Natalia V. Ivanova Paul D.N. Hebert EXPRESS BARCODES: Racing from Bugs to Identifications

Conventional DNA barcoding at CCDB Automation: 3 Biomek robots (Beckman Coulter ) xl DNA sequencers (ABI) Methods: Automated DNA extraction Pre-made frozen PCR and sequencing plates PCR with Platinum Taq E-gel PCR check No PCR cleanup Sequencing with 1/24 BigDye v. 3.1 Automated Agencourt sequencing cleanup 0.5M specimens 1M sequences per year

Outline of the analytical chain Extract DNA PCR AmplifySequence Specimen Tissue Sample Extract DNA PCR AmplifySequence Specimen Tissue Sample Photograph Collection Data Web-Accessible Data and DNA Barcode

Time required for conventional DNA barcoding Total: ~ 8 hours Extract DNA PCR AmplifySequence Specimen Tissue Sample DNA ExtractionPCRSequencingSequencing run PCR check Sequencing cleanup

Why do we need express DNA barcoding? Express Identification System: Monitoring of invasive species Identification of all life stages Simple Fast Portable

Keeping everything simple Merging Technology with a Simple Approach Basic equipment Fast DNA extraction Pre-made frozen or lyophilized reagents for PCR and sequencing Size-exclusion sequencing cleanup

Express DNA extraction – 5 min Alkaline Lysis (Whatman) Boil water Take a small bug fragment Place in a tube with alkaline buffer (0.1 N NaOH, 0.3 mM EDTA, pH 13.0) Incubate for 2 min at 95°C Add neutralization solution (0.1 M Tris-HCl pH 7.0)

PCR amplification TaKaRa Z-Taq QIAGEN Fast Cycling Kit Platinum Taq (control) Fresh moth DNA, ~300 bp: 5 min – 95 ºC 40 cycles: 5 sec – 98 ºC, 5 sec – 51 ºC, 10 sec – 68 ºC 1 min – 72 ºC

Z-Taq PCR optimization – how fast can it work? PCR optimization on moth legs extracted with alkaline lysis 5 sec – 98 ºC, 5 sec – 51 ºC, 10 sec – 68 ºC Template Dilution

Cycle sequencing optimization Cycle sequencing parameters to get ~300 bp: 2 min – 96 ºC 30 cycles: 5 sec – 96 ºC, 5 sec – 55 ºC, 20 sec – 60 ºC 26 minutes

Sequencing cleanup using size-exclusion Size-exclusion Sequencing Cleanup using Nanosep (PALL) Dilute sample with 0.5 mM EDTA Apply onto membrane, centrifuge Wash with 0.5 mM EDTA, centrifuge Add 0.5 mM EDTA, collect from the top and transfer to collection plate 10 min

Making lab procedures fast and simple < 2 hours ~ 8 hours European chafer Rhizotrogus (Amphimallon) majalis DNA ExtractionPCRSequencingSequencing run PCR check Sequencing cleanup

Portable PCR and sequencing devices available today PNAS | January 22, 2002 | vol. 99 | no. 2 | Microchip sequencing combined with on-chip cleanup could be completed in min

A place for a hand-held barcoder? Merging Technology with Express-barcoding Methods: Lyophilized reagents Quick on-a-chip alkaline lysis PCR on a microchip Sequencing on a microchip Connection to BOLD

Rick Turner Alex Borisenko Acknowledgements Jeremy deWaard Janet Topan and CCDB