Course Materials

Specimen and Sample Processing

Invertebrates
Vertebrates
Plants and Fungi

Data Management

Specimen arraying

Organizing specimens and, more specifically, arraying them in a set pattern that mirrors a 96-well plate is a key step in DNA barcoding protocols. Properly arrayed specimens have a higher barcoding success and are less prone to error and contamination, complementing a medium to high throughput barcoding pipeline.

Databasing

Complete and accurate specimen provenance information is important in multiple respects.  Submission of this information to the Barcode of Life Data Systems (BOLD) is required to meet barcode standards and it is also a prerequisite for submitting samples to our Laboratory Information Management System and commencing molecular analysis. In addition, these data are very useful at the publication stage.

Imaging

Images are a key component of DNA barcoding practices and there are standard protocols for submitting images to BOLD. In this course, we will review both SLR and microscopy imaging setups.

Sampling

Barcoding protocols are optimized for a small amount of tissue/DNA. Consequently, most organisms which do not go through voucher recovery require tissue sampling according to standard procedures to avoid contamination.

For a detailed overview of the sampling process, click here.

Data tracking

It is important to keep detailed data records throughout the specimen processing  procedure.

Voucher reference

A collection voucher specimen preserved in an internationally recognized collection repository needs to accompany each barcode record. This should be available for re-examination by interested experts and for reference should there be any issues with the accompanying data.

Click here for a webinar presentation by International Development lead Dr. Alex Borisenko.

Molecular Analysis

Good laboratory practices (GLPs)

We ensure that our laboratory staff and our visiting trainees follow GLPs not only for their safety but for the integrity, consistency, reliability, reproducibility and quality of the research conducted at BIO. Trainees go through a detailed briefing on GLPs before they start using the lab space, equipment and chemicals for their research.

Laboratory pipelines at the CCDB

 Use of LIMS

DNA extraction

CCDB has developed an automation-friendly DNA extraction method for high quality DNA. The membrane-based protocol is compatible with both manual and robotic extractions and matches the performance of the best commercial kits. Trainees receive hands-on experience with DNA extraction protocols adopted by the CCDB.

For a more detailed overview, click here.

PCR and primers

High amplification rates of the target genes and clean PCR products guarantee high barcoding success. PCR success depends on the quality ingredients and properly optimized cycling conditions. Accordingly, PCR protocols at the CCDB are optimized for barcode amplification from diverse taxonomic groups using PCR primers that range from taxon-specific to universal.

Gel electrophoresis/ E-gel

PCR products are analyzed using E-gels that are compatible with the 96-well protocol. A quick gel-run without running buffer, followed by image-view using a computerized gel-doc, is convenient and effective and provides a publication quality gel image.

DNA sequencing

After successful PCR and E-gel analysis, trainees receive hands-on experience with cycle sequencing reaction preparation and cycle sequencing thermocycling. The DNA sequencing facility at the CCDB cleans the cycle sequencing products and then generates the sequences using one of the facility’s Applied Biosystems 3730XL DNA sequencers. Sequencing results are generally available within 48 hours.

Quality control

As the sequences are generated, the results are analyzed for accuracy and quality prior to submission to BOLD. Trainees will meet the staff specialized in maintaining quality control.

Informatics

Barcode of Life Data Systems (BOLD)

All of the information on specimens and sequences is submitted to BOLD. BOLD is not only a database, it also provides basic analytical tools to explore barcode data.

Sequence assembly and editing with CodonCode

Sequence assembly and editing is conducted in CodonCode, the software of choice at the CCDB. Trainees receive experience with the use of CodonCode and have an opportunity to edit their barcode sequences.

Analyzing barcode data using third-party software

Multiple analytical tools are generally required to draw conclusions from barcode data. There are a number of software packages which are commonly used for sequence analysis, including MEGA, BioEdit, ABGD, and DAMBE.  As part of the training program, visitors will explore the range of built-in tools for data analysis offered by these software packages, with a particular emphasis on MEGA.