Oct 18, 2024
Version 2
DToL Tissue and Blood Sampling Standard Operating Procedure: Chordata: Vertebrata: Aves V.2
- Inez Januszczak1,
- Nancy Holroyd2
- 1Natural History Museum;
- 2Wellcome Sanger Institute
- Darwin Tree of Life
Protocol Citation: Inez Januszczak, Nancy Holroyd 2024. DToL Tissue and Blood Sampling Standard Operating Procedure: Chordata: Vertebrata: Aves. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3yedvzp/v2Version created by Inez Januszczak
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 03, 2023
Last Modified: October 18, 2024
Protocol Integer ID: 109454
Keywords: birds, bird, ave, sampling, protocol, Darwin Tree of Life, Wellcome Sanger Institute, Natural History Museum, Sample Operating Procedure, DToL, tissue, and, blood, Sampling protocol, whole genome, biobanking, sequencing
Abstract
This Standard Operating Procedure (SOP) contains guidance on how to sample tissue and blood from bird specimens submitted to the Darwin Tree of Life (DToL) project.
Please note this SOP covers sampling tissue and blood either during an appropriate veterinary procedure or post-mortem, with assumed veterinary knowledge.
It is applicable if the submitter/centre in question has opportunities to sample tissue from birds that are being either being euthanized for clinical purposes, undergoing veterinary procedures. or eligible biobanked carcasses (storage dependent).
This SOP does not cover procedures regarding bio banked, pre - dissected tissue or blood.
This SOP is covers birds specifically - please refer to the DToL Chordata: Vertebrata SOP for a general overview.
For vouchering and sampling fish specimens, with the idea of donating tissue and carcasses specifically to the Natural History Museum in London, please refer to the DToL Chordata: Vertebrata: Fish Vouchering SOP.
This is version two of this SOP, and may be subject to change in the future.
Attachments
Guidelines
Tissue/blood sampling post mortem
If the centre in question has opportunities to sample tissue and blood from birds that are being either being euthanized for clinical purposes, undergoing veterinary procedures or biobanked carcasses, such samples would be very useful for the Darwin Tree of Life (DToL) project.
Regulatory compliance considerations
Animal Welfare - the samples will only come from birds undergoing veterinary care, euthanized for health reasons or biobanked carcasses that are eligible for dissection/ tissue preparation.
Permissions may be requested depending on the species in question.
Note
Regarding frozen, whole carcasses
Please note the ideal storage conditions for a biobanked carcasses will be at -20 degrees or lower. Carcasses that have gone through multiple types of preservation should be handled on a case by case basis - please contact the Natural History Museum (NHM) and Sanger Sampling teams, listed at the end of the SOP.
Carcasses stored in ethanol (at room temperature or lower) will only be considered in exceptional circumstances, for example if it is a rare species.
Please note: If the frozen carcass is in exceptional condition, or of a rare or unusual species, the NHM will consider obtaining the carcass and performing the dissection. This is to comply with the voucher/ skin requirements of the NHM Birds curatorial staff.
Similarly if the submitter in question does not have dissecting facilities but does have a frozen carcass of a specimen of interest, the NHM will consider doing the dissection separately and liaise with the Sanger Institute on the behalf of the submitter.
This will require the submitted carcass to NOT be dissected and instead sent to the museum or to a relevant partner whole, and still frozen. The NHM could provide postage and shipping facilities to do this. Do enquire with the NHM sampling team and Sanger sampling team as appropriate (email addresses at the bottom of the SOP).
Logistical Considerations
A -80 freezer is required for tissue sampling (suggested brand Fryka B35-85 under bench, ETA 2-3 weeks 80kg. 58x76x54cm). Preserving the tissue directly on dry ice is also an option.
Prior to submission
1. It is important to check whether DToL would require the sample.
The submitter/centre in question would be shared a species target list. As this is opportunistic sampling, the list should be regularly checked to see what species are needed.
Note
Please ensure each submitted specimen has available all its relevant metadata (Date of Collection, Collector, Identifier, Collection location, Species, Specimen ID).
The Darwin Tree of Life project will not accept specimens which don't have this information.
