Non-human DNA Flashcards
LO
*** How can animal DNA be useful in a forensic context?
* How can we apply forensic techniques for DNA identification in the field of wildlife forensics?
* What non-human DNA research is Kings forensics undertaking?
**
Most DNA evidence collected from crime scenes is human, but sometimes the crimes directly involve non-human DNA, in what instance is this the case?
Wildlife poaching
Some evidence that can be left at a crime scene that is non-human can be what?
Animal, plant, microbial: Hairs, blood, tissue, fluid
Why is it important to distinguish between human and non-human DNA?
Exclude irrelevant samples
Link: scene- suspect- victim
Analyse non-human DNA which is relevant to case
What do we consider when talking about non-human DNA?
o Animal DNA
o Plant DNA
o Microbial DNA
When may plant DNA play a part in forensics?
- Identification of illegal plant material (plant DNA)
o Cannabis
o Timber: Ebony, rosewood, satinwood- illegal trading and logging - Pollen and other plant material at crime scenes- sticks to clothing so can help link forensically
When may microbial DNA play a part in forensics?
Microbial DNA
o Anthrax- on mail
It what areas is animal DNA important in forensics?
- Food fraud
- Post-mortem interval
- Wildlife forensics
- Another animal DNA
Whats meant by a DNA barcode?
Short, homologous sequences of DNA (vary between species but remains conserved within a species)
In what circumstances is considered ‘food fraud’?
- Charging a premium price
o Olive oil
o Caviar
o Mozzarella - Using cheaper ingredients
o Horsemeat rather than beef - Food adulteration
- GM crops
With the horsemeat scandal, what did eurofins offer?
When have blowflys typically been used in forensics?
Whats the method behind this?
Entomology has traditionally been used to establish PM interval of the deceased
Method:
o Collection of insects (e.g., maggots)
o Incubation of insects until species can be identified by microscopy
o Insects collected can be compared to its species’ life cycle
o Extrapolate age of insect at time of collection and PMI
Whats the limitations to blowfly entomology?
o Incubation of insects take time- need facilities and expertise
o Species identification by microscopy can be subjective
What is a molecular genetic tests thats being done on blowflies?
Sensitive methods to identify insect species
What is some research going on into blowflies?
DNA profiles from the deceased can be obtained from the gut of the insect
Tell me about DNA barcoding and some target markers
What do you want from your DNA barcodes?
- Method used for identifying species using a short, standardised sequence that varies between species but remains conserved within a species
**Target markers with DNA barcoding: **
o Cytochrome c oxidase I (COI or COX1) gene found in mtDNA genome- generally what is used for animals
o Internal transcribed spacer (ITS) rRNA, often used for fungi
o RuBisCO, often for plants
o 16S rRNA and 18s rRNA, often used for micro-organisms
*** What you want for DNA barcodes: **High taxonomic resolution, specific conservation between species
What is the process behind DNA barcoding?
With a blowfly species ID, what are most of the markers (and example) and where are they found?
- Most markers are mitochondrial markers
- Within the coding and non-coding regions of mtDNA
- Popular markers
o COI and COII
o Cyt b
o 12S
What is wildlife forensics?
What does this field use scientific procedures to do?
- Wildlife forensics is the application of science to legal cases involving wildlife
- This field uses scientific procedures to investigate wildlife- related crimes involving the exotic pet trade, poaching, other illegal hunting activities
What are endangered species at risk to?
* Trophies: tusks, horn, pelts, skins
* Food: illegal whale hunting, bush meat, caviar shark fin soup
* Medicines: bears gall bladder, rhino horn, tiger parts
* Sport: fox hunting, illegal killing of birds of prey
* Exotic pets: African grey parrots, marmosets, meerkats, poison dart frogs
* Fashions: furs
Wildlife forensics
International wildlife trafficking, or “wildlife and forest crime” (INTERPOL designation)
o Generates between $7 and $23 billion annually
o Grows at approximately x3 the growth rate of the global economy (between 5-7% a year)
o Typically defined as the smuggling, poaching, capture, and/or collection of protected or managed species, and products or derivatives thereof
What are some biosecurity threats?
