COI Barcoding Notes
I submitted my first samples for COI barcoding in March 2024. I'm recording the process here because, while it was easy, it definitely has several specific steps that I found useful to document for future submissions.
What is COI barcoding?
COI is the abbreviation for the Cytochrome Oxidase I mitochondrial gene, a widely-used taxonomic marker to identify animals. An easy gene to isolate and amplify, it has become the most sequenced gene for most animal groups, allowing for a large database to compare specimens and species. Barcoding of genes is like scanning a barcode on a grocery store item. The unique section of lines on a barcode of an apple tell the cash register that the item is a fuji apple. The unique genetic sequence of the COI gene isolated from the piece of fruit can identify that it is specifically a fuji apple.
COI is the abbreviation for the Cytochrome Oxidase I mitochondrial gene, a widely-used taxonomic marker to identify animals. An easy gene to isolate and amplify, it has become the most sequenced gene for most animal groups, allowing for a large database to compare specimens and species. Barcoding of genes is like scanning a barcode on a grocery store item. The unique section of lines on a barcode of an apple tell the cash register that the item is a fuji apple. The unique genetic sequence of the COI gene isolated from the piece of fruit can identify that it is specifically a fuji apple.
Sending in Sequences for Barcoding
While it may be cheaper to process your samples on campus, I did the easiest thing, which was to submit my samples to the Canadian Centre for DNA Barcoding (CCDB).
Send an email here: inf[email protected]
Include the general taxonomy (ex. ants and bees) and age of your samples. If you have many samples, you will be placing them in 96-well microplates. The last well will be empty, so think about your samples in sets of 95.
Step 1: Determining the number of samples and the sequencing price
There are two options: https://ccdb.ca/pricing/
1. Full cost recovery rate: is CAN $2,200 ($1,618 USD) for 95 specimens of animal tissue
2. Barcode library contribution rate*: CAN $1,250 ($919 USD) for 95 specimens of animal tissue
To qualify for the barcode library rate, you need to meet the following conditions:
* Barcode Library Contribution rate is available to Providers that align their research efforts with our mission to assemble a DNA barcode library for all eukaryotic species. Barcode Library Contribution rate applies only to submissions where all required data elements are uploaded to BOLD. To qualify, Providers must conform to the following metadata quality standards:
1. Collection location with GPS co-ordinates
2. Voucher specimen images (representative images are not acceptable, for more information please refer to the Photography Guide in the BOLD Handbook)
3. Museum voucher location and ID
4. Full taxonomy to the level of species or operational taxonomic unit
The Provider also must agree to data sharing in compliance with best practices established under iBOL Data Release Policy Phase 1 (Phase 2 data release will commence upon data publication, after 18 months from the date the sequence has been generated).
Once they know which rate will apply to your samples, they will send you a quote and one or more 96-well microplates (depending on the number of your samples) to sample into (one plate is used for 95 samples, and a negative control). You then follow the included guides for 1) tissue sampling specimens and 2) submitting the data and samples back to them.
While it may be cheaper to process your samples on campus, I did the easiest thing, which was to submit my samples to the Canadian Centre for DNA Barcoding (CCDB).
Send an email here: inf[email protected]
Include the general taxonomy (ex. ants and bees) and age of your samples. If you have many samples, you will be placing them in 96-well microplates. The last well will be empty, so think about your samples in sets of 95.
Step 1: Determining the number of samples and the sequencing price
There are two options: https://ccdb.ca/pricing/
1. Full cost recovery rate: is CAN $2,200 ($1,618 USD) for 95 specimens of animal tissue
2. Barcode library contribution rate*: CAN $1,250 ($919 USD) for 95 specimens of animal tissue
To qualify for the barcode library rate, you need to meet the following conditions:
* Barcode Library Contribution rate is available to Providers that align their research efforts with our mission to assemble a DNA barcode library for all eukaryotic species. Barcode Library Contribution rate applies only to submissions where all required data elements are uploaded to BOLD. To qualify, Providers must conform to the following metadata quality standards:
1. Collection location with GPS co-ordinates
2. Voucher specimen images (representative images are not acceptable, for more information please refer to the Photography Guide in the BOLD Handbook)
3. Museum voucher location and ID
4. Full taxonomy to the level of species or operational taxonomic unit
The Provider also must agree to data sharing in compliance with best practices established under iBOL Data Release Policy Phase 1 (Phase 2 data release will commence upon data publication, after 18 months from the date the sequence has been generated).
Once they know which rate will apply to your samples, they will send you a quote and one or more 96-well microplates (depending on the number of your samples) to sample into (one plate is used for 95 samples, and a negative control). You then follow the included guides for 1) tissue sampling specimens and 2) submitting the data and samples back to them.
