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Sequencing run

Starting the run Changing plates during the run Sequencing process Notes  Comments

NB! post-PCR operation. This protocol is for FRAGMENT library / M-P libraryM-P library / FRAGMENT library

Starting the run

1. check, that that there are enough HD-space:
     •  images: 1.34TB per cell;
     •  results: 0.2-0.8TB per cell;
2. empty waste canister;
3. prepare 1x Instrument buffer, store at 4°C for 10 days

   density 1L H2O 1g/ml 700ml 10x Instrument buffer 1.014g/ml 100ml / 101.4g / Glycerol 1.262g/ml 200ml / 252.4g /

4. check, that all buffers are installed (table below);



5. create run:
   • click "Create Run …";
   • select "Sequencing Run" & Next;
   • (optional) change name, insert comments & Next;
   • select position of each sample (orientation as for closed FlowCell) & Next;
   • select reference genome & Next;
   • check all parameters (reference human: 1000genomes) & Finish;
6. (optional) repeat procedure for second FlowCell;



7. install FlowCell(s);
8. set Focal range;
9. click "Start Run";

Changing plates during the run

• set up 4°C on centrifuge, start rotation to accelerate cooling;
• look through the previous results:
  NB! Restart the computer, if results of analysis does not shown: (i) close ICS and other programs; (ii) restart (do not turn off!); (iii) start ICS; (iv) check, that "fluidic" & "imaging" is online.
  • no Errors (red) messages in Run log;
  • "Cycle scan":
      •  >50% of "good & best beads";
      •  <2% of bad panels;
      •  color distribution is balanced;
      •  satays looks acceptable;
  • (at least once) write the number of beads, estimate the data output;



• prepare enzyme and buffer plates:
  • melt buffer plates if necessary;
  • spin down at 4°C, 2000 rpm for 1min (hold enzyme plate in cold);
  • write the flowcell numbers on plates;
• change plates in the instrument:
  • unlock doors;
  • change plates;
  • check rest volumes in old plates;



• change buffers according to the volume-list:
  • put paper towel on the top of the storage buffer / instrument buffer;
  • keep attention during unscrew bottle not to snap end of tube;

  Day Primer 1X Instrument Storage Reset Cleave 1 Cleave 2 1 Focal, P1 372/744 64/127 24/48 18/36 18/36 2 P2 346/693 59/117 12/24 18/36 18/36 3 P3 371/742 66/132 12/24 18/36 18/36 4 P4 371/742 66/132 12/24 18/36 18/36 5 P5 371/742 69/138 12/24 18/36 18/36 Total: 1.98/3.7L 323/647 72/144 90/180 90/180 buffer life [days] 10 10 5 5 5 bottle voluime 1L 1L 150ml 72ml 72ml handling first cycles: full; the last cycle: 132mm pour out till 95mm pour out till 58mm for 1-3: 69mm; for 4-5: 49mm for 1-3: 77mm; for 4-5: 52mm rest 13mm 13mm 8mm 8mm 8mm

  Day Primer 1X Instrument Storage Reset Cleave 1 Cleave 2 1 Focal, P1 271/543 48/97 24/48 12/24 12/24 2 P2 246/491 43/87 12/24 12/24 12/24 3 P3 270/540 51/102 12/24 12/24 12/24 4 P4 270/540 51/102 12/24 12/24 12/24 5 P5 270/540 54/108 12/24 12/24 12/24 Total: 1.33/2.66L 247/495 72/144 60/120 60/120 buffer life [days] 10 10 5 5 5 bottle voluime 1L 1L 150ml 72ml 72ml handling ??? ??? ??? ??? ??? rest 13mm 13mm 8mm 8mm 8mm




• start ICS: flowcell block should move diagonally [/] in the upper-right position. If flowcell block does not move, or move vertically or horizontally it is a program mistake: open doors (in program and in reality), close program, and restart it it again.
• restart run

Sequencing process

known sequence unknown sequence 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35               Prim A # # 1 # # 2 # # 3 # # 4 # # 5 # # 6 # # 7             Prim B # # 1 # # 2 # # 3 # # 4 # # 5 # # 6 # # 7             Prim C   # # 1 # # 2 # # 3 # # 4 # # 5 # # 6 # # 7   Prim D   # # 1 # # 2 # # 3 # # 4 # # 5 # # 6 # # 7   Prim E   # # 1 # # 2 # # 3 # # 4 # # 5 # # 6 # # 7

known sequence unknown sequence 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25               Prim A # # 1 # # 2 # # 3 # # 4 # # 5             Prim B # # 1 # # 2 # # 3 # # 4 # # 5             Prim C   # # 1 # # 2 # # 3 # # 4 # # 5   Prim D   # # 1 # # 2 # # 3 # # 4 # # 5   Prim E   # # 1 # # 2 # # 3 # # 4 # # 5





known sequence unknown sequence 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25               Prim A1/A2 # # 1 # # 2 # # 3 # # 4 # # 5             Prim B1/B2 # # 1 # # 2 # # 3 # # 4 # # 5             Prim C1/C2   # # 1 # # 2 # # 3 # # 4 # # 5   Prim D1/D2   # # 1 # # 2 # # 3 # # 4 # # 5   Prim E1/E2   # # 1 # # 2 # # 3 # # 4 # # 5




Primer time A / 1 (1,2) (6,7) (11,12) (16,17) (21,22) (26,27) (31,32) B / 2 (0,1) (5,6) (10,11) (15,16) (20,21) (25,26) (30,31) C / 3 (4,5) (9,10) (14,15) (19,20) (24,25) (29,30) (34,35) D / 4 (3,4) (8,9) (13,14) (18,19) (23,24) (28,29) (33,34) E / 5 (2,3) (7,8) (12,13) (17,18) (22,23) (27,28) (32,33)

Notes

• starting (restarting) run with two FlowCells should be done with at least 2 min interval between FC's;



• workflow:

  stage time enzyme & buffer plates in advance prepare dep. chambers in advance bead deposition ~3h set-up run ~10min buffers & waste ~15min install slide ~20min run ~6~10 days analysis ???