FRSC-4600 Lecture Notes

Lecture 1: Course Introduction and Overview

Instructor:

• Tristan Baecklund

• Office DNA B108.7

• Office Hours: Wed 1-3 pm

• tbaecklund@trentu.ca

• Demonstrator for MScFS program

• Recent graduate of ENLS program

• PhD in host population genetics in context of disease (arctic fox and bats)

Course Description:

• Applied: Know the potential applications

• Molecular: Know the molecular technique

• Genetics: Understand the target gene function and biology, whit it was selected

Learning Outcomes:

• Understand the fundamental principles of DNA marker characterization and profiling through a history of genetic and genomic marker development

• Understand how the characteristics of DNA markers can be used directly to applications in diverse fields and species

• Gain perspective on the pace of technological developments in this area will be assessed  by examining current news about genomics

• Understand how the area of molecular genetics is rapidly changing due to the emergence of novel technologies and be able to apply these underlying technologies to applications in diverse fields of study

Genetics Terminology:

• Heredity: The transmission of traits from one generation to the next

• Genetics: The scientific  study of heredity and variation in heredity

• Gregor Mendel documented discrete heritable units (genes) that were passed from one generation to the next

• Molecular genetics: The study of structure and function of genes at the molecular level

• Applied molecular genetics: Using this information for research, conservation, management, and law enforcement

Classical Genetics:

• Classical genetics: Refers to the use of crosses to breed new strains of organisms and to understand how traits are transmitted

• An important tool of classical genetics is the isolation of mutants affecting particular traits which can then be compared to normal strains

• The first genetic maps of genes on chromosomes were generated through this approach

• Common in health research

• Biological process → identify mutants → find the gene → biochemical function

• Phenotype → genotype

  • Discover new phenotypes

  • Show that is has genetic basis (heritable trait)

  • Find the gene that has mutated

  • Understand what/how the wild-type gene functions

Modern Genetics:

• Modern/reverse genetics: The study of genes at the molecular level

• It has been made possible because of advances in cloning and sequencing of genes or complete genomes

• Modern genetics has provided an enormous understanding about the physical nature of genes, their expression patterns, their role in development and disease, and how their products interact

• Gaining traction in human health and forensics:

  • 23 & Me

  • Commercially available profiling kits that encompass both STRs and informative loci (phenotypes, hair colour, eye colour, etc)

• Gene in hand → create mutants → phenotype? → biological process

• Genotype → phenotype

  • Change something in a known gene

  • Observe phenotypic effect

  • Find out why you see what you see

  • Understand what/how the wild type gene functions

  • In non-model organisms we often go directly from gene to phenotype

    • Does not have a published genome or is not an ideal species for lab research

Transmission of Genetic Information:

• The laws of inheritance were first elucidated by Gregor Mendel

• 1856-1863

• Mendel made careful crosses between pea plants that differed in particular characteristics (traits), counted the number of times the traits appeared in subsequent generations, and interpreted the results

• Law of segregation:

  • Mendel crossed the F1 hybrids with many of the F2 plants having purple flowers; however, some had white flowers

  • Mendel observed a ratio of 3:1, purple to white flowers in the F2 generation

  • From these observations he developed a model to explain inheritance pattern

    • Inherited characteristics are determined by indivisible factors (genes) and alternative versions of genes (alleles) account for variations in inherited characters

    • For each characteristic, a diploid organism inherits two alleles, one from each parent

    • If two alleles at a locus differ, then one (dominant) determines the organism’s appearance, and the other (recessive) has no noticeable effect on appearance

    • The two alleles for a heritable character separate (segregate) during gamete formation

  • Law of independent assortment:

    • Mendel identified his second law of inheritance by following two characters at the same time: seed colour (Y, y) and seed shape (R, r)

    • Crossing two true-breeding parents differing in two characters produces dihybrids in the F1 generation, heterozygous for both characters (YyRr)

    • A dihybrid cross between F1 dihybrids (YyRr) can determine whether two characters are transmitted to offspring dependently or independently

    • Each pair of alleles segregates independently during gamete formation

    • Applies to genes on different chromosomes or genes that are located very far apart on the same chromosome

    • Genes located near each other on the same chromosome tend to be inherited together

