HS read 1 run

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[править] Preliminary

required kits: TruSeqSBS Kit-HS (200 cycles) (Catalog # FC-401-1001); enough for 209 cycles or TruSeq SBS Kit-HS (50 Cycles) (catalog # FC-401-1002); enough for 58 cycles

long time storage: -80°C (lasting for 12 month from manufacturing date)

TruSeq Cluster Generation Kit contains an accessories kit: which includes eight single-use reagent funnel caps used on 250 ml SBS reagent bottles, and manifold gaskets used between the manifold and the flow cell on the HiSeq


TruSeqSBS Kit-HS (200 cycles)contains:
Box 1: Store at 2° to 8°C 1 x250 ml bottle PW1 Wash Buffer
1 x250 ml bottle ICR-200 cycles Incorporation Reagent
1 x250 ml bottle SB1-200 cycles High Salt Buffer
2 x250 ml bottle SB2-200 cycles Incorporation Buffer
1 x250 ml bottle SB3-200 cycles Cleavage Buffer
Box 2: Store at -15° to -25°C
1 x250 ml bottle CMR-200 cycles Cleavage Mix Reagent
1 x250 ml bottle SMR-200 cycles Scanning Mix Reagent
1 x5 ml tube LRP-200 cycles Long-Read DNA Polymerase
4 x5 ml tube LFN36 Long Read Nucleotide


  • at least one day before starting a new run transfer Box 1 to (4°C) and Box 2 to -20°C
  • check the disk space (should be ### on drive D and E) The HiSeq instrument computer has a storage capacity of 1.8 TB per flow cell, enough space to hold a 209 cycle run if images are not saved to disk. Data from flow cell A is stored on the D: drive, while data from flow cell B is stored on the E: drive
  • wash the HS and the table with a wet paper towel
  • perform the HS pre-run wash;
  • prepare FC with primer hybridization



[править] Thaw reagents

  • thaw reagents: place PW1, ICR, SB1, SB2, SB3, SMR in water bath filled with room temperature water or thaw over night at 4°C
  • LFN36 (nucleotides) thaw on ice when you are sure to start the HS
  • LRP 200 (Long Read DNA Polymerase) still keep on -20°C until using for mixing ICR
  • after complete thawing invert each bottle several times and inspect the reagents for ice crystals
  • store all components on ice ( except LRP 200-polymerase)
  • use the following components directly from 4°C storage:
• ICR (Incorporation Mix Reagent)
• SB1 (High Salt Buffer)
• SB2 (Incorporation Buffer)
• SB3 (Cleavage Buffer)
  • put CMR (cleavage mix!) in a seperate water bath for thawing! After every contact with CMR bottle change gloves!



[править] Preparing reagents

  • thawed reagents in 250ml conical bottles are ready for use with the exception of ICR (Incorporation mix)
  • ICR requires mixing prior to each read (activity of the polymerase!)
  • loading of other sequencing reagents is sufficient for Read 1 + Read 2
  • Preparing of ICR PE51-run:
  • split the volume of SB1, SB2, SB3, PW1, CMR, SMR into two equal portions
  • return the second half to 4°C storage: SB1, SB2, SB3, PW1
  • or to -20°C storage: CMR, SMR
  • split the volume of ICR-200 into two equal portions (47ml)
  • return the second bottle of ICR to 4°C
  • split the volume of the first half of ICR-200 into two equal portions (23,5ml), one bottle for Read 1 and the other bottle for Read 2
  • thaw one tube of LFN36 (storage -20°C)
  • add the content of one LFN36 tube to 23,5ml of ICR
  • rinse the tube of LFN with ICR to ensure that all LFN is transferred
  • add 550µl of LRP200 (storage -20°C) to the ICR/LFN36 mix
  • return the unused portion of LRP-200 to -20°C storage
  • Cap the bottle and invert ICR mix several times to mix and set on ice
  • Preparing of ICR PE101-run:
  • split the volume of ICR-200 into two equal portions (47ml), one bottle for Read 1 and the other bottle for Read 2
  • return the second bottle of ICR to 4°C storage until start of Read2
  • thaw two tubes of LFN36 (storage -20°C)
  • add the content of the two LFN36 tubes to the first half of ICR
  • rinse each tube of LFN with ICR to ensure that all LFN is transferred
  • add 1,1ml of LRP200 (storage -20°C) to the ICR/LFN36 mix
  • Cap the bottle and invert ICR mix several times to mix and set on ice
  • return the unused portion of LRP-200 to -20°C storage


» empty 250ml conical tube for ICR is in the Box HiSeq accessory:

Illumina recommends using the "spare" bottle of ICR first and storing the remaining ICR in the bottle with the original label

» record lot numbers and weight of each reagent on the lab tracking form: LabTrackingForm_Sequencing_HiSeq-PE51.pdf
» label the 250ml conical bottles with the sequencing reagents according their position in the HiSeq rack

