04/22/21
Rehybridization of primers to a flow cell allows the current sequencing read to be repeated from the first base of that read. Primer rehybridization might be required to resolve various issues, including fluidic issues that affect intensity for one or more lanes on a flow cell or when incorrect run parameters are entered during run setup.
Priming of the reagent lines might be necessary before rehybridization. The following tables provide requirements for priming before rehybridization and list the paired-end rack reagents required for the rehybridization.
Make sure that the remaining SBS reagents are sufficient to complete the sequencing run. Depending on how many cycles of the read were completed before the run was stopped, SBS reagents might need replenishing.
In addition to the SBS reagents, the rehybridization process requires additional primers and reagents, depending on the type of rehybridization performed. Rehybridization Kit details:
If a wash was not performed before rehybridization:
| Reagent Version | Read 1 | Index 1 (i7) |
Index 2 (i5) SR flow cell only |
Read 2 PE flow cell |
|
|---|---|---|---|---|---|
| TruSeq v3 | Required Reagents |
TruSeq v2 cBot Multi- Primer Re-Hybridization Kit (on cBot) |
HP8/HP12 - position 17* HP3 (diluted) - position 18 HT2 - position 19 |
HP9 - position 16 HP3 (diluted) - position 18 HT2 - position 19 |
HP7/HP11 - position 16* HP3 (diluted) - position 18 HT2 - position 19 |
| Required Priming | If HiSeq has been idle overnight, consider priming SBS | ||||
| HiSeq v4 | Required Reagents |
FDR - position 15 HP10 - position 18 |
FDR - position 15 HP12 - position 17 |
FDR - position 15 HP9 - position 16 |
FDR - position 15 HP11 - position 16 |
| Required Priming | If HiSeq has been idle overnight, consider priming SBS | ||||
| HiSeq Rapid v2 | Required Reagents |
FDR - position 15 HP10 - position 18 |
FDR - position 15 HP12 - position 17 |
FDR - position 15 HP9 - position 16 |
FDR - position 15 HP11 - position 16 |
| Required Priming | If HiSeq has been idle overnight, select prime for SBS | ||||
If a wash was performed before rehybridization:
| Reagent Version | Read 1 | Index 1 (i7) |
Index 2 (i5) SR flow cell only |
Read 2 PE flow cell |
|
|---|---|---|---|---|---|
| TruSeq v3 | Required Reagents |
TruSeq v2 cBot Multi- Primer Re-Hybridization Kit (on cBot) |
HP8/HP12 - position 17* HP3 (diluted) - position 18 HT2 - position 19 |
HP9 - position 16 HP3 (diluted) - position 18 HT2 - position 19 |
HP7/HP11 - position 16* HP3 (diluted) - position 18 HT2 - position 19 |
| Required Priming | Select Prime for SBS | Select Prime for SBS; Manually prime positions 18 and 19 with 250μL | |||
| HiSeq v4 | Required Reagents |
FDR - position 15 HP10 - position 18 |
FDR - position 15 HP12 - position 17 |
FDR - position 15 HP9 - position 16 |
FDR - position 15 HP11 - position 16 |
| Required Priming | Select Prime for SBS; Manually prime positions 15 with 250μL | ||||
| HiSeq Rapid v2 | Required Reagents |
FDR - position 15 HP10 - position 18 |
FDR - position 15 HP12 - position 17 |
FDR - position 15 HP9 - position 16 |
FDR - position 15 HP11 - position 16 |
| Required Priming | Manually prime position 15 (250μL) and all SBS positions except PW1 (2x250μL each) | ||||
*HP8 and HP7 are not compatible with Nextera libraries. HP12 and HP11 are compatible with all library types.
For more detailed information on performing the various types of rehybridization runs, see:
For more information on rehybridization runs using the HiSeq X, refer to the Read 1 Primer Rehybridization on a HiSeq X Flow Cell guide.
For information on rehybridization runs using the HiSeq 3000/4000, refer to the Read 1 Primer Rehybridization on a HiSeq 3000/4000 Flow Cell guide.