VascoElbrecht's video: Development of metabarcoding primers tutorial on primer degeneracy

This is your ultimate guide for the successful development of metabarcoding primers. Key is a well matching primer pair targeting the ecosystem or taxonomic groups under study. Without primer degeneracy (wobble bases) your primers will be affected by serious bias, in the worst case only amplifying a small proportion of biodiversity in your sample. Testing the primers you develop or pull from the literature is essential to the success of your metabarcoding project! In this tutorial we look at: 0:00 The difference between single specimen barcoding (sanger) a sequencing of environmental bulk samples with (HTS) 4:15 COI binding site variability, mismatches and how primer degeneracy can help 10:57 Experimental evidence for primer bias, and benefits of degenerated bases. 14:35 Overview of the primer design process Tutorial: Downloading reference sequences for primer design with PrimerMiner https://www.youtube.com/watch?v=bpFMJbu-18Q&list=PLGlB7fhZ-TeUwJrZ_Dppp4bwsan7lFQPp 17:35 Finding suitable primer binding regions and general primer design guidelines (considering degeneracy) 24:39 In silico evaluation of primer sets Wiki for PrimerMiner in silico evaluation: https://github.com/VascoElbrecht/PrimerMiner/wiki/6-Primer-evaluation-(in-silico) 26:15 Number of primer versions, and how all degenerate primers are incorporated in PCR due to fuzzy matching (few mismatches at a time) 32:20 Primer slippage and how to fix it 36:29 Summary Literature: Demonstration of primer bias (Elbrecht & Leese 2015): http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130324 Example primer evaluation and design of degenerated primers (Elbrecht et al. 2016): https://www.frontiersin.org/articles/10.3389/fenvs.2017.00011/full R package PrimerMiner for sequence processing and in silico evaluation http://onlinelibrary.wiley.com/doi/10.1111/2041-210X.12687/full Stay up to date by following me on Twitter https://twitter.com/VascoElbrecht