Cluster generation on cBot

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  • Please note that this is a protocol for the Cluster generation kit v2-HS which is compatible to the HiSeq Control Software version v1.1 and later and the TruSeq SBS kit v1.
  • There is a new kit available. Cluster generation kit v3-HS which is compatible to the HiSeq Control Software version v1.4 and later and the TruSeq SBS kit v3.


NB! post-PCR operation

Содержание





[править] Time plan

  • Cluster Generation on a cBot takes ~4 hours. Hands-on time - 30 min in the beginning.
  • After the run is complete, FC is kept at 20°C - FC may remain on the instrument overnight.
  • FC may be stored in storage buffer in the original tube at 2° to 8°C for several months.
  • When stored longer than 10 days - primer hybridization should be repeated.




[править] Preliminary

  • kits required : Cluster Generation kits are available for the Genome Analyzer and the HiSeq™

  Single-Read Cluster Generation Kit v2-HS (Catalog # GD-401-2001):

Kit componentsStorage requirements
HiSeq Single-Read Flow Cell2° to 8°C
cBot Manifold for HiSeq Flow CellRoom temperature
Single-read Reagent Plate (Box 1)-15° to -25°C
HT1 (Hybridization Buffer) for diluting templates and primers-15° to -25°C
Multiplexing Reagent Kit (used with sequenzing protocols on the HiSeq)(Box2)-15° to -25°C
HiSeq Accessories Kit (contains gaskets, bottle caps and ICR instrument bottle used with sequencing protocols on the HiSeq)Room temperature


  Paired-End Cluster Generation Kit v2-HS (Catalog # PE-401-2001):

Kit componentsStorage requirements
HiSeq Paired-End Flow Cell2° to 8°C
cBot Manifold for HiSeq Flow CellRoom temperature
Paired-End Reagent Plate (Box 1)-15° to -25°C
HT1 (Hybridization Buffer) for diluting templates and primers-15° to -25°C
Paired-End Reagent Kit (used with sequenzing protocols on the HiSeq)(Box 2)-15° to -25°C
Multiplexing Reagent Kit (used with sequenzing protocols on the HiSeq) (Box 3)-15° to -25°C
HiSeq Accessories Kit (contains gaskets, bottle caps and ICR instrument bottle used with sequencing protocols on the HiSeq)Room temperature


  Single-Read Cluster Generation Kit v2-GA (Catalog # GD-300-2001):

Kit componentsStorage requirements
Genome Analyzer Single-Read Flow Cell v42° to 8°C
cBot Manifold for Flow Cell v4Room temperature
Single-read Reagent Plate-15° to -25°C
HT1 (Hybridization Buffer) for diluting templates and primers-15° to -25°C


  Paired-End Cluster Generation Kit v2-GA (Catalog # PE-300-2001):

Kit componentsStorage requirements
Genome Analyzer Paired-End Flow Cell v42° to 8°C
cBot Manifold for Flow Cell v4Room temperature
Paired-End Reagent Plate-15° to -25°C
HT1 (Hybridization Buffer) for diluting templates and primers-15° to -25°C
Multiplexing Reagent Kit (used with sequenzing protocols on the Genome Analyzer)-15° to -25°C
Paired-End Reagent Kit (used with sequenzing protocols on the Genome Analyzer)-15° to -25°C


  • determine loading concentration for libraries by RT-PCR(to obtain necessary number of Clusters)
  • clean your labdesk
  • thaw reagent plate in a 96-well plate (at RT or use RT water bath, thawing takes 1h)
  • write the date on 96-well reagent plate
  • fill in the Lab Tracking Form (upload on the site and make a link) lot numbers of reagents
  • thaw the tube with HT1 (at RT or use RT water bath)
  • thaw 2N NaOH (HP3) at RT
  • (if not already done) thaw stocks of 10nM library solutions by finger tapping and place in ice
  • prepare EB buffer aliquots Qiagen (stored at RT) -- how much per library
  • NB! During reagents thawing prepare the DNA templates — see next step
  • calculate dilution recipe for each library (library is first denatured in NaOH, then diluted in Hybridization buffer):

--- insert calculation form

NB! max. 8 μl of NaOH-denatured DNA sample per 1ml of hybridization buffer -- NaOH concentration > 800µM in diluted samples inhibits the formation of clusters
Denaturation (in low binding tubes 0,5ml)Dilution in low binding tubes 1,5ml
LaneLib-IDlibrary nameEB in µl10nM lib in µlHP3(2N NaOH) 1:2 in EBsumConc. of denat. libraryDilution in HT1(denat. lib.:HT1)Required final conc. of libr.
11µl10µl3:
21µl10µl3:
31µl10µl3:
41µl10µl3:
51µl10µl3:
61µl10µl3:
71µl10µl3:
81µl10µl3:
e.g.1950pelib 2347µl2µl1µl10µl2000pM8pM



