Title: Module 2: Visual and DNA Methods

Welcome to Module 2 of the Wildlife Forensic Science course.

Click Read to begin learning.

Welcome

Welcome to Module 2: Visual and DNA Methods.

The module enables the participants to achieve the following objectives:

- Explain how DNA forensic testing is used to identify wildlife.
- Assess the strengths and limitations of wildlife DNA forensic evidence.

Content for this resource was developed by TRACE Wildlife Forensics Network. Funding for the digitization of this content was provided through the USAID Combatting Wildlife Crime in Southern Africa Activity (VukaNow) in South Africa Contract No. 72067418C00005

The contents are the sole responsibility of the authors and do not necessarily reflect the views of USAID’s VukaNow Activity, USAID, or the United States Government.

Introduction

To prosecute or adjudicate over criminal cases in which wildlife forensic evidence is submitted, it is important for you to have a level of understanding regarding the forensic science techniques that have been used to produce the evidence. Wildlife forensics utilizes a broad spectrum of analytical methods and reference materials to identify biological evidence*. As mentioned in Module 1, the primary techniques used in wildlife forensics are visual (morphological) analysis and DNA analysis. Here, we look at these methods in more detail and the key considerations in their use when generating evidence for court.

* Jane E. Huffman and John R. Wallace, eds., Wildlife Forensics: Methods and Applications. Chichester, West Sussex, UK: John Wiley & Sons, Ltd., 2012; Eleanor E. Dormontt, et al, “Forensic timber identification. Biological Conservation,” Biological Conservation no. 191 (2015): 790-798

GLOSSARY

This glossary includes key terms relating to wildlife forensics.

Click each term to reveal its definition.

Approach

A high-level description of the science being used – for example, DNA analysis.

Deoxyribonucleic acid (DNA)

A long molecule found inside the cells of every animal and plant. A DNA molecule contains genetic code that holds instructions for cells to make proteins.

DNA barcoding

A standardized approach to comparing DNA sequences generated from an unknown specimen with a DNA sequence reference database in order to identify its species of origin.

DNA base pairs

Letters of the genetic code. There are four bases or ‘building blocks’ in DNA: adenine (A), cytosine (C), guanine (G) and thymine (T). To form base pairs, adenine always pairs with thymine (A-T) and cytosine always pairs with guanine (C-G).

DNA marker

A specific individual section of DNA.

DNA profiling

A process where short regions of repeated DNA (1-6 bases long) are measured at markers around the genome and the results from multiple markers are combined to create a profile for the individual sample. This is also known as individualization.

DNA sequencing

A process where the genetic code is determined at a DNA marker.

Evidence security

A documented process for ensuring that evidence is kept demonstrably secure from crime scene to court room, through to subsequent storage or disposal.

Good Laboratory Practice (GLP)

General quality assurance (QA) requirements for laboratory testing.

Meta-data

In the context of forensic reference samples, meta-data refers to the non-genetic data that may be available regarding the sample, such as the time and location of sampling, information about the individual sampled, etc.

Method

The protocol being followed, for example, to generate a DNA sequence.

Method validation

A procedure to verify that an analytical method is suitable for its intended purpose and can generate robust, reliable data.

Polymerase chain reaction (PCR) amplification

A process for making millions of copies of specific individual sections of DNA (known as DNA markers).

Quality assurance (QA)

The management process that ensures that the delivery of a product or service, for example a laboratory test, is quality controlled.

Quality management system (QMS)

A documentation system that describes all of the steps involved in the delivery of a quality assured service or product.

Technique

The specific approach being used, for example, DNA sequencing.

Scientific test

What the scientist is actually attempting to do; for example, generating a DNA sequence to identify the species.

In the next sections, we will explore the analytical methods used in wildlife forensic science.

