Q: Where should voucher specimens collected for the "Ant Barcode of Life" campaign be housed? In cases where the donors do not need the specimens shipped back, should all of these donated specimens be curated and housed in one location? Will housing DNA voucher specimens in different locations (under different databases) make things easier or harder for future students/researchers wanting access to them (to describe new species, re-check ID's, work on associations, look at specimens for phylogenetic work, etc.)?
A: The majority of the ant specimens contributed to barcoding will go back to the original donors. Alternatively, if the donors do not need to keep the specimens, specimens will be deposited at Guelph. However, BIO does not have the capacity to store and curate a large amount of specimens, especially pinned materials. So we encourage our collaborators to take the responsibility to store specimens that are used for DNA barcoding. We appreciate the extra efforts needed on the collaborators' side to cooperate with barcoding initiatives. Hopefully, we can come up with a reasonable system that will reduce the investment of time of the collaborators while meeting the basic requirements for DNA barcoding (that is to store barcoded specimens individually, each attached with a unique sample ID). Also, an effective tracking system should be developed so that people who needs to reach a specific individual specimen is able to retrieve that information from the Barcode of Life Data System (BOLD).
Currently, Matrix boxes are most frequently used for transferring tissues. For example, insect legs are routinely assembled into these boxes while the pinned specimens stay in the original museums. However, storing voucher specimens individually will take up extra space and efforts. At the moment, we can offer two kinds of containers, Matrix Box (12.6cm X 8.5cm X 5.8cm) and Matrix Plate (12.6cm X 8.5cm X 2.7cm), both in 96-matrix format and can hold up to 94 specimens. Matrix Boxes have longer tubes than Matrix Plates (5.2 cm vs. 2.1 cm), and are more suitable for big specimens. On the other hand, some collaborators prefer to move the barcoded specimens back to the original vials from which they were taken. In this case, the small tubes in Matrix Plates can be separated and stored in bigger container while each tube remains self-sealed.
Detailed information for all barcoded specimens are deposited on BOLD, including SampleID and donor's contact information. Using this system, we should be able to trace all individual specimens that are used for barcoding. Of course, our collaborators may have their own systems on their ends to make easy access to these specimens.
Q: Who will ultimately "owns" the DNA? For example, many (if not most) of the ant species are known from only a very few specimens. Some of these may be old. Some of these may be from recently collected material, itself the result of much effort to collect, curate, identify, store, etc. Let's say we ""sacrifice" a specimen of one of these few individuals for DNA barcoding. You extract the DNA and sequence the COI barcode region. What happens to the rest of the DNA? What if one of my students wanted to use that DNA to sequence other gene regions for phylogenetic research? Would they have access to it?
A: The Biodiversity Institute of Ontario is committed to respecting the intellectual and physical property of its contributors, particularly including internal tissue policies of contributing museums. There are three ways the donor may choose to provide the tissue: donation, permanent loan, or temporary loan. These conditions are outlined in more detail in the BIO Tissue Policy statement and are specified by the tissue provider in the Biological Materials Transfer Agreement. Unless donated, tissue and any DNA extracted from it remain the property of its original donor and will not be redistributed to third parties without the donor's approval. It is particularly recommended that samples sent to BIO as temporary loans contain only the minimum amount of tissue required for one DNA extraction, thereby allowing consumptive analysis. For quality control purposes, BIO intends to keep residual DNA extracts from all tissues submitted for the duration of iBOL. These extracts will be subject to the same loan conditions as original tissue and, unless donated, will not be redistributed to third parties or used for non-barcoding analyses without permission from the donor. An aliquot of the DNA may be shipped back to the donor upon request.
We suggest that participants in the Formicidae Barcode of Life initiative consider the merits of donating the tissue samples and DNA extracts associated with their projects. This will help to facilitate research into the future, e.g. enabling the sequencing of additional genes in the future as sequencing costs are ever diminishing. Every attempt will be made to ensure that priority is given to follow-up requests for new tissue usages from the original donors.
