Spiking custom primers into the Illumina sequencing primers

07/28/21


Libraries prepared with custom sequencing primers require optimization to perform successfully on Illumina sequencing platforms. Combining (or “spiking”) custom primers into the Illumina primers is necessary when including PhiX or other standard Illumina libraries with the custom sample. Illumina cannot guarantee the performance or compatibility of custom primers, and it is up to the customer to validate primers for sequencing on a specific platform. When using custom sequencing primers, it is critical to know the recommended platform-dependent loading concentration, total volume, loading positions, and primer annealing temperatures. This bulletin provides information for spiking custom primers into the Illumina primers.

iSeq™ 100 | MiniSeq™ | MiSeq™ | NextSeq™ 500/550 | NextSeq 1000/2000 | HiSeq™ 1000/1500/2000/2500 | HiSeq 3000/4000 | NovaSeq™ 6000

 

Spiking custom primers into Illumina sequencing primers

  • The cartridge position, total volumes, and final concentration of custom primers for each platform are provided in the tables below.
  • Calculate the volume of the custom primer to add to the Illumina primer cartridge position based on the final concentration of the custom primer in the cartridge.*
  • After spiking in the custom primer, adjust with an appropriate pipette to half of the final volume, and at the cartridge position gently pipette up and down five times to mix thoroughly.**
  • For MiSeq, MiniSeq, NextSeq™ 500/550 and NovaSeq™ 6000 platforms, do not check the “custom primer” box position in the sample sheet or during the run setup.
    *To calculate how much custom primer to spike into the well, use the (C1)(V1) = (C2)(V2) equation where:
    C1 = the concentration of your custom primer (when using high concentration primer stock, such as 100uM, the small additional volume to the final volume is negligible).
    V1 = solve for the volume of the custom primer to be spiked in
    C2 = the recommended custom primer final concentration from the chart below
    V2 = total volume of Illumina primer in the charts below
     
    Example: (100uM * V1) = (0.5uM * 680uL)
                                     V1 = 3.4 µl
     
    **MiSeq, NextSeq and MiniSeq cartridges: to increase successful measurement of the Illumina sequencing primers, the addition and mixing of custom primers, it is recommended to use slimmer tips or larger volumes of lower stock concentration custom primers.

Important Note: The guidelines below are based on the current primer volumes. To ensure accuracy, measure total primer volumes in the cartridge with a pipette before proceeding with setup.

 

iSeq 100
Due to the construction of the iSeq 100 cartridge, it is not possible to load and use custom primers.

 

MiniSeq
Because of the unique dual-indexing workflow on the MiniSeq, combinations of custom primers and Illumina primers are limited. Be aware of the custom primer dependencies outlined here.

Kit version Illumina Primer (name) Cartridge
Position
Total
Volume (µL)
Custom primer final
concentration (µM)
Rapid Run
100 Cycles
Read 1 (BP10) 24 590 0.3
Read 2 (BP11) 25 530 0.3
Index 1 (i7) and Index 2 (i5) (BP14) 28 740 0.3
High Output
75 Cycles
Read 1 (BP10) 24 550 0.3
Read 2 (BP11) 25 610 0.3
Index 1 (i7) and Index 2 (i5) (BP14) 28 820 0.3
High Output
150 Cycles
Read 1 (BP10) 24 550 0.3
Read 2 (BP11) 25 610 0.3
Index 1 (i7) and Index 2 (i5) (BP14) 28 857 0.3
High Output
300 Cycles
Read 1 (BP10) 24 550 0.3
Read 2 (BP11) 25 610 0.3
Index 1 (i7) and Index 2 (i5) (BP14) 28 820 0.3
Mid Output
300 Cycles
Read 1 (BP10) 24 550 0.3
Read 2 (BP11) 25 610 0.3
Index 1 (i7) and Index 2 (i5) (BP14) 28 820 0.3
 

MiSeq

Kit version Illumina Primer (name) Cartridge
Position
Total
Volume (µL)
Custom primer final
concentration (µM)
v2 and v3 Read 1 (HP10) 12 680 0.5
Index 1 (i7) (HP12)* 13 680 0.5
Read 2 (HP11) 14 680 0.5

* There is no option for a custom Index 2 (i5) primer since the template uses the grafted P5 primer on the surface of the flow cell.

 

NextSeq 500/550
Because of the unique dual-indexing workflow on the NextSeq 500/550, combinations of custom primers and Illumina primers are limited. Be aware of the custom primer dependencies outlined here.