Analytical Methods in Wildlife Forensic Science

VISUAL (MORPHOLOGICAL) IDENTIFICATION

Intuitively, using visible physical characteristics is the simplest approach to identifying wildlife and wildlife products in trade. Indeed, in cases where the evidence retains sufficient characteristics and appropriate expertise exists, visual identification is usually the quickest and least expensive form of forensic analysis.

Visual identification, technically referred to as “morphological identification”, is normally applied to determine the taxon of origin. In some instances, morphology can be used to identify the species of origin. But more often, this method is limited to identifying a higher taxonomic rank of a sample, from its genus to kingdom.

Click on the markers to see the taxonomic classification for a lion.

Kingdom

Animalia

Phylum

Chordata

Class

Mammalia

Order

Carnivora

Family

Felidae

Genus

Panthera

Species

Panthera leo

Source: 2015 Encyclopeaedia Britannica, Inc.

Visual identification is extremely effective as a rapid, affordable screening test, as it is possible to train law enforcement officers in the use of identification keys in the field, or to obtain initial opinions through the use of digital photography and mobile communications.

The availability of experts capable of performing a detailed, objective analysis that is accepted in court limits the use and application of visual identification in forensic analysis. This limitation is particularly acute in countries lacking well-established natural history institutions with the appropriate reference collections and expert staff.

Click here to access the World Wildlife Fund ivory identification guide, developed by the United States Fish and Wildlife Service National Forensic Lab.

GENETIC IDENTIFICATION (DNA ANALYSIS)

Observed genetic differences among species, populations and individuals can be used to identify and categorize biological evidence. The use of DNA analysis in wildlife forensics is most developed for the process of species identification (including "DNA barcoding"), where it is now routinely used to identify the taxon of origin. However, with the availability of species-specific DNA profiling systems and reference data, DNA analysis can also be used to match samples at an individual level or support/refute parentage claims.

There are four steps common to all types of DNA analysis:

DNA is a molecule found inside the cells of every animal and plant. To analyze DNA, it is necessary to isolate it in a purified form. This process is known as DNA extraction. During DNA extraction, the cells within the biological evidence item (for example, blood, meat, leaves, horn) are broken down, the unwanted cellular components are removed, and the DNA is retained.

The DNA extract usually contains a relatively low number of copies of the whole genome of the individual plant or animal sampled. For DNA analysis, we only need to focus on a small part of the genome, but we need lots of copies to allow us to measure the DNA (next step). PCR (polymerase chain reaction) amplification is a process for making millions of copies of specific individual sections of DNA (known as DNA markers). It is rather like photocopying a single page from a book millions of times.

After PCR amplification, we have millions of copies of our target DNA marker that we need to measure. There are two main ways to measure genetic variation: DNA sequencing or DNA profiling.

The DNA results from a forensic evidence sample have to be compared with reference materials to be understood. Comparison with reference data allows forensic scientists to identify the species, population or individual that the evidence item originated from. This last stage requires the most experience and expertise from the forensic scientist.

Now that you are familiar with the general approach to DNA analysis, let’s look more closely at the two techniques for DNA measurement: DNA sequencing and DNA profiling.

DNA SEQUENCING FOR SPECIES IDENTIFICATION

DNA sequencing is where the genetic code (ACGT pattern) is determined at a DNA marker. DNA sequences for wildlife forensics are usually around 300-500 bases (DNA letters) in length.

Following DNA extraction, the DNA is subject to PCR amplification which targets species diagnostic DNA sections of DNA. The resulting PCR product is subject to DNA sequencing. The sequences are analyzed and assessed for quality. Good quality sequences are then compared electronically against large databases of reference sequence data from multiple species. The sequence similarity is recorded for the most similar sequence and for sequences from the next nearest species. Results are interpreted and a species identification is made where possible.

In DNA sequencing for species identification, the target DNA marker, which displays sequence variation among species, is amplified via PCR. The resulting PCR product is sequenced to generate around 400 base pairs of sequence data for the unknown sample. This is then compared to a reference data base to identify the species of the unknown sample, based on sequence similarity analysis.