2. Sampling
Aside from the specimen itself, labelled cryotubes that are able to withstand -80 degrees cold storage will be required (for the tissue) as well as ziplock/ plastic bags to store the tubes and carcasses.
Each specimen requires two identical ID numbers; one will be used to label the ziplock bag of tubes containing the samples, the other to label the remaining carcass (if applicable).
The NHM is able to provide unique ID numbers, otherwise any matching ID will be appropriate, preferably a series of numbers.
The NHM is able to provide cryotubes with unique IDs if this aids the archiving.
Note
Frozen carcasses will need to be partially defrosted prior to tissue preparation. The most frozen part of the tissue should thus be removed and preserved.
If the specimen (in any of the listed scenarios) is suitable:
a. Take a photo of the specimen – a dorsal and ventral photograph, with the specimen ID clearly visible in the frame of the photo. This photo will act as a photographic voucher if it is not possible to retain to the rest of the carcass post dissection.
b. Dissect out multiple samples per tissue (listed below), maximum 0.5g per tube (around pea-sized) as is practical from the size and condition of specimen.
Ideally minimum of 3 and up to 8 pieces as a ballpark amount, although if more tissue can be sampled this can be used for other future research.
Inject out blood IF POSSIBLE (see Notes for more details)
Muscle
Heart
Liver
Brain
Gonads
Please note listed tissues are ranked in order of priority.
Frozen carcasses will need to be partially defrosted prior to tissue preparation. The most frozen part of the tissue should thus be removed and preserved.
Note
Regarding blood – 200ul is the ideal amount, as it provides enough material for HiC, PacBio and RNA.
However, sometimes the bird is too small to provide this.
In that case, the absolute minimum required would be 25ul, which would allow for one extraction and HiC attempt.
Up to 50ul would allow for two extractions and two HiC attempts, thus this would be the recommended lowest volume of blood to be obtained.
After the blood is removed, it must be flash frozen at -80 degrees with molecular grade ethanol (100%).
The ethanol to bird blood ratio should be 3:1.
If this is not possible, flash frozen without ethanol is preferred.
If flash freezing the blood is not an option, it can be stored in ethanol only (molecular grade).
c. Put the tissue immediately into clearly labelled tube (with a unique tube ID) and place immediately into the -80 freezer/dry ice. Place all tubes into a ziplock bag along with tissue information- keeping tissues separate can also help with the labeling. Ensure each ziplock bag of samples has its own specimen ID label.
The NHM is able to provide tubes with unique IDs if this helps with the archiving.
d. Put the rest of the carcass into a ziplock bag, double bag the carcass, then label with the second identical specimen ID.
e. Add the appropriate data to an Excel spreadsheet, clearly linking the specimen ID, tube barcode and tissue type. A draft spreadsheet is attached to this protocol.
f. Email the DToL NHM sampling coordinator regarding the samples obtained.
The NHM is interested in obtaining the rest of the carcass as a specimen voucher, regardless of its condition after post-mortem. If it is possible to store the rest of the carcass in minimum –20 conditions, dry frozen, then it should be stored. The carcass must thus also remain frozen throughout its transportation to the museum.
3. Submission
Once the submitter/center has collected at least 5 sets of samples from 5 specimens, and thus potentially 5 carcasses, they should contact NHM and Sanger DToL Sampling teams who will arrange for their collection.
Note
Please note - an Excel spreadsheet with Specimen ID, Species, tube ID and tissue type of all submitting samples must also be kept (including whether a carcass was kept or not).
Each tube ID must match the tissue sampled.
Ensure that samples from separate animals are kept separate.
This spreadsheet must also be emailed to the NHM and Sanger DToL Sampling team. Any photographs of the specimens must also be saved and emailed to the DToL NHM Sampling coordinator.
When submitting/ considering a submission, please contact:
Safety warnings
Please note - it is assumed all appropriate zoonotic disease checks have been carried out prior to any post mortem or veterinary procedure.
Ethics statement
Regulatory Compliance Considerations
Animal Welfare - samples will only be accepted if they come from animals undergoing veterinary care. euthanized for health reasons or from a valid biobank collection.