  • The relationship between genotype and phenotype is rarely as simple as in the pea plant characteristics Mendel studied

  • Most heritable traits are not determined by a single gene (today referred to as one gene of large effect) rather many genes of small effect

  • However, the basic principles of segregation and independent assortment apply even to more complex patterns of inheritance

Genetic Disorders Following Mendelian Inheritance:

• Cystic fibrosis: Recessive

• Sickle-cell anemia: Recessive

• Tay-Sachs disease: Recessive

• Phenylketonuria: Recessive

• Hemophilia: Sex-linked recessive

• Huntington’s disease: Dominant

• Muscular dystrophy (Duchenne): Sex-linked recessive

• Congenital hypothyroidism: Recessive

• Hypercholesterolemia: Dominant

DNA as the Genetic Material:

• DNA is a polymer of nucleotides, each consisting of a nitrogenous base, sugar, and phosphate group

• 1950: Chargaff reported that DNA composition varies from one species to the next

• Two findings became known as Chargaff’s “rules”

  • The base composition of DNA varies between species

  • In any species, the number of A and T bases are equal and the number of G and C bases are equal

Building a Structural Model of DNA:

• After DNA was accepted as the genetic material, the challenge was to determine how its structure accounts for its role in heredity

• Wilkins and Franklin were using a technique called X-ray crystallography to study molecular structure

• 1952: Franklin produced a picture of the DNA molecule using this technique

• Watson and Crick thought the bases paired like with like (A with A and so on), but such pairings did not result in uniform width

• Purine = Adenine and Guanine

• Pyrimidine = Cytosine & Thymine

• Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray data

• They determined that adenine (A) paired only with thymine (T) and guanine (G) paired only with cytosine (C)

• The Watson-Crick model explains Chargraff’s rules: in any organism the amount of A = T and G = C

• 1953: Watson and Crick introduced the double-helical model for the structure of DNA

• DNA, the substance of inheritance, is the most celebrated molecule of our time

• Hereditary information is encoded in DNA and reproduced in all cells of the body

• DNA strands are antiparallel and their backbones are negatively charged

Sources of Genetic Variation:

• Many phenotypic differences between organisms are the results of differences in the genes they carry

• Genetic  differences arise through the process of:

  • Mutations: Change in genetic material (substitutions, deletions, insertions, translocations)

  • Recombination and crossing over: Exchange of chromosomal material between homologous chromosomes at meiosis

  • Random fertilization: billions of gametes – laws of segregation and assortment

• Mutations:

  • Mutations are changes in genetic material of a cell or virus

  • Spontaneous mutations can occur during DNA replication, recombination, or repair

  • Approximately 110 mutations introduced to your offspring

  • Mutagens are physical or chemical agents that can cause mutations

  • Germline mutations: occur in gametes

    • Significant because they can be transmitted to offspring

    • Most from paternal line

  • Somatic mutations: occur in other cells of the body

    • May have little effect on the organism because they are confined to just one cell and its daughter cells

    • Cannot be passed onto offspring

• The behaviour of chromosomes during meiosis and fertilization is responsible for most of the variation that arises in each generation

• Three mechanisms contribute to genetic variation:

  • Independent assortment of chromosomes:

    • Each pair of chromosomes sorts maternal and paternal homologs into daughter cells independently (randomly) of the other pairs at metaphase I of meiosis

    • The number of combinations possible when chromosomes assort independently into gametes is 2^n, where n is the haploid number

    • For humans n = 23, there are more than 8.4 million (2^23) possible combinations of chromosomes

  • Random fertilization:

    • Adds genetic variation because any sperm can fuse with any unfertilized egg

    • The fusion of two gametes produces a zygote with any of about 70 trillion diploid combinations

    • Crossing over adds even more variation; each zygote has a unique genetic identity

Replication Variation in SARS-CoV2:

• 1: Spike protein on the virion binds to ACE2, a cell-surface protein

• 2: The virion releases its RNA

• 3: Some RNA is translated into proteins by the cell’s machinery

• 4: Some of these proteins form a replication complex to make more RNA

• Proteins and RNA are assembled into a new virion in the golgi

• 6: New virions are released

• This is non-mendelian transmission

• Drug treatments typically target spike protein binding (1), RNA translation (3), and replication complex formation (4)