Principle
DNA species identification is based on the principle that variation in the genetic code of each individual is associated with its species of origin. By sequencing DNA markers that show this type of variation, we can match an unknown sample to its species of origin, based on the similarity of its DNA sequence to reference sequences from multiple species in a database. The process is sometimes referred to as DNA barcoding.

The DNA markers used for species identification are usually located in the mitochondrial genome, a special kind of DNA only inherited from the female parent and found in every cell. Mitochondrial DNA has several characteristics that make it very useful for forensics.

There are several gene regions within mitochondrial DNA in which the DNA sequence is typically conserved within species (all members of the species have very similar or identical sequences) but vary among species. Examples include genes such as cytochrome b and cytochrome oxidase 1, which are often used for species identification. The length of the DNA sequence influences the strength of species identification. Ideally, DNA sequences longer than 400 base pairs (letters of the genetic code) are generated, but species identification is also possible with shorter sequences (for example, 200 – 400 base pairs).

Mitochondrial DNA is present in multiple copies within each cell and all copies are identical, which makes it relatively easy to sequence in the laboratory. This method has been comprehensively validated and used in law enforcement for over 30 years.

Interpretation of DNA sequence results
The DNA sequence from the evidence will either be a 100 percent match for a species in the reference database or will be less than 100 percent match.

100 percent matches

If there is a 100 percent match, it is likely that the species of the evidence item is the same as the species of the matching reference sample.

However, there are two key points to consider:

1. Do you trust the accuracy and authenticity of the reference data?
2. What is the species gap? i.e. What is the percentage difference between the sequences of the top matching species to the next closest species? Can they be confidently separated? Note a species gap of more than two percent is preferred.

Answer the True/False question below about the implications of this.

Multiple Choice

Q: If there is a 100 percent match between an evidence item and a sample in the reference database, the court can conclude that they are from the same individual.
- True (Incorrect! DNA sequencing matches an evidence item to its species of origin. It is likely that the species of the evidence item is the same as the species of the matching reference sample.)
- False (correct) (Correct! DNA sequencing matches an evidence item to its species of origin. It is likely that the species of the evidence item is the same as the species of the matching reference sample.)

Less than 100 percent matches

There are no absolute rules for how to deal with this common situation. The following helps with interpretation, but ultimately, the court must rely on the expertise of the forensic scientist:

- >98 percent match — likely to be the identified species or very close relative (for example, Mountain zebra versus Plains zebra).
- 95<percent<98 match — likely to be the same genus.
- <95 percent — could be a relatively unrelated species.

Note the following points to consider:

- The taxonomy of the species group should be examined to see if all related species have reference sequences.
- The gap in sequence similarity to the next closest species should be evaluated and, ideally, be more than two percent.
- What taxonomic level needs to be reported? If genus level identification is sufficient then this may be a more robust result than a species identification. For example, the scientist may be certain the sample is from a zebra but may not know which species of zebra.

Answer the True/False question below about the implications of this.

Multiple Choice

Q: If there is a match greater than 98 percent between an evidence item and a sample in the reference database, the species gap needs to be evaluated before concluding that the species of the evidence item is the same as the species of the matching reference sample.
- True (correct) (Correct! The percentage difference between the sequence of the evidence item and the sequences of top matching species to the next closest species should ideally be greater than two percent.)
- False (Incorrect! The percentage difference between the sequence of the evidence item and the sequences of top matching species to the next closest species should ideally be greater than two percent.)

Now, let us look at the process of DNA profiling.

DNA PROFILING (INDIVIDUALIZATION)

DNA profiling is where short regions of repeated DNA (1-6 bases long) are measured at markers around the genome and the results from multiple markers are combined to create a profile for the individual sample.

Following DNA extraction, PCR amplification is conducted targeting an identical set of multiple DNA markers in the two samples considered to possibly match. The PCR products are then detected and measured using a genetic analyzer, which is used to produce DNA profiles. The data are checked for quality and DNA profiles are then recorded for each sample. These DNA profiles are then compared.