As a general policy, we are willing to share aliquots of DNA with collaborators who donated the specimens from which the DNA was extracted. The routine protocol of DNA extraction at BIO produces 40 ul of genomic DNA, 2 ul of which is needed for each PCR amplification. Typically, there should be enough DNA extractions left for sharing after the barcode is generated. Protocols of shipping DNA in dried condition under room temperature has been developed, making exchange of DNA samples easy via regular mail carriers.
Q: What is the procedure for ant workers to get more DNA data for specimens besides the COI? If someone is interested in sequencing other gene fragments for phylogenetic or other purposes, can they provide funding to Guelph to have the sequencing done there?
A: The major focus of the barcoding initiatives is mitochondrial COI. However, if common interests arise along projects process and multi-gene analysis is needed, running non-COI genes is possible especially if some funds can be allocated to BIO to compensate chemical consumables and technicians' time. However, the feasibility will depend on the availability of the facilities at BIO and sufficient funding and should be negotiated with the campaign coordinator on a case-by-case basis. Such projects are usually given lower priority in the lab, compared to routine DNA barcoding analysis.
Q: What is the policy on data sharing and authorship? Let's say we send you all of the species in a genus of ant, and you sequence the COI region and publish on it. Would we share authorship? On the other hand, let's say you get the COI sequence you want, and we use the remaining DNA aliquot to sequence other regions for phylogenetic study. Would you or someone at your institution demand authorship on the phylogenetic paper that results? The main thing I am thinking about is the tremendous amount of effort (in time and money) it has taken us (and taxonomists in general) to collect, curate, maintain, identify, etc. all of this material - as well as the commitment to maintain the voucher specimens from your DNA barcoding effort - as this effort relates to the use and sharing of the data that result.
A: Because of the efforts made by collaborators (collecting, curating, identifying, and etc.), they are considered as co-authors of the resultant DNA barcoding publications. On the other hand, we do not automatically demand authorship for other publications, such as taxonomic or phylogenetic works, that are generated merely using the DNA we produce. Of course, if the collaborators have agreements with people in our institute, and we do make further contributions to the publication (for example if the co-author at Guelph agrees to contribute personal time to sequence additional genes with allocation of some funds from the collaborators to compensate chemicals, in which way the sequencing cost can be greatly reduced using Guelph facilities), we would be honored to share authorship. Of course, that will only happen upon reciprocal agreements. In addition, as we mentioned in the proposal, sequence data will be shared among collaborators even before they are published.
Q: Will the DNA be automatically made public or would it wait until I had published it?
A: Data that are currently in BOLD will follow the prior policy on data release, which is that COI sequences are not be publicly accessible unless the project coordinator submits a specific request to release them. Normally, the collaborator and the project coordinator come up with agreements on how and when to publish the data. The collaborator can also share data with other people he/she wants to include in the project. The sequence data and progress are kept confidential between collaborators and project coordinator for a particular project. Starting July 1st, 2009, a new data release policy will be enacted, which is in line with typical genomics project requirements.
As the producer of a community resource (the DNA barcode library), the CCDB is expected to make genomic data publicly available within one week following their generation (see http://www.ncbi.nlm.nih.gov/). The information disclosed includes the DNA sequence, associated sequence trace files, BOLD Process ID (individual accession number automatically generated by BOLD), taxonomic position down to the ordinal level, and the country of origin. Information on associated voucher specimens, their detailed geographic origin, and detailed taxonomic position remains confidential at this pre-publication stage. The rapid release principle applies to all genomic information generated by the CCDB through support from its key finding agencies. In the genomics community, this data release model has worked well, and both publication and sequence annotation follow this preliminary release of raw sequence data.
All sequence data contained in BOLD (including unpublished projects) are used by the BOLD identification engine to provide DNA-based taxonomic identifications to public users submitting DNA barcode sequences. Reports generated by the BOLD identification engine include probability scores and tree-based identification with branch labels containing detailed taxonomic names, broad geographic localization (to province level), and corresponding BOLD Process ID's. Information on individual specimens (museum catalogue numbers and place of voucher deposition) and their detailed geographic origin are not disclosed through the BOLD identification engine.