[править] Prepare reagents

  1. [  ]  put the thawed H1 (hybr. buffer) on ice until required
  2. [  ]  take the thawed reagent plate from the water bath:
    " dry with paper towels
    " remove each tube strip from the reagent plate base and mix the reagents by inverting each tube strip
    " visually check that volume is equal in each tube of the strip --> label reagent level in the tubes --> insert strips back
    " check that the tubes are securely seated in the reagent plate
    " pulse-spin the plate (or shake down)
    " store the reagent plate at RT (
    not on ice — formamide!
    ), proceed to setting up the run immediately
  3. [  ]  denature library:
    " protocol:
    EB17µl
    Library2µl
    NaOH 2N1µl


    " mix EB and library in 0.5ml LB tube, vortex, spin down
    " add NaOH, vortex, spin down, incubate 5min at RT (not longer!!!)
    " prepare 1.5ml LB tubes with pre-chilled hybr. buffer HT1 (volumes calculated previously; one tube per library)
    " transfer 3µl of denatured library into corresponding tubes with HT!
    " vortex, spin down, transfer 120µl of diluted template per channel to the signed eight-tube strip
    " keep the strip with templates on ice till loading on the FC
    " record each sample position and concentration on the Lab Tracking Form
  4. [  ]  take following things to the cBot:
    • cBot manifold (specific adaptor for GAII FC)
    • gloves
    • empty FC
    • Wash Buffer (mQ water) for pre-run wash
    • paper towels (low dust, name of the towels Kimberley??)
    • strip with ready-to-load libraries (on ice)
    • thawed reagent plate
    • Lab Tracking Form



[править] Cluster generation

  1. [  ]  turn on the cBot and follow instructions on the touch screen
    • type in your name ⇒ Enter
  2. [  ]  perform pre-run wash
    • raise the lid by gently lifting from the top-right corner
    • fill the wash reservoir with aprox. 12ml mQ water (from 15ml falcon tube)
    • close the lid and follow the touch screen
    • after the wash is finished, wipe the remaining water with paper towel dry (don`t rub the outlet ports)

  3. [  ]  select the protocol
    • type in the Experiment name
    • select the program, e.g. protocol: PE_Amp_Lin_Block_Hyb_v7.0 (complete cluster generation run, including steps for amplification, linearization, blocking, and primer hybridization)[1]

  4. [  ]  load reagents
    • remove plastic lid from the reagent plate
    • make sure strips are securely seated in the plate
    • use the barcode scanner for the reagent ID
    • remove the red foil seal from HP5 in row 10 and make sure the stripe is seated correctly
    • follow the touch screen and place the reagent plate (see picture1, A) in the cBot (positioned with row 1 facing to the front)
    • make sure that the beveled corner of the plate is positioned in the front-right corner

  5. [  ]  load flow cell
    • write date on FC-tube and in Lab Tracking Form
    • follow instructions on the screen how to load the FC correctly
    • lift the FC clamp
    • wash the adapter plate on the thermal stage with a small amount of deionised water, and wipe dry with a cleaning tissue.
    • scan flow cell-ID to activate the barcode scanner or enter the reagent ID using the keyboard
    • remove the FC from the storage tube using plastic forceps and dry the FC
    • rinse the FC with deionised water and dry the flow cell with a cleaning tissue
    • place the flow cell on the thermal stage with holes facing upward ( barcode on the FC is facing down and not visible)
    • !!!lane orientation is lane 8-1, from left to right!!!
    • touch the checkbox to indicate that you have loaded the FC

  6. [  ]  load manifold
    • fill in the manifold number in the Lab Tracking Form
    • use the right adaptor plate the for fc (GA ob HS)
    • set the manifold over the FC and touch the check box to indicate that the manifold is seated correctly
    • close the FC clamp to look the manifold and follow the checkbox
    • connect the outlet end of the manifold to the outlet port in the wash reservoir
    • snap the outlet clamp closed to secure the outlet end of the manifold
    • touch the checkbox to being sure that you all thinks connect correctly
    • align the sipper comb with the two metal guide pins on the front edge of the thermal stage
    • snap the sipper comb into place using the plastic tabs on either side of the sipper comb.
    • when the sipper comb is secure, the system checks the final checkbox
    • ensure that the sipper comb sippers are straight and perpendicular to the reagent plate and touch next

  7. [  ]  template loading
    • type in the template id and follow the screen
    • load the eight-tube strip with templates in the second row of the strip holder (see picture1, C)
    • !!! for GA : lane 1 on left side and lane 8 on the right side, for HS lane 8 on left side and lane 1 on the right side!!!
    • touch the checkbox to indicate that you have loaded templates

    A — 96-Well Reagent Plate; B — Reagent Plate Lever; C — Templates; D — Custom Primers.



