Custom Primer Requirements for the Illumina® DNA PCR-Free Prep, Tagmentation Kit

11/12/20


Illumina DNA PCR-Free Prep, Tagmentation libraries require custom sequencing primers for successful sequencing on Illumina platforms because these libraries use novel chemistry. Failure to use the specified custom sequencing primers for these libraries results in sequencing failure on Illumina instruments. This bulletin reviews the custom sequencing primer kits contents, custom primer loading volume and position requirements per instrument, and how to set up runs using custom primers.

The custom sequencing primers, sold separately, are provided at final concentration and require no further dilution. They are loaded into the dedicated custom primer wells, rather than spiked into the Illumina primer wells. For full instructions, refer to the Custom Primers Guide for the respective instrument, listed in the “Resources” section of this bulletin.

These custom sequencing primers are compatible with Illumina DNA PCR-Free libraries, PhiX control library (Cat. No. FC-110-3001), and all currently supported/compatible Illumina libraries.

The required custom sequencing primers are available in two kit configurations, and are sold separately from the Illumina DNA PCR-Free library preparation kit:

  • Illumina® DNA PCR-Free Prep Sequencing Primers Read 1 (Cat. No. 20041796)
    • Contains VP10 Custom Read 1 Primer (4 tubes X 7.5 ml)
    • For use on the NovaSeq™ 6000 (SBS v1.0 only), MiSeq™, and HiSeq™ 1000/1500/2000/2500
  • Illumina® DNA PCR-Free Prep Sequencing Primers Read 1 + Index 2 (Cat. No. 20041797)
    • Contains VP10 Custom Read 1 Primer (2 tubes X 7.5 ml) and VP14 Custom Index 2 Primer (2 tubes X 10.5 ml)
    • For use on MiniSeq™, NextSeq™ 500/550, NextSeq 1000/2000 and HiSeq 3000/4000

Custom primer loading volume and position requirements per instrument:

    Read 1 Primer Index 2 Primer
Instrument Custom Primer
kit to purchase
VP10 volume
to load
Cartridge
position
VP14 volume
to load
Cartridge
position
iSeq™ 100 Not compatiblea
MiniSeq 20041797 1 ml #15 1 ml #13
MiSeq 20041796 600 µl #18 n/a
NextSeq 500/550 20041797 2 ml #7 2 ml #9
NextSeq 1000/2000b 20041797 600 µl #1 600 µl #2
HiSeq 1000/1500/
2000/2500
(PE flow cells)
Rapid Run
v2
20041796 2 ml #18 n/a
High Output
v3
20041796 150 µl / tube
(8-strip tube)
cBot primer
row 11
n/a
High Output
v4
20041796 150 µl / tube
(8-strip tube)
cBot primer
row 2
n/a
HiSeq 3000/4000
(PE flow cells)
20041797 150 µl / tube
(8-strip tube)
cBot primer
row 11
5 ml PE rack
#17
HiSeq X Not Supported
NovaSeq 6000 SBS v1.0 20041796 SP/S1/S2: 2 ml
S4: 3.5 ml
#5 n/a
SBS v1.5 n/ac
  1. iSeq 100 instruments do not support use of custom sequencing primers
  2. Requires NCS2k v1.2 or higher and P2 v3 or P3 v3 flow cells. Contact Technical Support for additional instructions for using P2 v2 flow cells.
  3. Primers required for sequencing Illumina DNA PCR-free Prep, Tagmentation libraries are included with NovaSeq v1.5 SBS cartridges, which require NovaSeq Control Software v1.7 or higher

Run set up instructions per software:

Software Applicable instrument Sample sheet? Library Prep
Kit Selection
Custom Primer
Run Setup
Read Lengths
Control software
(on instrument)
NovaSeq w/ SBS v1.0 Not required n/a Manually select
custom Read 1
151, 10, 10, 151
NovaSeq w/ SBS v1.5 Not required n/a No selection of
custom primers
151, 10, 10, 151
HiSeq 1000/1500/2000/2500
& 3000/4000
Not required n/a n/a 151, 10, 10, 151
NextSeq 1000/2000
(NCS2k ≥ v1.2)
Not required n/a Manually select appropriate custom Read1 and Index2 wellsa 151, 10, 10, 151
Local Run Manager
(LRM)
MiniSeq (MNCS v2)
NextSeq 500/550 (NCS v4)
In LRM Download library prep kit definition file and import as custom library Manually select custom
Read 1 and Index 2
149, 10, 10, 149b
MiSeq
(MCS ≥ v3.0)
In LRM Download library prep kit definition file and import as custom library Manually select
custom Read 1
151, 10, 10, 151
Illumina Experiment Manager
(IEM)
MiSeq
(MCS ≤ v2.6c)
Required IEM ≥ 1.19:
Built in
IEM ≥ 1.19:
Built in selection
of custom Read 1
151, 10, 10, 151
IEM ≤ 1.18:
Unavailable
IEM ≤ 1.18:
Manually select
custom Read 1
151, 10, 10, 151
BaseSpace Instrument Run Setup NextSeq 1000/2000
(NCS2k ≥ v1.2)
Required Illumina DNA PCR-Free Manually select appropriate custom Read1 and Index2 wells in instrument control softwarea 151, 10, 10, 151
PrepTabd MiniSeq (MNCS v1)
NextSeq 500/550 (NCS v2)
Not tested
  1. Leave “Denature and Dilute Onboard” box checked. For more details on appropriate well selection, please see the NextSeq 1000 and 2000 Custom Primers Guide. Contact Technical Support for additional instructions for using P2 v2 flow cells.
  2. Read length is limited by the number of SBS cycles available in reagent kits, maximum read length for Illumina sequencing platforms, and 10 bp dual indexes
  3. IEM sample sheet creation is required only for MiSeq run setup. IEM can create sample sheets for demultiplexing purposes for other instruments 
  4. Recommend run setup in LRM or standalone mode

Resources: