Dane.Kouttron
[5.4.17] 454 FLX Gene Sequencer Tear-down & Camera Re-purposing
What? |
First Power-On |
Sequencing Theory of Operation | Camera Extraction | Hardware Upgrades | First Images | Conclusion | Image Directory |
What now? |
454 lifesciences / Roche recently discontinued [link] the FLX series of genome seuencer. These contraptions are ~5-6 years old and use a process called pyro-sequencing to sequence dna fragments. Pyro-sequencing results in light being emitted in presence or absence of a sequential associated nucleic acid based upon which a sensitive camera looks for photon emissions and uses that data to generate sequences from DNA fragments. More of how this happens is included below. The discontinued machines are similar to a Polaroid camera, after the film is no longer manufactured, they dont really do anything. Its uncertain if Roche will have a 3rd party make equivalent reagents available. Given that the sequencer will not 'sequence' anything without reagents, why not get the camera up and running and use it for, astro-photography, particle detectors or charged particle imagers. Tune in below for more! |
Pre-Disassembly: Firing up
the Sequencer to see what works |
After the machine ran through all of its startup preperations, a 454 Life Sciences prompt was displayed, requesting login information. I did a bit of hunting through every manual I could find for this machine and found a reference to The default login being 'adminrig' for both user / password. Fortunately for this machine, that had not been changed. And lo it awoke, and after a bit of struggling came up to the main desktop |
So for whatever reason, the onboad ports on this IBM board for SATA were unusable, as in, the bios did not support the use of media connected, nor could it pickup a good drive. Whats bizarre is this same IBM machine came with the option of hot-swap sata instead of hot-swap SCSI. I attempted to clone one of the drives to a larger SSD, but alas, it would be hella slow over USB2.0, so i opted to copy off as much as possible manually. |
Sequencer Theory of operation |
EXCELLENT QUESTION: First off, here's some reference files, local copy in case direct link to manufacturer sites change. * GLS FLX Owners manual for an upgraded version of this machine [link] * GLS FLX Software manual [link] * GLS Sequencing method [link] * Spectral Camera 800 Series manual [link] * IBM Server that the GLS FX uses [link] * Bizarro gigabit fiber interconnect used on newer version of camera [link] |
![]() Start with a genomic sample, fragment it into smaller chunks, or segmented samples. There's a few mechanisms to do this: * Physical-shearing approach: (Acoustic, Sonication, Hydrodynamic) * Enzyme-shearing : Using a restriction endonuclease or Transposase * Chemical-shearing: Heat and a divalent cation are used for longer |
![]() Add adapters. 'A' and 'B' adapters are binded to the ends of the DNA fragments. The fragments are then 'unzipped' to form unzipped, adapter tagged strands. |
![]() Clonal amplification of fragments (take a single sample and make a whole pile of them). This can be done with PCR, and you end up with a pile of cloned segments with associated A and B adapters. |
![]() Pump clonally amplified fragments into the P2P plate (part that sits on the optics path). Inside the P2P plate are a bunch of 'wells' or small containers. Inside each of these wells is a 'bead' or solid location for segments to bind to. |
![]() Single strands bind to 'bead' in PTP wells. Shown is one 'well'. After binding has completed, wells are 'packed' with holding material (tiny clear beads). This prevents things from moving about, or from the bead falling out during the washing / pumping phases below. |
![]() A bead, covered in clonally amplified dna segments is subjected to a nucleic acid binded to AP5 (adenosine 5 phosphosulfate). If the nucleic acid binds to the dna corresponding segment, a side reaction occurs, ATP is synthesized. The ATP is consumed in a Luciferin + Luciferase enzyme reaction and light is emitted. Simplified: Adenine+ AP5 is poured into the chamber with the clonally amplified segments. If Guanine is present on the FIRST open base pair location, light is emitted, if it is not, no light is emitted. This whole process is called 'pyro-sequencing' |
![]() yes, suprisingy yes, At the end of each cycle the PTP well is 'washed', the bead and single strand sequence fragments stay in place, but any dissolved AP5 is washed out. This doesnt just happen once, it happens quite a number of times in parallel, depending on the PTP plate size and number of wells. The sequencer pumps in 'nucleic acid + AP5' observes on all wells if pyro-sequencing has occured (if light is emitted) as well as how much light, and keeps a log of these events. After cycling through wash and 'other nucleic acids + AP5' it does a pile of data processing looking for similar sequences. These similar sequences get sewn back together. This re-assembles a sequence that was physically sheared into smaller sequences. Because the same sequence occurs in parallel, a higher probability of accurate data is obtained. |
![]() in each chemical pumping cycle the camera analyzes a well, if 3x the brightness in comparison to a single event is found it is categorized as three binding locations. Nominally, the camera images reaction plate and records pyrosequence photon emissions, The machine washes out reaction well of remaining nucletoide and switches to another nucleotide. Cycle continues until base pairs filled. |
![]() Super-simplified, the A T C G and WASH tanks are each tied to a small peristaltic pump. The tanks and sipper tubes are actually visible (right). The whole sipper tube assembly rises up out of the solutions. The 'waste' container is actually shaped around the pocket that holsters the reagents. When removing, take care, it may be full of liquid. |
(There's other
photos in the photo gallery)
Comments: |
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(be
careful, im not responsible for whatever weird biotics were
leftover in your surplus sequencer, wear safety gear!)
Dane.Kouttron
Rensselaer Polytechnic
Institute
Electrical & Electrical
Power
631.978.1650