Microfluidic Enrichment Barcoding (MEBarcoding): a new method for high throughput plant DNA barcoding

DNA barcoding has become a valuable tool to support species identification with a broad range of applications in fields such as traditional taxonomy, ecology, forensics, food analysis, and environmental science. We introduce Microfluidics Enrichment Barcoding (MEBarcoding) for plant DNA Barcoding, a cost-effective method for high throughput DNA barcoding. MEBarcoding uses the Fluidigm Access Array™ to simultaneously amplify targeted regions for 48 DNA samples and hundreds of PCR primer pairs (a total of 23,040 PCR products) during a single thermal cycling protocol. A second generation instrument from Fluidigm, called the Juno™, can accommodate 192 DNA samples simultaneously. As a proof of concept, we developed a microfluidic PCR workflow using the Fluidigm Access Array™ and Illumina MiSeq to generate new sequences from 96 samples for each of the four primary DNA barcode loci in plants: rbcL, matK, trnH-psbA, and ITS (384 total sequences). This workflow was used to build a reference library that includes 78 families and 96 genera from all major plant lineages, including bryophytes, ferns and lycophytes, gymnosperms, and all major groups of angiosperms, which are currently lacking in public databases. Our results demonstrate that this technique offers a highly efficient alternative method to traditional PCR and Sanger sequencing by increasing the estimated number of plant DNA barcodes that can be sequenced by a single technician in one week by 800%, at a reduced cost, and by generating a barcode library with a more comprehensive taxonomic coverage.