Reagents for PE 51 run
Reagentbottle 1 bottle 2
1 ICR47ml 47ml
3 SMR107ml107ml
4 SB1100ml 100ml
5 SB262ml 62ml
6 SB262ml 62ml
7 CMR107ml 107ml
8 SB3110ml 110ml



[править] Starting the Run

this step includes: loading and priming reagents, loading the flow cell with cluster, and perform a fluidics check

[править] Enter Run Parameters

  1. [  ]  from the start screen, select sequence
  2. [  ]  tipe in the following parameters for the run:
    » a. scan or enter the flow cell ID
    » b. enter your experiment and user name
    » c. select the flow cell type from the list
    » d. select none for control
    » e. choose drive D (Fc A) and drive E (Fc B) for your run folder and enter the name.
  3. [  ]  select advanced for additional settings:
    » a. select first base report
    » b. select saving thumbnail images
    » c. select the use of existing recipe or allow the software to create your recipe based on information you provide on the run setup screens and press next
  4. [  ]  enter the following recipe parameters:
    » a. enter the number of cycles for read 1, 0 for index read and number of cycles for read 2
    » b. select the SBS chemistry from the dropdown list
    » c. for PE run, select the chemistry from the dropdown list and press next
  5. [  ]  enter the following reagent parameters:
    » a. scan or enter the reagent kit IDs
    » b. select the reagent kit type from the dropdown list
    » c. specify 101 cycles or less. The software will ask to load fresh ICR and PE reagents after the last cycle of read 1 completes
  6. [  ]  select next
  7. [  ]  review the run information; for correct informations select next. If not, select back.



[править] Loading of Sequencing Reagents

  1. [  ]  record the weight on the lab tracking form.
  2. [  ]  open the reagent door and remove the reagent rack
  3. [  ]  replace the cap from ICR, SMR, SB1, SB2, and SB3 with a funnel cap. (funnel caps are provided in the HiSeq Accessories Kit, part of the HiSeq Cluster Generation Kit)
  4. [  ]  remove the cap from the CMR bottle and replace it with a funnel cap and change gloves
  5. [  ]  place the reagent bottles onto the instrument and make sure that the rack is placed centered
  6. [  ]  close the door and press next



[править] Clean the Flow Cell Holder

  1. [  ]  open the flow cell compartment door
  2. [  ]  make sure that the flow cell lever is in the OFF position.
  3. [  ]  put on gloves and wipe with tissue paper with ethanol carefully the surface of the flow cell holder NB! use only lens cleaning tissue; fold it to use the clean, untouched part each time remove the old FC



[править] Load Used Flow Cell

  1. [  ]  clean used flow cell with alcohol and cleaning tissue and place it in the flow cell holder (barcode on the right)
  2. [  ]  move the flow cell lever slowly to position 1. to engages the vacuum first the flow cell lever turns yellow, and then quickly blinks green. (green: the vacuum is engaged. in case of yellow: the vacuum is not secure, repeat the cleaning steps and reload the flow cell)
  3. [  ]  wait for about five seconds, and then move the flow cell lever slowly to position 2 (far-right). This raises the manifolds, allowing the gaskets to make contact with the flow cell. When the flow cell lever is solid green, the flow cell is ready for use
  4. [  ]  follow the screen and choose next.



[править] Confirm Proper Flow

  1. [  ]  select solution 5 (SB2) from the dropdown list
  2. [  ]  enter the following default values: • Volume: 250 • Aspiration Rate: 250 • Dispense Rate: 2,000 and select pump
  3. [  ]  check the flow cell for bubbles passing through the lanes (in case of bubbles check or change the gaskets) and leaks near the manifolds
  4. [  ]  In case of bubbles reduce the aspiration rate to 100, and pump 250 μl of water to the flow cell or contact Illumina Technical Support



[править] Prepare for Priming

  1. [  ]  collect the waste of priming (place each waste tubing into a empty 15 ml tube, one line per tube)
  2. [  ]  select next. The priming screen opens and the priming step starts automatically
  3. [  ]  meassure the collected priming waste and confirm that the volume is 1.75 ml from each lane. Record the results on the lab tracking form. If the measured volume differs from the expected volume by more than 10%, repeat the priming step
  4. [  ]  return the waste tubing to the waste container before proceeding and select next



[править] Remove the used Flow Cell

  1. [  ]  open the flow cell compartment door and slowly move the flow cell lever to position 1 to disengage the manifolds
  2. [  ]  slowly move the flow cell lever to the OFF position to disengage the vacuum seal
  3. [  ]  lift the used flow cell and discard the used gaskets



[править] Clean the Flow Cell Holder

  1. [  ]  put on gloves and wipe with tissue paper with ethanol carefully the surface of the flow cell holder NB! use only lens cleaning tissue; fold it to use the clean, untouched part each time
  2. [  ]  let the surface air dry and positionate two new gaskets in the slots on the front end and back end of the flow cell holder



