Target Primer Design

The IMAP kit does not provide target-specific primers. You are responsible for design and sourcing of your target primers. You can use primer sets that are available to the research community. Amplicon lengths of 400 base pairs are recommended. Longer amplicons may be necessary with some targets.

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A program (such as Primalscheme), for primer design for tiling across adjacent amplicons, for applications such as whole genome viral sequencing is required. The program must design sets of primers that cover the entire target input sequence, with amplicons arranged end to end across the target. Use each primer set designed by the program to generate two amplicon pools, and combine at the tagmentation step. This is the method used with ARTIC primers for SARS-CoV-2 sequencing.

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You can use other primer design programs for non-tiling targeted sequencing applications. Follow the recommended design parameters for the chosen design program. Design the primers for a multiplex PCR assay.

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Aim for primer annealing temperatures of 63–65 °C and try to keep the predicted temperatures consistent across amplicon primer pairs in a set. 63°C is the recommended starting temperature. Depending on the target, adjust the desired annealing temperature of the primers to optimize PCR amplification.

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Dilute the primer pools to 10 µM. Use 4.3 µl of each primer pool per sample or a total of 207 µl of each pool for 48 total samples.