Scripts/How to use Demultiplexing script

When using Oxford Nanopore or Illumina, we mix (pool) many samples into a single library. Once the sequencing is finished, we then need to sort the reads (obtained DNA sequenced) back to the original samples. This is called demultiplexing, and it is done with help of sample-specific unique tags that are attached to forward and reverse primers.

To make demultiplexing possible, we need to make a list of all samples and their sample-specific primers+tags. This is what this script prepares.

The script takes the following data:

• position of each sample (which plate and where on the plate)
• forward primer for each plate
• position of reverse primer in the plate
• sequence of each primer and its unique tag

and combines all this data into the list that gives

• name of the sample
• sequence and tag of its forward primer
• sequence and tag of its reverse primer

Script and example files to download

You can download the script here.

Example file with data needed to prepare demultiplex file: Data_for_demultiplex_EXAMPLE.xlsx

Needed files

List of plates Overview of sequencing plates with their names, amplicon lengths, and assigned forward primers.	Plate maps Layout of samples across plates, showing which sample code occupies each well position (A1–H12).
Reverse primer map Map of reverse primers (HCO_13 series) assigned to each well position across the plate.	List of primers Full primer list including tag sequences, basic primer sequences, and the combined tagged primer sequences used for demultiplexing.