Step 2: Preparing specimen tissue samples
Once you receive the 96-well microplate from the center, you fill it with your specimens according to their methods sheet. The final bottom right well, H12, is blank (no ethanol either). For my specimens, I used a leg from each bee or ant, but only a part of the leg for the very large bees. The methods include photos of sizes for tissue samples. I had mostly samples that had been in 95% ethanol (see right, yellow), as well as some pinned specimens (green and red). I filled every specimen well with 30uL of 95% ethanol. This is also clarified in the microplate instructions. The tissue sampling methods are below under "Instructions_Microplate." Upload your sample data to the CCDB sample datasheet, and BOLD (see xls datasheet below), the online barcoding database. |
instructions_microplate.pdf |
CCDB datasheet |
BOLD_specimen_datasheet.xls |
Step 3: Printing shipping information
Once you've filled up your sample plates, and input all the data into both the CCDB datasheet (sequencing center's data) and the BOLD datasheet (online searchable database), you're ready to mail your samples back to CCDB!
Make and print a shipping label that includes your return address. Also print out the checklist and BMAA (biological materials aggreement), and check off and sign these, respectively.
Once you've filled up your sample plates, and input all the data into both the CCDB datasheet (sequencing center's data) and the BOLD datasheet (online searchable database), you're ready to mail your samples back to CCDB!
Make and print a shipping label that includes your return address. Also print out the checklist and BMAA (biological materials aggreement), and check off and sign these, respectively.
to_print1_CCDB_ShippingLabel |
to_print2_CCDB_checklist.pdf |
to_print3_CCDB_BMAA.pdf |
Step 4: Packing samples
It is important to get this step right. Take your time and double-check each step.
These samples are 1) biological materials 2) with ethanol 3) going over international borders. If you fail to package them properly, they may get stopped or lost at the border, and not every mail carrier is allowed to carry ethanol specimens, even if its only a tiny amount.
Inside the package:
1. Make sure the microplate lids are completely pressed down
2. Wrap the microplate in paper towels (or cheesecloth, or other absorbent material)*
3. Place the wrapped microplate(s) in a plastic bag
4. Heat-seal the open end of the bag
5. Place the heat-sealed bag(s) into a larger bag, with more paper towels
6. Heat-seal this larger bag
7. Line a shipping box with bubble wrap, shipping paper, or other cushioning material
8. Place your samples in the box and ensure that they are cushioned in the shipping box
9. Add the checklist PDF and the BMAA PDF to the box
10. Seal the box with packing tape and tape the shipping label to it
11. Deliver the box to FedEx. The UTC FedEx can ship this. My single microplate cost $32 for standard shipping.
*If your samples are in vials, separate them so that no vial and/or bag contains more than 30mL of liquid. Total box content may not exceed 1L of ethanol.
As long as you are not working with infectious samples, or federally endangered/ threatened specimens, you are "Shipping Preserved Specimens (Non-Infectious) via IATA Special Provision A180."
FedEx is the most reliable carrier for this. I include it on the shipping label, but when you are there they will have you write “Scientific research specimens, not restricted, Special Provision A180 applies.”
It is important to get this step right. Take your time and double-check each step.
These samples are 1) biological materials 2) with ethanol 3) going over international borders. If you fail to package them properly, they may get stopped or lost at the border, and not every mail carrier is allowed to carry ethanol specimens, even if its only a tiny amount.
Inside the package:
1. Make sure the microplate lids are completely pressed down
2. Wrap the microplate in paper towels (or cheesecloth, or other absorbent material)*
3. Place the wrapped microplate(s) in a plastic bag
4. Heat-seal the open end of the bag
5. Place the heat-sealed bag(s) into a larger bag, with more paper towels
6. Heat-seal this larger bag
7. Line a shipping box with bubble wrap, shipping paper, or other cushioning material
8. Place your samples in the box and ensure that they are cushioned in the shipping box
9. Add the checklist PDF and the BMAA PDF to the box
10. Seal the box with packing tape and tape the shipping label to it
11. Deliver the box to FedEx. The UTC FedEx can ship this. My single microplate cost $32 for standard shipping.
*If your samples are in vials, separate them so that no vial and/or bag contains more than 30mL of liquid. Total box content may not exceed 1L of ethanol.
As long as you are not working with infectious samples, or federally endangered/ threatened specimens, you are "Shipping Preserved Specimens (Non-Infectious) via IATA Special Provision A180."
FedEx is the most reliable carrier for this. I include it on the shipping label, but when you are there they will have you write “Scientific research specimens, not restricted, Special Provision A180 applies.”
osu_ehs_shipping_instructions.pdf |
airline_special_provision_a180_info.pdf |
All done! You should get confirmations from the CCDB when your specimens have arrived. If you have any questions, feel free to contact the representative that responds to your original email! They are responsive and kind.