Principle
The DNA in every cell of an individual is identical and the DNA from different individuals is always different (except for twins). This means that if we compare the DNA from two samples and we observe differences between them then we know that they came from different individuals and a single source for the evidence can be excluded.

DNA profiling, or individualization, is where we investigate whether two samples definitely did, or did not, come from the same individual. Stating that samples do not have a single source is known as “exclusion” and is relatively straightforward to achieve.

Interpretation of DNA Results
So, we have two possible explanations for matching DNA profiles:

1. They come from the same individual.
2. They come from two individuals that share the same profile by chance.

To assess which explanation is more likely, we need to estimate the probability of observing matching profiles under each explanation.

If we propose that the profiles match because they come from the same individual, then the probability of observing this result is 1 (it is a certainty).

If we propose that the profiles match because they come from two individuals that share the same profile by chance, then the probability of observing this result depends on the frequency of the DNA profile in the population.

To evaluate this probability, we need to know about the genetic diversity of the population as well as having DNA profiles for the two samples.

We do not need profiles from every individual, but it is preferable to have samples from at least 100 individual animals or plants from the species in question.

The number required will vary depending on the size and structure of the different populations within the species in question. The population reference data allows us to calculate the probability of seeing the same profile twice in different individuals in the population.

The overall implication of this, is that DNA profiling systems for identification (sample matching) are quite difficult to develop and are not available for many wildlife species. But there are exceptions to this, such as white and black rhinoceros.

For these species, for example, a horn can be matched back to its carcass with a very high degree of confidence.

EXAMPLE OF PROFILE MATCHING

Imagine you found a dead animal that appears to have been illegally killed. You next identify a suspect for the killing and discover blood stains on their clothing.

By taking a reference DNA sample from the animal, you generate a reference DNA profile for that individual animal. Then you analyze the blood-stained clothing, first to confirm that the blood originated from the same species of the dead animal (via DNA sequencing), next to generate a DNA profile. The two DNA profiles are then compared.

Click on the arrows to learn how DNA profiling is used to determine the probability of the sample taken from the suspect’s clothing matching the dead animal.

Now that we are familiar with visual and genetic analysis, let us look at when each analytical method is applied in wildlife forensic science.

USES OF VISUAL AND GENETIC IDENTIFICATION

Below is a generalized overview of how visual and DNA analysis techniques can be applied to investigative questions in wildlife forensics. Note that all analyses are implemented on a case-by-case basis and factors such as the evidence item (type/condition/age) and specific point to prove in an investigation will dictate whether any particular technique will be suitable.

Click on the markers to learn more.

But see research on determining geographic provenance of African elephant ivory as a working example.

Parentage testing requires a DNA profiling system and a system of recording legitimate captive bred relationships.

DNA profiling systems exist for certain species, for example, rhinoceros, tiger etc., and can be readily developed for additional species.

Next, we will look at the importance of reference materials and databases in wildlife forensic science.

Reference Materials and Databases

Wildlife forensic science depends heavily on the existence and accessibility of authenticated reference materials against which to compare analytical results. Reference materials are typically in the form of physical samples but may also be held digitally as reference data.

PHYSICAL SAMPLES

For use in wildlife forensic science, every reference sample must have an unambiguous taxonomic identification along with meta-data appropriate to its use, for example, a specific collection location for geographic origin testing, or a specific age for radiocarbon dating.

The collection and curation of reference materials is a huge task and has historically been the role of natural history museums and herbariums around the world.

While many such institutions actively support wildlife crime investigations, challenges exist when attempting to use such collections as reference materials in wildlife forensic investigations.

The challenges include:

- Ambiguous taxonomy (there may be current or historic differences in how species are named)
- Reluctance by the institution to allow destructive sampling
- Difficulties in transfer of reference materials
- Suitability of the sample type available

To overcome many of these issues, digital reference data, particularly DNA sequence data, is now routinely used as reference material for wildlife DNA forensics.

