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We recommend that you also consult the relevant Custom Sequencing Primer Guides from Illumina and follow those instructions when preparing your CSP for use. If you have any questions regarding CSP use please contact our technical support team via support@lexogen.com.
ATTENTION: If you are sequencing QuantSeq REV libraries with a service provider or sequencing facility, please provide the following relevant information to the facility, ALONG WITH THE CSP Version 5!
General Guidelines
QuantSeq REV libraries should not be sequenced together with libraries prepared with any other different kit. This is due to the use of the CSP Version 5, which will not prime other library types correctly.
PhiX spike-in should not be used for QuantSeq REV runs as it will not be primed by the CSP Version 5.
If loading amount guidelines are not provided (see here) please use the upper range of loading amounts specified by Illumina for the instrument type.
The CSP should never be mixed together with the standard Illumina Read 1 Sequencing primer. A primer mixture would result in low clusters calls and the resulting reads would be contaminated by poly(T) stretches.
NextSeq 500 / 550
The CSP Version 5 is optimized for use on NextSeq 500 / 550 instruments.
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The prepared CSP / HT1 solution (CSP final concentration, 0.3 µM) can be then loaded into Position 7 of the NextSeq Reagent Cartridge.
ATTENTION! When sequencing dual-indexed QuantSeq REV libraries on the NextSEq 500 or 550 instruments an additional Custom Index 2 Primer (CIP) is also required. This includes QuantSeq REV libraries prepared with 12 nt UDIs, or the 6 nt i5 Dual Indexing Add-on Kits (Cat. No. 047). custom index primers are not required. The standard illumina index read 1 and 2 primers should be selected in the software during run setup.
Please refer to the NextSeq System Custom Primers Guide (Illumina) for for all details of custom primer restrictions, concentrations and loading guidelines.
NOTE: The former CSP Version 2 is not compatible with the NextSeq instruments. Please check the label on the tube for the CSP version number prior to preparing your sequencing run.
NextSeq 1000 / 2000
Use the HT1 reagent to prepare the CSP Version 5. Follow the instructions for Illumina libraries (not for “third-party libraries alongside PhiX or illumina libraries!”).
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Please also, alwaysconsult the NextSeq 1000 and 2000 Custom Primers Guide (Illumina) for all details of custom primer restrictions, concentrations and loading guidelines.
NovaSeq
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Please consult the NovaSeq Series Custom Primers Guide (Illumina) for all details including loading volumes and concentrations.
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The CSP Version 5 should be added to position 5 of the reagent cartridge. The volume of CSP Version 5 to add depends on the flow cell type in use.
Reagent Kit v1.5
ATTENTION! Sequencing dual-indexed QuantSeq REV libraries on the NovaSeq 600 using v1.5 reagents instruments requires an additional Custom Index custom index primers are not required. The standard illumina index read 1 and Index 2 Primer mixture. This includes QuantSeq REV libraries prepared with 12 nt UDIs, or the 6 nt i5 Dual Indexing Add-on Kits (Cat. No. 047)2 primers should be selected in the software during run setup.
Please consult the NovaSeq Series Custom Primers Guide (Illumina) for all details including custom primer restrictions, loading volumes and concentrations.
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CSP Version 5 can be placed directly into the cBot Cluster Generation Reagent Plate.
ATTENTION: The standard Illumina Multiplex Read 1 Sequencing Primer solution HP10 (for V4 chemistry located in row 2) provided in the cBot Cluster Generation Reagent Plate has to be REMOVED first!
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Usage of a custom sequencing primer is currently not supported on HiSeq 3000 and 4000 machines. A work around as described for the HiSeq2500 (CSP Version 5 REPLACES HP10 in the cBot Cluster Generation Reagent Plate) is possible though.
ATTENTION: Do not add the CSP Version 5 to the HP10 solution! A primer mixture would result in low clusters calls and the resulting reads would be contaminated by poly(T) stretches. Always use fresh HT1 and add the CSP Version 5 / HT1 dilution to the empty and rinsed well.
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Custom primer use is not supported on the iSeq 100 instruments. Therefore QuantSeq REV cannot be sequenced on this platform.
AVITI
For sequencing QuantSeq REV libraries on AVITI with CSP, please contact support@lexogen.com for further details.