conditions.

                I suppose you could argue that our movements and actions are all a result of the programs coded into our DNA, but traveling down that path ends up in big philosophical debates about the nature of decisions, choices, and consciousness.

                --
                Ask me about Sequencing DNA in front of Linus Torvalds [youtube.com]
            • (Score: 1) by anubi on Wednesday May 18 2016, @03:09AM

              by anubi (2828) on Wednesday May 18 2016, @03:09AM (#347640) Journal

              There were a couple of guys out in cyberspace I extremely highly respected... +Fravia and +ORC ( Old Red Cracker ).

              They seemed to have a really uncanny sense of how to reverse software given no glimpse of the source.

              I sure wish those two were still around and took an interest in cracking the genome. If I wasn't so damn old, I would love to get into it myself.

              This is one puzzle I could really enjoy because the results of solving it will be very meaningful. I am not so fond of "guessing-games", where I feel I am just wasting my time - those are more like filling out tax forms. No sense of accomplishment whatsoever for doing anything meaningful, but a relief to have the little box of things people expect from me ticked. However, cracking the genome - and conversely learning how to reassemble it in various ways to do specific things... WOW!

              I can already see the replacement of damned near every petrochemical plant on the planet with biological equivalents - all powered from photosynthesis. Custom trees, if you will, who manufacture via photosynthesis any desired chemical structure.

              --
              "Prove all things; hold fast that which is good." [KJV: I Thessalonians 5:21]