REFERENCE DATA

The use of digital analytical result data as a reference standard can circumvent many of the issues of physical sample access and allow wildlife forensic scientists in multiple labs to make use of a single reference sample.

Such remote access to reference data is very useful in wildlife forensic science, but its use must be managed very carefully and may not always be appropriate, where, for example, the quality of the reference data is suspect.

Most databases usually contain some errors, some types of data are very difficult to exchange between laboratories and some countries do not allow the use of reference data generated by third parties in a forensic investigation.

To address these problems, several initiatives have started to produce digital reference data specifically for wildlife forensics, creating quality assured databases that can be used in sequence analysis for forensic casework. Use of these databases is gradually starting across Africa and should increase over time. Whatever database is used, it is very important that the source of reference data is critically assessed during the interpretation of the casework results and disclosed in forensic casework reports.

DATABASES

A database is an organized collection of data. The availability of databases is essential in order to enable the comparison of the test results with reference materials. Examples of databases used in forensic investigation are provided below.

Click each term to learn more.

Species database

A database of species-specific reference DNA sequences that can be reliably used to distinguish all individuals of a species from any other species in the database.

Population database

A database of georeferenced DNA profiles of multiple individuals from each location sampled across the species range. It can be used for both geographic origin assignment and to enable statistical calculations in the event of DNA profile matches.

Searchable profile database

A database of forensic case-related samples (for example, DNA, fingerprints) recovered from known individuals that can be searched for unique matches with profiles recovered at a crime scene. This is currently only available for humans and rhinoceros.

Now that you are familiar with the role of reference materials and databases in forensic investigations, we will explore how laboratories quality assure the processes involved in DNA analysis.

Quality Assurance

QA is usually maintained through a Quality Management System (QMS), which describes a series of control processes and protocols surrounding the implementation of a test. The aspects of test implementation that QMSs cover include:

While a QMS is designed to be specific to a laboratory, general standards have been developed, such as ISO17025 or Good Laboratory Practice (GLP) which describe general QA requirements for laboratory testing and under which individual laboratories may choose to become formally accredited.

While a number of wildlife forensic laboratories do hold ISO17025 accreditation, an absolute requirement for laboratory accreditation to this level in wildlife forensic science is an unrealistic expectation at this time.

To address this issue, the Society for Wildlife Forensic Science (SWFS), has established a set of Standards and Guidelines specifically for several disciplines within wildlife forensics.

To access the SWFS Standards and Guidelines (2018), click here.

Individual test methods should be controlled by Standard Operating Procedures (SOP), documents that precisely define how a test should be performed and how the results should be analyzed and interpreted. This should include consideration of possible sources of error and conditions that may affect the accuracy of the results.

All individual test methods for wildlife forensics should be validated prior to use. This involves the performance of a validation study, a series of experimental tests conducted to assess the method. Validation protocols and reports should also be available for inspection. Test methods should ideally be published in peer-reviewed scientific journals before use in forensic analysis. Test methods may also be accredited against internationally recognized standards (for example, ISO17025, GLP).

Further reading
Click here to learn more on ISO17025.
Click here to learn more on Good Laboratory Practice.

Conclusion

Wildlife forensic science can provide valuable evidence to support wildlife law enforcement. However, it is important for prosecutors and judiciary to understand the fundamental principles of visual and genetic identification, their appropriate application, and key areas of interpretation.

For effective investigation and prosecution, it is crucial to:

- Be aware of the limitations of visual and genetic analytical methods.
- Understand the principles of DNA sequencing and DNA profiling, and implications of testing results.
- Ensure that validated, quality assured processes are consistently implemented.

To complete this module, advance to the Summary page, and click Submit report.

For more information on the use of DNA sequencing for geographic origin identification, download the appendix here.