Abstract
DNA barcoding is a valuable tool to support species identification with broad applications from traditional taxonomy, ecology, forensics, food analysis, and environmental science. We introduce Microfluidic 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 (producing up to 23,040 PCR products) during a single thermal cycling protocol. As a proof of concept, we developed a microfluidic PCR workflow using the Fluidigm Access Array and Illumina MiSeq. We tested 96 samples for each of the four primary DNA barcode loci in plants: rbcL, matK, trnH-psbA, and ITS. This workflow was used to build a reference library for 78 families and 96 genera from all major plant lineages – many currently lacking in public databases. Our results show that this technique is an efficient alternative to traditional PCR and Sanger sequencing to generate large amounts of plant DNA barcodes and build more comprehensive barcode databases.
Highlights
A laboratory working with a full-time technician, that sequences plant DNA barcodes from 96 samples per week for forty weeks would spend more than $100,000 USD using traditional Sanger sequencing protocols, whereas the same number of samples could be processed in just five weeks at a cost estimated at $12,500 using MEBarcoding on the Juno system
All primer pairs tested in our primer validation produced amplicons as anticipated and were used in our Access Array workflow. 96/96 samples tested for MEBarcoding produced at least one amplicon that was successfully sequenced
Results in this study show the capacity of a first-generation microfluidic instrument, the Fluidigm Access Array
Summary
Using the MEBarcoding approach described here on the Access Array or Juno systems plant DNA barcodes could be generated for 384 or 768+ samples, respectively. A laboratory working with a full-time technician, that sequences plant DNA barcodes from 96 samples per week for forty weeks would spend more than $100,000 USD using traditional Sanger sequencing protocols (see Supplementary Table 1), whereas the same number of samples could be processed in just five weeks at a cost estimated at $12,500 using MEBarcoding on the Juno system.
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