• Rapid antigen tests detect spike proteins (1) and qPCR tests target proteins in the replication complex (4)

• Variants arise from the replication complex (4)

Importance of Basic Concepts:

• Our understanding of inheritance and genetics has evolved over the last 170 years

• Understanding patterns of inheritance and generation of variation is fundamental to its application

• Multiple disciplines exist that focus on improving our understanding in this area

• Gene-phenotype receiving lots of attention in basic and applied research

• Molecular genetics employs known principles of DNA structure and function to investigate the molecular basis for genotype-directed phenotypes under normal and pathological conditions

• The term applied molecular genetics is used to describe the set of laboratory-based research tools that exploit the information potential or organismal DNA and use them for a variety of applications

• Databases play an important role when it comes to application
  

Lecture 2: DNA Markers and Platforms

Key Terminology:

• Gene: Any region of the genome (not restricted to protein coding)

• Allele: Variation of a gene

• Genotype: A combination of alleles

• Haplotype: A combination of linked alleles

• PCR: Polymerase chain reaction – makes millions of copies of a gene

• Primer: Used to tell the PCR what gene to amplify

Goal of Molecular Methods:

• Genetic methods simply aim to visualize and quantify differences

The Molecular Era:

• Before the mid-1960s, no methods were available for directly assessing molecular genetic variation

• In the mid 1960s, protein electrophoresis was invented

• Direct assessment of heritable variation became possible

Protein Electrophoresis and Allozyme Markers:

• Proteins move in the electric field (in gel medium)

• Relative speed (distance) depends on the charge, size, and shape of the protein

• Early protein electrophoresis studied revealed that the extent of genetic variation is much higher than previously thought

• An early application was a study on northern elephant seals

  • Uniform homozygosity among 21 proteins encoded by 24 loci identified allele fixation and helped explain the decimation of the species and vulnerability of the species

Genetic Marker Acronyms:

• Restriction enzyme and length variation:

  • RFLPs: Restriction Fragment Length Polymorphisms

  • AFLPs: Amplified Fragment Length Polymorphisms

• Repetitive DNA (size of fragment = # repeats):

  • Minisatellites/VNTRs: Variable Number of Tandem Repeats

  • Microsatellites/STRs: Short Tandem Repeats

• Direct assessment of nucleotide variation:

  • SNPs: Single Nucleotide Polymorphisms

  • Mitochondrial sequence

  • Chloroplast sequence

REs and DNA:

• Restriction enzymes recognize sequence motifs and cut DNA at the motif

• Cleavage products can be Sticky or Blunt:

  • 5’ protruding ends (sticky)

  • 3’ protruding ends (sticky)

  • Blunt

• How enzymes cleave nucleotides can be leveraged for downstream applications

  • Making sequencing competent molecules

• In an idealized genome:

  • RE cuts every 4^length of motif

  • This is a base of 4 because there are 4 nucleotides

  • RE that has a 4bp motif cuts every 4^4 of 1 in every 356 bp

• Some REs do not recognize methylated sites and cutting is often not 100%; there is variation in repeatability

• Genomes vary in nucleotide composition, which is important for experimental design and genome coverage

The RFLP Principle:

• RFLPs were detected by hybridizing radioactively labelled probes to DNA, transferred from a gel to a filter “Southern Blotting”

  • DNA restriction fragments

  • Agarose gel electrophoresis

  • Fragments separated by size

  • Transfer of DNA fragments from gel to nitrocellulose filter

  • Radioactive DNA probe hybridized to fragments on filter

  • Autoradiograph showing positions of hybrids DNA

• Parentage test with RFLP:

  • RFLPs were detected by hybridizing radioactively labelled probes to DNA, transferred from a gel to a filter (“southern blotting”)

AFLP (and RAPDs):

• AFLPs are essentially RFLPs but followed by a PCR step to be more selective

• Random Amplified Polymorphic DNA: No enzyme, rather a random set of primers and PCR

• Produces a profile for small fragments

• RAPDs rely on primers; a string of nucleotides

• Given what we know about RE motifs, how often would we expect to find a matching 10 bp primer across a genome? 4^10 = every 1 million

Minisatellites:

• Repeat unit > 9 bp, usually 30 bp

• Number of repeat units are scored when they are used as genetic markers

• Among the first markers to be used for DNA fingerprinting

Microsatellites:

