Curriculum

Training Modules in DNA Barcoding

Offered by the Biodiversity Institute of Ontario – Centre for Biodiversity Genomics

Below is an outline of the curriculum of the standard four-week hands-on course offered by the BIO-CBG Research Training Program.

Introductory Modules

Introduction to DNA barcoding
  • The DNA barcoding concept
  • The utility and societal benefits of DNA barcoding
  • Strategic goals of the international barcoding community
  • Scientific questions that can and cannot be studied using DNA barcodes
  • Overview of research themes in DNA barcoding
Introduction to front-end processing for high-volume molecular analyses
  • The logistical framework of DNA barcoding: interfacing between collections and barcoding centres
  • The importance of front-end logistics in facilitating high throughput molecular analyses
  • Front-end stages: specimen arraying, databasing, imaging, sampling
  • Pre-lab stages: challenges and solutions
  • Making collections compliant with molecular analyses: DNA-friendly preservation, data tracking, voucher reference
Introduction to DNA barcoding molecular protocols
  • High, medium, and low throughput protocols
  • Laboratory pipelines at the CCDB
  • DNA extraction (animals and plants)
  • PCR reactions and PCR primers
  • DNA sequencing
  • DNA storage and transportation
  • Good laboratory practices
  • Quality control
Introduction to BOLD Systems
  • BOLD: the global online repository for barcode data
  • General organization of BOLD (campaigns, containers, projects)
  • Project security and user access
  • What constitutes a BOLD record: sample page + sequence page
  • Data submissions: collection data, images
  • Editing and updating BOLD records
  • Data analyses: BOLD NJ trees, summary stats, image preview, mapping
  • The Barcode Index Number (BIN) concept and its applications
  • Accessory functions: file attachments, taxonomy curation
  • External connectivity: GenBank submissions, Google Earth, etc.
  • BOLD data release policies

Concluding Modules

  • Management of DNA barcoding results and making data publication-ready
  • Interpreting DNA barcode data: questions that can and cannot be answered using standard barcoding approaches
  • Retrieval of sequence data from BOLD and converting it for analysis using third-party software (MEGA)
  • Sequence alignment, model testing, bootstrapping, and generation of publication-friendly neighbour joining trees (MEGA)
  • Evaluation of phylogenetic signal based on transition saturation estimates (DAMBE)
  • Estimating distance thresholds for species delimitation
  • Simple mapping solutions for illustrating geographic sampling coverage
  • Reference management software and reference resources for DNA barcoding publications

Practical Modules

Specimen collection
  • Data spreadsheets: field collecting and museum sampling
  • Collecting events, specimen lots, and specimens
  • Data conversions and data standards
  • Aggregation of specimen data in a BOLD-compliant format
  • Hands-on training in digitization and conversion of specimen provenance information; population of specimen data sheets (iBOL data templates – optional)
  • Assembly of specimen arrays and population of array record files (plate records)
  • Preparation of specimen data for submission to BOLD (requirements and hints)
Specimen processing
  • Specimen labeling: high throughput pipeline (array-based approach)
  • Specimen imaging: high throughput pipeline (array-based approach)
  • SLR photo setups for pinned insects, fluid specimens, and study skins
  • Microscopy imaging setups for dry and fluid-preserved specimens; Z-axis stacking
  • Preparation of specimen images for upload to BOLD (requirements and hints)
  • CCDB sampling kits and media (tube rack, microplate, blotting card)
  • Box/plate/card records
  • Hands-on tissue sampling for select groups of organisms (animals or plants)
Molecular analyses
  • Preparation of reagents for DNA extraction
  • Tissue sampling (brief refresher) and tissue lysis
  • DNA extraction
  • PCR reagents and master mixes
  • PCR set-up and programming thermocyclers
  • PCR success validation using E-gels
  • Cycle sequencing and sequencing
  • Sequencing electropherograms (quality control)
  • Sequence assembly and editing using CodonCode Aligner software
Bioinformatics (BOLD Systems)
  • BOLD user interface and basic structure
  • Creation of BOLD user accounts, levels of user access
  • Creation of BOLD projects, changing project properties
  • Uploading and downloading specimen data and images
  • Searching, filtering, and aggregating BOLD data
  • Introduction to software tools used in barcoding (MEGA, BioEdit)
  • Common problems with assembled sequences; sequence alignment
  • Checking sequence quality and troubleshooting
  • Sequence upload to BOLD; downloading sequences from BOLD
  • BOLD tree-building and its applications for data validation/curation
  • BINs and their applications for data validation/curation
  • Other BOLD tools for analyzing and presenting sequence data
  • Publication of BOLD data: creation and publication of BOLD datasets

Other Discussion Topics

  • Conceptual questions related to DNA barcoding
  • Overview of the essential barcode requirements related to samples and data (standard sampling instructions, BOLD manual, etc.)
  • Overview of available tools and protocols to facilitate high throughput front-end processing
  • Discussion on legal and ethical considerations related to DNA barcoding: access and benefit sharing, biological material transfer agreements, etc.
  • Discussion on data troubleshooting, project management, and project planning
  • Discussion on designing ‘barcode-friendly’ field collecting and curatorial protocols
  • Discussion on equipment/reagent procurement specifics and the logistics of setting up DNA barcoding workstations and workflows in different countries