Once the initial specimen data submission has been made to BOLD, provenance information and images become partially available to the public online through the BOLD Taxonomy Browser at http://www.barcodinglife.org/views/taxbrowser_root.php. This information is used to generate summary statistics and illustrative distribution maps and does not disclose the contents of individual research projects and specimen data records.
Q: What are the obligations of external collaborators in order to have samples sequenced at Guelph?
A: All specimens processed at Guelph must have the goal of being "barcode compliant", which means that certain standards for data quality and completeness are achieved. Collaborators and internal project managers work together to ensure that this happens, and the distribution of tasks should be discussed at the beginning of each project. All specimens must be destined for permanent storage in a major collection and must be accessible for future research. Some specimen data fields are required for all specimens, including taxonomic identification (at various taxonomic levels depending upon the project), collector name, collection date, country, major administrative unit, and locality with GPS coordinates. Fragmentary information is acceptable only in the case of valuable museum material when that is all that is available. Biological information such as life stage (age), sex, and mode of reproduction are a valuable asset. All specimens must be photographed and the photos uploaded to BOLD. Finally, barcode-compliant records involve sequences that are at least 500 base pairs long; core laboratory personnel work towards this goal, but appreciate input from external collaborators regarding primer choice and any trouble-shooting that is needed. External collaborators are also asked to assist in resolving any data discrepancies that arise.
Q: What happens in the case that errors or discrepancies arise?
A: Because the CCDB core analytical facility is highly automated, analytical error rates are very low and are primarily due to human error at the early stages. Taxonomic misidentifications are not uncommon and are usually rectified by re-examining the photographs on BOLD or the original specimens. Tissues can be been placed into the wrong wells (mixed up), or cross-contamination between wells can occur during sampling. Such cases are the most problematic. Generally, records in BOLD should be annotated when this occurs and the sequences removed. Re-sampling from the original specimens may be performed. Contamination can occasionally involve DNA from outside organisms. (For example, if the specimens were prepared in an environment in which lepidopteran samples were also processed, then scales can occasionally get into other plates; this will be minimized through choosing clean preparation areas.) Annotation of records should be performed and specimens re-sampled. General contaminants such as human or Wolbachia (in insects) are easily detected and can be overcome through using taxon-specific primers.
As much as possible, discrepancies should be resolved by re-examining specimens and updating records. In cases where resolution is not possible, re-sampling from the original specimens may be performed. Always, it is good to evaluate whether this is a common species which already has several high-quality records. Putting this effort towards obtaining barcode records for new species, rather than repeating each failed specimen of common species, may be more productive. Close communication between CCDB staff and external collaborators is essential for quickly rectifying any discrepancies that occur.
Q: What are your permit requirements for collecting and export/import?
A: It is the sender's responsibility to ensure that biological materials are shipped to the Biodiversity Institute of Ontario in compliance with any applicable shipping regulations, that they have been obtained under appropriate collection and animal care permits in their country of origin, and that the necessary export/import documentation required by Canadian and International customs and conservation authorities has been provided, including, but not limited to:
a) Export permit and/or zoosanitary certificate from the country of origin (if applicable);
b) CITES registry certificate for the provider institution (if applicable);
c) Canadian Food Inspection Agency import permit (if applicable).
The Biodiversity Institute of Ontario cannot be held responsible in the event the provider fails to supply proper shipping documentation, causing the shipment to be held up in customs, or any penalties resulting thereof. Upon request, we can advise on Canadian import requirements and assist in obtaining relevant permits. The Biodiversity Institute of Ontario is a CITES-registered institution (registry certificate CA022). It is advisable that any international transactions involving CITES-listed mammal species are facilitated by a CITES-registered institution, whereby they will qualify as scientific exchange, thus reducing the amount of required paperwork.