Whats CITES?
- Convention on international trade in endangered species of wild fauna and flora
- An international agreement between governments. Its aims is to ensure that international trade in specimens of wild animals and plants does not threaten their survival
- More than 38,700 species of flora and fauna are categorised as endangered
Can you enforce legal action against poachers if the animal part/ remains found are beyond recognition?
e.g., carcass, meat, horn
No
Over 1/3 of IWT crimes do not result in prosecution due to improper identification of species
What can be done in order to identify the species?
- Microscopy of hair
o Classification of hair features under magnification - Immunological testing of blood
o HPLC
o Electrospray ionisation mass spectrometric analysis
What are the requirements for CITES to investigate?
The investigative questions to address the identification of animals and plants, or their parts and derivatives, can generally be categorised into 5 groups:
1. The species involved
2. The geographic origin of a specimen
3. The wild or captive/ cultivates source of a specimen
4. The individual origin of a specimen
5. The age of a specimen
Tell me about the Rhodis database
- Poaching is soaring
o Rumour that a former politician in Vietnam was cured of cancer using a rhino horn remedy
*** RhODIS- Rhino DNA Index System **
- Contains DNA profiles of individual rhinos
o 3,968 individual rhinoceros DNA in database
o 22 STRs + sex marker
o Enables the linking of rhino remains to a particular rhino
What are the stages to STR profiling in non-humans?
- Same concepts as standards (human) STR profiling:
o DNA extraction
o Amplification
o CE
How is Rhino DNA obtained?
Tell me about the Ursaplex database
- STR and sex determining panel for individual genetic identification of North American Black bears
- Human-wildlife conflict and poaching applications
Birds of prey
Why have MPS multiplexes been developed?
- Conservation genetics
- Wildlife crime forensics
- Leverage MPS technology (paralleling human forensics)
- Exploit newly emerging whole genome reference sequences
- Improve on existing markers
How were markers for non-humans discovered and whats the filtering criteria?
*** STR finding tools **
o RepeatMasker
o Msatcommander
o GMATA
*** Filtering criteria **
o Amplicon length: 100-400 bps
o Appropriate flank and primer design sites
o Repeat motif length: 4-6 inclusive
o Number of repeats: optimum 11-16
MPS data analysis
Birds of prey
Historical case investigating a falconer who sold allegedly captive-bred young
o Applies 14 STR multiplex
o 49 birds tested
o CE and MPS: additional 8 alleles were observed with MPS
o Relationship testing to compare genotypes to the breeding pairs owned by the accused
o 23 young birds were clearly excluded as being the offspring of any of his breeding pairs
Tell me what the source for taxonomic ID is
- Taxonomic source= species, genus, etc
- Regions from the mitochondrial genome are the primary choice for forensic taxonomic identification
What did taxonomic ID from ivory tell us?
- Cartier at al., 2020
o 21 specimens submitted for testing - Morphological analysis revealed it was impossible to determine where the ivory was elephant or mammoth
- Next generation sequencing:
o Higher throughout and increased sensitivity means better recovery of full target sequence from poor sample
o Better for mixed samples as individual reads can be assigned to separate taxa
Traditional medicines
- Global annual market of $83 billion (WHO, 2013)
- Often contain multiple species
- DNA identification is challenging due to presence of PCR inhibitors (protein, lipids, etc) and degradation through processing
o Extraction protocol important
o Arulandu et al 2019
Whats Metabarcoding?
What method does it use and what does this enable?
- Target several barcodes for simultaneous detection of multiple species in complex samples
*** Massively parallel sequencing enables this: **
o Reads aligned
o Sequences BLAST searched against NCBI for taxonomic classification
o “Mini-barcodes” useful for degraded samples
Whats the minion sequencer and what are the benefits and limitations?
