digitalMLPA is compatible with sequencing platforms using Sequencing by Synthesis (SBS) and XLEAP-SBS chemistries. The optimum library concentration is dependent on the NGS instrument and reagent kit type used. This article provides platform-specific recommendations for preparing and loading digitalMLPA libraries on sequencing instruments that have been evaluated by MRC Holland. These recommendations are based on validated workflows but may need further optimization.
Note
The absence of a sequencing platform from this article does not indicate incompatibility with digitalMLPA. For information on platform compatibility, please consult this article or contact MRC Holland support.
Illumina NGS instruments
NextSeq 1000
Please note that this dilution is to be used in combination with the NextSeq ‘onboard denature and dilute’.
- Mix 10 µl of combined PCR product with 990 µl RSB with Tween 20. This results in a 2 nM library.
- Mix 7.8 µl of the 2 nM library with 16.2 µl RSB with Tween 20. This results in a 650 pM library.
- Load 20 μl of the 650 pM library in the Library reservoir of the NextSeq reagent cartridge.
MiSeq
- Mix 5 µl of combined PCR product with 95 µl ultrapure water. This results in a 10 nM library.
- Mix 5 µl of the 10 nM library with 5 µl of NaOH (0.2 M). Incubate for 5 minutes at room temperature. This results in a 5 nM library.
- Mix 10 µl of the 5 nM library with 990 µl pre-chilled HT1 buffer. This results in a 50 pM library.
- Load 600 µl of the 50 pM library in the Load Samples reservoir of the MiSeq reagent cartridge.
GeneMind NGS Instruments
FASTASeq 300
For library prep and loading instructions, see this application note.
SURFSeq 5000
For library prep and loading instructions, see this application note.