[править] Clean the Flow Cell

  1. [  ]  hold the edges of the flow cell with two fingers. Ensure the inlet and outlet ports are facing up
  2. [  ]  fold lens cleaning tissue to use the clean, untouched part each time. Repeat until the flow cell is completely clean and dry the flow cell using a new, dry lens cleaning tissue



[править] Load the new Flow Cell

  1. [  ]  clean the new flow cell with alcohol and cleaning tissue and place it in the flow cell holder (barcode on the right)
  2. [  ]  move the flow cell lever slowly to position 1. to engages the vacuum first the flow cell lever turns yellow, and then quickly blinks green. (green: the vacuum is engaged. in case of yellow: the vacuum is not secure, repeat the cleaning steps and reload the flow cell)
  3. [  ]  wait for about five seconds, and then move the flow cell lever slowly to position 2 (far-right). This raises the manifolds, allowing the gaskets to make contact with the flow cell. When the flow cell lever is solid green, the flow cell is ready for use.
  4. [  ]  follow the screen and choose next



[править] Check for proper flow with new Flow Cell using the fluidics check screen

  1. [  ]  select solution 5 (SB2) from the dropdown list.
  2. [  ]  enter the following default values: • Volume: 250 • Aspiration Rate: 250 • Dispense Rate: 2,000 and select pump
  3. [  ]  check the flow cell for bubbles passing through the lanes (in case of bubbles check or change the gaskets) and leaks near the manifolds
  4. [  ]  In case of bubbles reduce the aspiration rate to 100, and pump 250 μl of water to the flow cell or contact Illumina Technical Support
  5. [  ]  select next and follow the screen. Ensure the flow cell lever is green (vacuum engaged) and close the flow cell door
  6. [  ]  select start to start the sequencing run.



[править] First Base Incorporation

The first base report appears automatically after the first cycle is complete. If the results are satisfactory, select continue to continue your run.

The following exmple is a typical first base report, followed by the RTA summary for the same run after template generation . These examples are only guidelines and should not be used as an exact conversion

First Base Report

Top Surface
MetricLane 1Lane 2Lane 3Lane 4Lane 5Lane 6Lane 7Lane 8
Cluster Density (k/mm²)1125.122361.432406.872435.812546.442239.082268.212251.75
A Intensity24845.7525534.5625551.62256912611926657.4426555.3826499.06
C Intensity26842.6926434.9426498.526553.7527190.6227601.6927478.9427418.75
G Intensity12918.691231211908.6911610.5614641.1915180.5914736.7814497.16
T Intensity25775.8126291.882601526151.4427523.7529577.1529252.3129271.62
A Focus Store58.3563.6264.916463.7761.7262.3162.25
C Focus Store57.8163.1464.263.3863.2861.3361.7761.72
G Focus Store60.9165.4766.6665.6765.3463.3863.9863.92
T Focus Store58.4963.3664.5863.663.2961.3862.0161.92


RTA Summary

Read #1
LaneTilesClu. Dens. (#/mm²)% PF ClustersClusters PF (#/mm²)% Phas./Preph.1st
132128K +/- 9.2K0.0 +/- 0.00.0K +/- .00K0.306 / 0.2796551 +/- 167.0
232445K +/- 55.5K0.0 +/- 0.00.0K +/- .00K0.284 / 0.2276162 +/- 158.4
332462K +/- 43.0K0.0 +/- 0.00.0K +/- .00K0.281 / 0.2516090 +/- 162.1
432477K +/- 58.2K0.0 +/- 0.00.0K +/- .00K0.280 / 0.2146139 +/- 154.2
532472K +/- 58.6K0.0 +/- 0.00.0K +/- .00K0.288 / 0.2166233 +/- 169.6
632388K +/- 51.3K0.0 +/- 0.00.0K +/- .00K0.291 / 0.2246516 +/- 112.2
732404K +/- 50.4K0.0 +/- 0.00.0K +/- .00K0.286 / 0.2286429 +/- 143.5
832404K +/- 50.2K0.0 +/- 0.00.0K +/- .00K0.288 / 0.2326442 +/- 119.1


As a guideline of what you can expect, the following table lists expected ranges for each of the parameters in the first base report. This is not a guideline to terminating your run. (HiSeq™2000 User Guide, January 2011)

ParameterExpected Range
Cluster Density (K/mm²)1,700-3,000
A Intensity15,000-30,000
C Intensity20,000-40,000
G Intensity 5,000-15,000
T Intensity15,000-30,000
A Focus Score55-80
C Focus Score55-80
G Focus Score55-80
T Focus Score55-80



[править] Notes

  • contact clean FC only with clean (!) gloves or with a lens cleaning tissue. Do not rub surfaces, clean by blotting or one-way movements
  • fold lens cleaning tissue accurately and use new clean area for every wipe;
  • channels of the FC should not be dried out: contact holes with lens cleaning tissue VERY ACCURATELY (especially if FC is oriented vertically). If some channel will dry out, put a drop of High Salt Buffer on the hole with a 200µl pipette — channel will be filled by capillary force;


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