- Portable NGS solution
- Benefits:
o Long-read output
o Low initial start-up cost ($1000)
o Simple, rapid library preparation protocols - Limitations
o High error rate: 5-25% compared to 0.01% on MiSeq
What are the challenges to non-human DNA?
Reference samples and database
Quality control
Cost and availability of testing
Tell me about the challenge with **Reference samples and database **
- Human forensic genetics
o Databases e.g., NCBI, EMBL, 1000 genome project
o Reference genomes e.g., rCRS - Wildlife forensics genetics
o Many different species
o Mostly only have databases for “appealing” animals - EarthBioGenome Project and ForCytDNA
Tell me about the challenge with quality control
- Human forensic genetics
o Accreditation and ISO standards
o Rigorous and curated databases lie EMPOP or THRD - Wildlife forensic genetics
o Adhered to international quality standards often not priority - OSAC wildlife forensic biology subcommittee and society for wildlife forensic science
Tell me about the challenge with cost and availability of testing
- Sending samples off-site= expensive and complicated
- MPS remains out of reach for many labs doing wildlife forensics
- African Wildlife Forensics Network: network of wildlife forensics stakeholders for the implementation of forensic science and crime scene services across Africa
- MinION protable technology?
DNA evidence can also be utilised in relation to other types of forensic evidence collected from a scene, give two examples
*** Ballistics **
o DNA profiles can be obtained from touch DNA or biological evidence recovered from cartridge casings and weapons
*** Fingerprints **
o DNA profiles can be obtained from touch DNA
Tell me about obtaining fingerprints from ivory,
whats the aim, a probelm encountered and the outcome
- Low tech equipment needs at police departments, ports and in the field
- AIM: to recover finger-mark friction ridge detail from seized wildlife items using effective, cheap and easily deployable technology
- Partnership: The metropolitan police and the international Fund for Animal Welfare (IFAW)
- Together delivered a fingerprinting kit that works on ivory and horn
Fingerprints on ivory continued…
How can domestic animals be involved in forensics?
- Cats and dogs most common
- Crimes can take place in settings where animals are present
- Crimes can directly involve animals:
o Animal attacks e.g., badger baiting, fox hunting
o Dog fighting
How can Domestic pets be analysed?
- Sample concepts as standard (human) STR profiling
o DNA extraction
o Amplification
o CE - Examples “MeowPlex”
o 11 polymorphic tetranucleotide loci
o Identify cats
When barcoding for mixtures what is used and what are the advantages of MPS here?
- 16S rRNA
- Advantages of MPS:
1. Short PCR amplicons
2. Universal PCR-primers i.e., no need for prior species information
3. High capacity for sample multiplexing
4. Deep sequencing
When using metabarcoding and trying to identify multiple species from mixed sources, what are the three mtDNA targets used?
- Three mtDNA targets:
**o 16S rRNA
o Cytochrome b
o Cytochrome c oxidase I ** - In silico studies show they can differentiate vast majority of species (4500 tested)
- Tested on real cases: bite marks in murder case identified to be from a dog
Whats are the limitations to next-generation signalling?
- With great sensitivity comes **great contamination **
o Validations must address acceptable contamination levels - Inhibitors commonly co-extracted with forensic samples can negatively impact ligation of indices or adaptors during library prep
- Substantial **cost **investment to bring NGS into casework
-
Storage and analysis of large amount of sequence data
o One illumine MiSeq v3 run generates up to 15GB of data
With Forensic genetic identification of Sharks involved in human attacks what two genetic approaches are used?
o Barcoding to identify shark species
o Micro satellite testing to confirm species and for individual identification
**Conclusions **
- Animals can be perpetrators e.g., animal attacks
- Animals can be victims e.g., wildlife forensics, animal abuse
- Animals as evidence: e.g., food fraud, blowflies for PMI, hairs at a crime scene
- Depending on case we must be able to:
o Identify specific animal: STR and mrDNA testing
o Identify species of animal(s): Barcoding and metabarcoding