Kit version Illumina Primer (name) Cartridge
Position
Total
Volume (mL)
Custom primer final
concentration (µM)
High Output
75 cycles
Read 1 (BP10) 20 1.72 0.3
Read 2 (BP11) 21 1.98 0.3
Index 1 (i7) and Index 2 (i5) (BP14) 22 2.83 0.3
High Output
150 Cycles
Read 1 (BP10) 20 1.73 0.3
Read 2 (BP11) 21 1.98 0.3
Index 1 (i7) and Index 2 (i5) (BP14) 22 2.83 0.3
High Output
300 Cycles
Read 1 (BP10) 20 1.73 0.3
Read 2 (BP11) 21 1.98 0.3
Index 1 (i7) and Index 2 (i5) (BP14) 22 2.83 0.3
Mid Output
150 Cycles
Read 1 (BP10) 20 1.34 0.3
Read 2 (BP11) 21 1.51 0.3
Index 1 (i7) and Index 2 (i5) (BP14) 22 2.09 0.3
Mid Output
300 Cycles
Read 1 (BP10) 20 1.33 0.3
Read 2 (BP11) 21 1.52 0.3
Index 1 (i7) and Index 2 (i5) (BP14) 22 2.09 0.3
 

NextSeq 1000/2000

Due to the construction of the NextSeq 1000/2000 cartridge, it is not possible to load custom primers into the Illumina primers.

If Illumina primers are needed to sequence PhiX during a run, a NextSeq 1000/2000 Custom Primer kit is required. Refer to the NextSeq 1000/2000 Custom Primers Guide for instructions.

 

HiSeq 1000/1500/2000/2500

Kit version Illumina Primer (name) Position Total
Volume (mL)
Custom primer final
concentration (µM)
High Output v4 Read 1 (HP10) cBot plate - Row 2 0.3 per tube 0.5
Index 1 (i7) (HP12) PE rack - 17 2.5 0.5
Index 2 (i5) (HP9)
on a single read run*
PE rack - 16 2.5 0.5
Read 2 (HP11) PE rack - 16 2.5 0.5
High Output v3 Read 1 (HP6) cBot plate - Row 11 0.3 per tube 0.5
Index 1 (i7) (HP8 or HP12) PE rack - 17 3.15 0.5
Index 2 (i5) (HP9)
on a single read run*
PE rack - 16 3.15 0.5
Read 2 (HP7 or HP11) PE rack - 16 3.15 0.5
Rapid Run v2 Read 1 (HP10) PE rack - 18 1.93 0.5
Index 1 (i7) (HP12) PE rack - 17 1.93 0.5
Index 2 (i5) (HP9)
on a single read run*
PE rack - 16 1.93 0.5
Read 2 (HP11) PE rack - 16 1.93 0.5

* There is no option for a custom Index 2 (i5) primer since the template uses the grafted P5 primer on the surface of the flow cell.

 

HiSeq 3000/4000

Kit version Illumina Primer (name) Position Total
Volume (mL)
Custom primer final
concentration (µM)
High Output Read 1 (HP10) cBot plate - Row 11 0.39 per tube 0.5
Index 1 (i7) and Index 2 (i5)
(HP14)
PE rack - 17 4.31 0.5
Read 2 (HP11) PE rack - 16 2.64 0.5
 

NovaSeq
Because of the primer design and dual-indexing workflow on the NovaSeq, combinations of custom primers and Illumina primers are limited. Be aware of the custom primer dependencies outlined here.

NovaSeq v1.0 Consumables

Kit version Illumina Primer (name) Cartridge
Position
Total
Volume (mL)
Custom primer final
concentration (µM)
SP
100/200/300/500 cycles
Read 1 (BP10) 24 4 0.3
Index 1 (i7) (BP14)* 23 5 0.3
Read 2 (BP11) 13 2 0.3
S1 and S2
100/200/300 cycles
Read 1 (BP10) 24 4 0.3
Index 1 (i7) (BP14)* 23 5 0.3
Read 2 (BP11) 13 2 0.3
S4
200/300 cycles
Read 1 (BP10) 24 7.3 0.3
Index 1 (i7) (BP14)* 23 5 0.3
Read 2 (BP11) 13 3.5 0.3

* There is no option for a custom Index 2 (i5) primer since the template uses the grafted P5 primer on the surface of the flow cell.

NovaSeq v1.5 Consumables

Kit version Illumina Primer (name) Cartridge
Position
Total
Volume (mL)
Custom primer final
concentration (µM)
SP
100/200/300/500 cycles
Read 1 (VP10) 24 4 0.3
Index 1 (i7) and Index 2 (i5) (VP14) 23 5 0.3
Read 2 (VP11) 13 2 0.3
S1 and S2
100/200/300 cycles
Read 1 (VP10) 24 4 0.3
Index 1 (i7) and Index 2 (i5) (VP14) 23 5 0.3
Read 2 (VP11) 13 2 0.3
S4
200/300 cycles
Read 1 (VP10) 24 7.3 0.3
Index 1 (i7) and Index 2 (i5) (VP14) 23 5 0.3
Read 2 (VP11) 13 3.5 0.3

 

Using Custom Primers in Place of Illumina Primers

If only the custom primer is needed, custom primers can be used in place of the Illumina primers. For instructions on how to set up a run using only custom primers, see the following guides:

For more information on the indexing strategies for each instrument, refer to the Indexed Sequencing Overview Guide.