Developing fusion primers for DNA metabarcoding – sample indexing / tagging (Illumina)

In this tutorial we are looking a fusion primer design, to include amplification primers, inline tagging, and Illumina sequencing adapters/flow cell binds in a single primer set, to reduce tag switching and allow for target amplification and library preparation in the same single PCR step. 0:01 quick introduction into DNA metabarcoding 2:41 Illumina index system explained 7:00 Short fusion primer introduction 8:11 Skip the index read step when sequencing with fusion primers on MiSeq / HiSeq 9:16 Exploring fusion primers, sequence shifting and parallel sequencing (adds diversity) 14:57 Why is sequence diversity important, how to design inline tags 18:50 Visualisation (shifting and parallel sequencing) 22:20 Primer combinations 32:20 One and two-step PCR approaches 27:10 Summary and pro/con

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