  8. [  ]  load primers custom primers:
    • close the cBot lid and touch next to proceed to the pre-run check
    specialty primers:
    • type in the primer name and primer id and follow the screen
    • load the eight-tube strip with primer in the first row of the strip holder (see picture1, D)
    • !!! for GA : lane 1 on left side and lane 8 on the right side, for HS lane 8 on left side and lane 1 on the right side!!!
    • touch the checkbox to indicate that you have loaded primers
    • close lid and touch next, the system automatically performs a pre-run check

  9. [  ]  perform pre-run-check (~3 min) this is a flow check using bubble sensors to detect air in the lines if the pre-run check is successful: the start button appears and the run can begin if the pre-run check fails:
    • check components are correctly loaded, inspect the manifold seeded cover the fc, clap is closed correctly and ensure the outlet end of the manifold is evenly seated in the outlet port and the clamp is closed
    • check row #1 of the reagent plate is fully thawed and contains reagent
    • check that the sippers are straight and that the foil-sealed tubes in row #1 of the reagent plate are pierced
    • touch rerun check to repeat the check and rerun the sensor check

  10. [  ]  run (~4 hours)
    • complete cluster generation run, including steps for amplification, linearization, blocking, and primer hybridization

    1. Protocol Steps

    <Step Name = "Template Hybridization">

    TempRamp (20°C, 0.9°C/sec)
    Pump Reagent=HT1, AspirationRate=60µl/m, Volume=120µl
    TempRamp (96°C, 0.9°C/sec)
    Pump Reagent=Template, AspirationRate=15µl/m, Volume=75µl
    Pump Reagent=Template, AspirationRate=100µl/m Volume=10µl
    Wait Duration=30000ms
    TempRamp (40°C, 0.05°C/sec)
    Pump Reagent=HT2, AspirationRate=15µl/m, Volume=75µl

    <Step Name="Amplification">

    "Extension-Amplification"
    Pump Reagent=APM, AspirationRate=15µl/m, Volume=70µl
    Pump Reagent=HFE, AspirationRate=60µl/m, Volume=95µl
    TempRamp (74°C, 0.9°C/sec)
    Wait Duration=90000ms
    TempRamp (20°C, 0.9°C/sec
    Pump Reagent=HP5, AspirationRate=60µl/m, Volume=75µl
    Pump Reagent=HT2, AspirationRate=60µl/m, Volume=95µl
    TempRamp (60°C, 0.9°C/sec)
    "Amplification 1", Cycles=17
    Pump Reagent=AT1, AspirationRate=30µl/m, Volume=20µl
    Pump Reagent=APM, AspirationRate=30µl/m, Volume=20µl
    Pump Reagent=AMX1, AspirationRate=30µl/m Volume=32µl
    "Amplification 2", Cycles=18
    Pump Reagent=AT1, AspirationRate=30µl/m, Volume=20µl
    Pump Reagent=APM, AspirationRate=30µl/m, Volume=20µl
    Pump Reagent=AMX1, AspirationRate=30µl/m, Volume=32µl
    "Amplification-Wash"
    Pump Reagent=HT2, AspirationRate=30µl/m, Volume=120µl
    TempRamp (20°C, 0.9°C/sec)
    Pump Reagent=HT1, AspirationRate=60µl/m, Volume=95µl

    <Step Name="Linearization PE">

    TempRamp (20°C, 0.9°C/sec)
    Pump Reagent=LMX1, AspirationRate=60µl/m, Volume=95µl
    TempRamp (37.9°C, 0.9°C/sec)
    Wait Duration=1800000ms
    TempRamp (20°C, 0.9°C/sec)
    Pump Reagent=HT2, AspirationRate=60µl/m, Volume=95µl

