What is the CSP and how do I use it?
A Custom Sequencing Primer (CSP Version 5, included in the kit) is required to achieve cluster calling on Illumina machines for QuantSeq REV libraries. This is to ensure the poly(T) stretch that is present at the beginning of the Read 1 insert sequence is covered by the sequencing primer, to avoid quality drops and registration errors.
The provided CSP covers the poly(T) stretch and replaces the Multiplex Read 1 Sequencing primer.
Please find specific information below for several Illumina instruments regarding the correct use of the CSP V5. These guidelines are subject to change should Illumina sequencing reagents or platforms be updated.
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.
Spin down the provided tube of CSP before use.
Add 6 µl of 100 µM CSP to 1,994 µl of HT1 buffer (final volume 2,000 µl). Mix well and spin down.
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 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!”).
The CSP Version 5 is then added to the reagent cartridge in Custom 1 well or Custom 2 well (the well used must be specified in the software during run setup).
Please also, always consult the NextSeq 1000 and 2000 Custom Primers Guide (Illumina) for all details of custom primer restrictions, concentrations and loading guidelines.
NovaSeq
Please consult the NovaSeq Series Custom Primers Guide (Illumina) for all details including loading volumes and concentrations.
Reagent Kit v1.0
These reagent cartridges support Custom Sequencing Primer use for Read 1, without the need for additional custom index primer addition.
Prepare the CSP Version 5 in the HT1 Buffer (must be purchased separately).
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 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 consult the NovaSeq Series Custom Primers Guide (Illumina) for all details including custom primer restrictions, loading volumes and concentrations.
MiSeq
Clustering is performed on the machine. The MiSeq uses a reservoir of 600 µl with 0.5 µM sequencing primer final concentration, i.e., 3 µl of 100 µM CSP Version 5 in 597 µl HT1.
The CSP Version 5 in HT1 should be added to Reservoir 18.
Please consult the MiSeq System Custom Primers Guide (Illumina) for all details including loading volumes and concentrations.
HiSeq 2000 / 2500 / 3000 / 4000
Please consult the HiSeq System Custom Primers Guide (Illumina) for further updated details of CSP useage on the various instrument platforms.
HiSeq 2000, HiSeq 2500 (CSP Version 5 added on cBot)
CSP Version 5 should be provided in a tube strip at 0.5 µM final concentration in a volume of 120 µl (final concentration 0.5 µM, to be diluted in HT1 = Hybridization buffer). Take 0.6 µl of 100 µM CSP Version 5 and add 119.4 µl of HT1 buffer per sequencing lane. Place the 8-tube strip into the cBot position labeled primers.
HiSeq 2500 (CSP Version 5 replaces HP10 in cBot Cluster Generation Reagent Plate)
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!
The Illumina V4 chemistry cBot Cluster Generation Reagent Plate only has 8 rows filled. A simple trick is to have the empty rows facing towards you, this way if you want to use a CSP in lane 1, you have to remove the HP10 solution from well 1 (first one on the far left) of the 2nd row, rinse the well a couple of times with HT1 and then add the diluted CSP Version 5. For this take 1.25 µl of 100 µM CSP Version 5 and add 248.75 µl of HT1 buffer per sequencing lane. The CSP should be at 0.5 µM final concentration in a volume of 250 µl (final concentration 0.5 µM, to be diluted in HT1 = Hybridization buffer). ATTENTION: Do not add the CSP to the Standard Illumina Multiplex Read 1 Sequencing Primer = HP10 solution! Always use fresh HT1 and add the CSP / HT1 dilution to the empty and rinsed well.
HiSeq 2500 – Rapid Run
Add 12.5 µl of 100 µM CSP Version 5 to 2,487.5 µl HT1 = Hybridization buffer, resulting in a total volume of 2.5 ml and a final CSP concentration of 0.5 µM. In a rapid run, both lanes will use the same sequencing primer. It is not possible to run the two lanes with different sequencing primers.
HiSeq 3000, HiSeq 4000 (CSP Version 5 replaces HP10 in cBot Cluster Generation Reagent Plate)
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.
iSeq 100
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.