Mar 13, 2022
Rapid Run v2 Primer Rehyb
- 1Whitehead Institute Genome Technology Core
Protocol Citation: A Chilaka, Sumeet Gupta 2022. Rapid Run v2 Primer Rehyb. protocols.io https://dx.doi.org/10.17504/protocols.io.b46nqzde
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 16, 2022
Last Modified: March 13, 2022
Protocol Integer ID: 58286
Abstract
Switch the flowcell to another sequencer if first base fails - HiSeq
Guidelines
If a rapid run has failed at first base and you want to switch the flowcell to another side or sequencer, follow these steps (ONLY applicable to single read runs, changing sequencer for paired end run is not possible)
These steps are to be performed on the HiSeq where the flow cell is being moved.
Safety warnings
ONLY applicable to single read runs, changing sequencer for paired end run is not possible
Rapid Run v2 Primer Rehyb
Rapid Run v2 Primer Rehyb
2h 16m 30s
2h 16m 30s
Wash the sequencer that will be used (MWB and then water). [Link to wash protocol]
1m
Load the SBS rack (with reagents) to the sequencer.
1m
Load water in the Paired End rack and the template tubes.
1m
Place a dummy flowcell on the deck. Engage vacuum.
30s
Setup the run as normally you would, but select “cluster generation on cbot.” even if it was onboard clustering. This will save an additional hour of time.
1m
Enter the other parameters used for the flowcell, such as read lengths, flowcell ID, etc...This will create the files needed to allow you to do a rehyb later.
1m
Start the run and wait for the first base report to come up. End the run.
1h 30m
Thaw a Rehybridization kit and replace or top-off the reagents according to the “HiSeq Rapid Primer Rehybridization Reference Guide.” A general summary is below.
30m
For Single-Read Runs, replace/top-off the following reagents with these from the Rehyb kit:
| Position | Reagent | Description | |
| 5 | USB | Add to current USB bottle | |
| 18 | HP10 | Replaces Read1 primer | |
| 17 | HP12 | Replaces Index1 primer (i7) | |
| 16 | HP9 | Replaces Index2 primer (i5) |
5m
Leave the dummy flowcell on the sequencer. You will prime following reagent twice:
- FDR (position 15) - aspiration rate of 1500, dispense rate of 2000, and using 250uL
- Flow USB (5) - aspiration rate of 1500, dispense rate of 2000, and using 250uL
5m
Return to the Main screen and select “Sequence,” then “Rehyb Run.”
Replace the dummy flowcell with sequencing flowcell and proceed with rehybridization.
1m