Progress
specimens barcoded:  197536
 
species barcoded:  8553
 
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clusters found: 
3173
 
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FAQs
Q:    What are the factors that can possibly decrease or increase the success rate of DNA barcoding for my samples? My specimens went through a relaxing container before being mounted; does that make them unsuitable for DNA extraction?
A:    Basically, DNA degradation – which results in fragmentation of the DNA double helix – occurs through time. It is accentuated in wet and warm conditions, as well as by direct exposure to UV light. Development of bacteria or molds also accelerates the degradation process. For that reason, it is better to pull up legs before relaxing the specimens, though many relaxed specimens proved to provide good quality DNA as well. Small-sized specimens often get negative results after humidity exposure longer than 24 hours.

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Q:    How old can be the specimens sampled if to expect a reasonable success rate?
A:    From our experience with thousands of samples of Lepidoptera processed so far, you can expect high success rate (>80%) for specimens collected within the past 10 years, both for collection specimens and papered specimens; the latter should work better though, especially if they had been kept frozen. We strongly encourage our collaborators to give priority to samples not older than 10 years old, reserving others for these species/specimens which can hardly be found elsewhere as fresher material. Please advise the campaign or campaigns coordinators if you wish to sample a large quantity of relatively old specimens as we may adopt a special procedure for them.

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Q:    What’s the oldest specimen you did successfully sequence in your laboratory?
A:    Using a specifically designed protocol, we managed to assemble complete DNA barcode sequences for specimens more than two centuries old. This is especially interesting to address taxonomic questions for which only very old type specimens can bring the answer. However, because the protocol is much more demanding in term of cost and time, we reserve it for very specific case studies and it requires dedicated funding. Nevertheless, our current workflow includes a failure tracking step targeting shorter fragments and permitting to produce DNA barcodes with a fairly high success for specimens several decades old.

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Q:    Does the method used to kill the moth or butterfly affect the success of recovering DNA sequences?
A:    From our experience, specimens killed by injection of ammonia or ethanol, or by exposure to cyanide gas are perfectly suitable for DNA barcoding. If you do use a different method and have a doubt about its potential impact on DNA quality, please refer to the campaign or campaign coordinator; if necessary, a test will be run to evaluate this impact.

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Q:    How much tissue do you need? Is a single leg enough?
A:    Yes, a single leg is enough for most moths, though the smallest Microlepidoptera may require two or more legs to increase the DNA concentration and the chances to produce a good quality barcode, especially if the specimens are not freshly collected. As we encourage our collaborators to sample the legs directly in 96-wells lysis plates, it is not necessary to sample the whole leg for larger moths for which the tarsomeres or tibia + tarsomeres are sufficient. On the other hand, tiny tissue samples may “jump” when using these plates because of static electricity. We then usually recommend that you add a small volume of ethanol to the wells; if you mention it when you request the plates from us, we can send them already filled with small volume of ethanol.

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Q:    Is your extraction protocol destructive? If I send abdomens in the wells instead of legs, can you extract DNA and retrieve them for future examination of the genitalia?
A:    Our extraction protocol is not destructive and tissues can be retrieved. However, it requires a time-consuming additional step that we cannot ensure on a large scale. A specific request should be sent to the campaign or campaign coordinator for a limited number of samples/plates.

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Q:    Who will ultimately "own" 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:    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 the CCDB produces 40 ul of genomic DNA, 2 ul of which is needed for each PCR amplification. Typically, there should be enough DNA extract left for sharing after the barcode is generated. Protocols for shipment of dehydrated DNA samples have been developed, ensuring long term stability at room temperature and making exchange of DNA samples easy via regular mail carriers.

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Q:    What is the procedure to get more DNA data for specimens besides the DNA barcode fragment? If someone is interested in sequencing other gene fragments for phylogenetic or other purposes, can they provide funding to the CCDB to have the amplification and sequencing done there?
A:    The major focus of the barcoding initiatives is the standard fragment of the mitochondrial gene COI used as a DNA barcode. However, if common interests arise along the project process and multi-gene analysis is needed, running non-COI genes is possible especially if some funds can be allocated to the CCDB to compensate chemical consumables and technicians' time. However, the feasibility will depend on the availability of the facilities at the CCDB and the time that the person at the lab end can contribute.

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Q:    What is the policy on authorship and data sharing?
A:    There are many different situations related to publication of DNA barcoding data. Collaborators can take the initiative of leading some publication projects using their own DNA barcoding data and possibly those in other projects they have access to (upon agreement, and possibly co-authorship with managers and/or contributors to these other projects). Depending on their role, campaign coordinators may or may not be associated to these publications. On the other hand, project coordinators having a broader overview of DNA barcoding projects can lead the assembly of manuscripts with a broader scope and will then invite all involved collaborators for co-authorship. Reciprocal agreements should be arranged at the early stages of publication projects to avoid misunderstandings and make the process as smooth and as fair as possible for all participants.
Data sharing is made easy by the structure of BOLD projects. Its implementation is the responsibility of campaign coordinators and project managers.

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Q:    Will the DNA data be automatically made public or would it wait until publication?
A:    Within the context of the iBOL Lepidoptera campaign, all the records and the related DNA barcodes will fall under the iBOL data and release policy. Basically, sequence data (sequences and trace files) will be released immediately along with geographic data and basic taxonomic information.

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Q:    What is the typical turnaround time for processing specimens at the CCDB in Guelph?
A:    All specimens will go through a chain of processes until the sequences reach the BOLD system. Typically, it takes 1-2 weeks from the DNA extraction step to the upload of the sequences resulting from the first PCR amplification (bidirectional full length DNA barcode). Samples requiring failure tracking and a second pass of amplification are usually processed within the next 2-4 weeks. Collaborators are stressed to take much care of the completeness and accuracy of the data they submit (in particular images and GPS coordinates) since it is not unusual that the pre-processing steps take enormous amount of extra time.

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