    <Step Name="Blocking">

    "CombinedBlocking 1"
    TempRamp (38°C, 0.9°C/sec
    Pump Reagent=BMX, AspirationRate=60µl/m, Volume=95µl
    "CombinedBlocking-CyclicPumping", Cycles=9
    Pump Reagent=BMX, AspirationRate=15µl/m, Volume=5µl
    Wait Duration=180000ms
    "CombinedBlocking 2"
    TempRamp (60°C, 0.9°C/sec)
    Pump Reagent=BMX, AspirationRate=60µl/m, Volume=25µl
    Wait Duration=900000ms
    "CombinedBlocking-Wash"
    TempRamp (20°C, 0.9°C/sec
    Pump Reagent=HT2, AspirationRate=60µl/m, Volume=95µl
    Pump Reagent=HT1, AspirationRate=60µl/m, Volume=95µl

    <Step Name="Hybridization">

    "Denaturation-Hybridization"
    TempRamp (20°C, 0.9°C/sec)
    Pump Reagent=HP5, AspirationRate=60µl/m, Volume=95µl
    Pump Reagent=HT2, AspirationRate=60µl/m, Volume=95µl
    Pump Reagent=HP1, AspirationRate=60µl/m, Volume=95l
    TempRamp (60°C, 0.9°C/sec)
    Wait Duration=300000ms
    TempRamp (40°C, 0.9°C/sec)
    Pump Reagent=HT2, AspirationRate=60µl/m, Volume=95µl
    TempRamp (20°C, 0.9°C/sec)
    Pump Reagent=HT1, AspirationRate=60µl/m, Volume=95µl

    <Step Name="Drain Sippers">

    Pump Reagent=Air, AspirationRate=60µl/m, Volume=40µl



  11. [  ]  post-run-check:
    • touch unload and raise the cBot lid
    • check reagent and template uniform delivery
    NB!only when the delivery volume is ok (approx. equal remaining volume in each tube of the strip) start thawing of nucleotides for sequencing(if seq. run start is planned for the same day)
    • check that all reagent tube seals were pierced (see fig. 2)
    • check the eight-tube strips containing primers, if you used custom primers
    • unload the FC:
    " first step release the outlet clamp securing the outlet end of the manifold
    " disconnect the outlet end of the manifold from the outlet port in the wash reservoir
    " release the flow cell clamp
    " remove the sipper comb from the metal guide pins using the plastic tabs on either side of the sipper comb
    " when the sipper comb is removed, the system selects the checkbox indicating that the manifold is removed
    " remove the manifold from the cBot
    " carefully lift the flow cell from the thermal stage
    " pull the reagent plate lever toward you to release the reagent plate
    " remove the reagent plate from the reagent stage and cover the strips of the reagent plate with parafilm and store the plate at 4°C
    " remove the eight-tube strip containing the templates and additional primers
    " empty waste bottle at the cBot
    " the wash button becomes active when all components have been removed
    " touch wash to proceed to the post-run wash


    File:Reagent_tubes.jpg‎

  12. [  ]  perform post-run wash:
    • wash the plate on the thermal stage with mQwater (to remove any salt) and dry with cleaning tissue.
    • fill the wash reservoir with approximately 12 ml deionised water and close the cBot lid
    • touch the checkbox on the screen and then the wash bottom
    • when the wash is complete, dry the wash reservoir (don’t rub the outlet ports)
    • empty and clean waste bottle at the cBot
    • touch the checkbox and then the exit bottom
    • the start screen appears and the cBot is ready for next run

  13. [  ]  registration of cluster generation on cBot:
    • register prepared flowcell in run list of Napoleon database!
    • register used library volumes in the list: ngscore > projects > sample overview > library preparation overview > remaining amplified library
    • register the remaining library in the right project list!
    finish with rehybridization
    for rehybridisation step in case of low intensities in the first base report of the sequencing run
    • after the run a run data report automatically appears :Protocol name, Flow cell ID, Reagent ID,Template name, Start time and finish time



[править] Notes

Do not toggle the Power Switch on the right side panel to the OFF position.
Use the Start Switch on the left of the waste bottle compartment door to start/close the software.


  • Reagent plate scheme:
row 1 — HT1     (Hybridization Buffer)
row 2 — HFE     (1xPhusion Master Mix, Finnzymes Oy)
row 3 — APM1    (AMX1 Premix)
row 4 — AMX1    (Amplification Mix)
row 5 — AT1     (100% Formamide)
row 6 — AMX1    (Amplification Mix)
row 7 — HT2     (Wash Buffer)
row 8 — LS1     (Linearisation Solution)
row 9 — BMX     (Blocking Mix)
row 10 — HP5     (0.1N NaOH)
row 11 — HP6     (Sequencing Primer Mix 1)
row 12 — empty


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