Yes, it is possible to combine samples that have been tested with different digitalMLPA probemixes or different probemix versions into a single run. It is also possible to combine different experiments with the same probemix (version). For a successful run it is important to take the points listed below into account.

Unique barcodes

To allow reliable sample recognition, a barcode can only be used for one sample in a sequencing run, regardless of the probemix (version) used. This means that each reaction must have a unique barcode.

Proportions

All reactions in the sequencing run must be mixed in the correct proportions.

1. For each probemix (version) or experiment, mix 5 μl of each PCR reaction in a vial (resulting in a separate vial per probemix (version) or experiment).
2. For each of these PCR-product pools, calculate the required amount as follows:
   
3. Mix the calculated amounts of the PCR-product pools into a single vial.
4. Dilute this mixture as described in the section on library preparation of the digitalMLPA General Protocol.

Example

20 samples are tested with probemix A (575 probes) + 56 samples with probemix B (300 probes). Combine the PCR reactions of each probemix (step 1), resulting in two vials: one with 100 μl of all reactions with probemix A, and one with 280 μl of all reactions with probemix B. Mix  of the PCR-product pool of probemix A with  of the PCR-product pool of probemix B (step 2/3). Proceed with the library preparation as described in the digitalMLPA General Protocol.

Required number of reads

When combining different experiments into a run, it is important to know how many reads are required. This can be calculated as follows:

1. Multiply the number of probes in a probemix (version) by the number of samples tested with this probemix (version). Do this for each probemix (version).
2. Sum these calculated numbers and multiply with the read depth of 500.

Example

20 samples are tested with probemix A (575 probes) + 56 samples with probemix B (300 probes). The required number of reads is .

Make sure not to exceed the capacity of the Illumina reagent kit when combining experiments in one run. If the required number of reads is higher than the capacity of the Illumina kit, use a kit with a higher capacity or lower the number of samples in the run.