• Di, tri, tetra… repeat units

• Shorter than VNTRs

• Humans vs non-humans

Replication Slippage:

• Microsatellites arise through polymerase slippage during DNA replication and a higher rate of replication slippage with more repeats

• Dissociation and subsequent mispairing gives change in number of repeats

  • The length of strand increases the risk of slippage

  • Point mutations can terminate replication

Scoring Microsatellites in Practice:

• You score the number of repeats of a particular repeat unit

• You do not know (or care) if it's an imperfect repeat

• Score both alleles (or variants) in each individual based on size

• Allele or variants gets named and scored in individuals

Huge Number of Applications:

• Abundant in the genome (human genome contains > 400,000); highly variable and neutral with respect to fitness (phenotype); simple and cheap to genotype

• Many microsatellites give you a genotype; variation in genotypes leads to population structure; variation in genotypes leads to population structure (non-random mating)

Microsatellites as Genetic Markers:

• Some problems with using microsatellites as genetic markers include:

  • Time and labour-consuming to develop primers from non-model species

  • Often do not transfer well between species (may not amplify or may not be variable)

  • Null alleles or PCR-induced mutations can cause problems

  • Difficulties in modeling the mutation process poses problems for population genetics

Basic Principles of DNA Profiling:

• The genome of each individual is unique with the exception of most identical twins

• Probe subset of genetic variation in order to differentiate between individuals; statistical probabilities of a random match are used

• DNA typing must be performed efficiently and reproducibly as information must hold up in court

• Current standard DNA tests do not look at genes: they give little to no information about predisposal to disease or phenotypical information is obtained

DNA Typing Applications:

• Forensic science:

  • Human and non-human

  • Solving crime (compare suspect with evidence)

  • Identifying accident victims, soldiers, missing persons, mass disasters

  • Paternity testing

• Conservations genetics

• Deciphering evolutionary relationships

• Pathogen detection and monitoring

• Diagnosis of disease

• Breeding in agriculture

DNA Typing in Forensics:

• A DNA profile by itself is fairly useless because it has no context

• DNA analysis for identity only works by comparison, you need a reference sample (databases)

  • Crime scene evidence compared to suspect(s)

  • Child compared to alleged father

  • Victim’s remains compared to biological relative

  • Soldier’s remains compared to direct reference sample

• Three DNA typing outcomes:

  • Exclusion (no match)

  • Non-exclusion (inclusion)

  • Inconclusive result (no result or complex mixture)

Single Nucleotide Polymorphisms:

• A SNP is a single nucleotide variation at a specific location in the genome

• Common in most genomes: In humans, average 1 SNP per 1000 bp across the genome

• Fewer SNPs in coding regions, wide variation in SNP density across most genomes

  • Genes must be highly conserved, vital to organism

• SNPs are popular genetic markers because:

  • They are abundant

  • They can be genotyped in a high throughput manner

  • The mutation mechanism is well established

• Most often are biallelic

• SNPs are frequently used in associations studies and in population and evolutionary genetics

  • Humans, agricultural animals

• You need to know where your SNPs are; first do SNP identification

• SNPs are usually identified by sequencing a limited pool of individuals

  • By this procedure, only common SNPs are found

  • This can create an “ascertainment bias”

• Less power of discrimination per locus for individual identification compared to microsatellites generally need more SNPs compared to microsatellites

• Can give you clues into phenotypes

Mitochondrial Sequence:

• Haploid and maternally inherited

• Approximately 17000 BP (37 genes)

• All SNPs (alleles) are linked and inherited together

  • mtDNA halotypes

• Variation in haplotypes is very useful for species identification and provenance

  • Cytochrome b is used for mammalian ID

• Control region (faster evolving, species and provenance)

Chloroplast Sequence:

• dsDNA

• Plants and algae

• 120-247kbp (most are small)

• 100+ genes

• IR sequence

  • Repetitive regions

  • Vary in size and position between species

Why SNPs Might Not Replace STRs

• Large databases containing STR information; would need to replace data on existing samples with new DNA markers

  • Databases for STRs are already established

• Mixture detection and interpretation benefits from marker systems with alleles; SNPs only have two alleles and three genotype possibilities

• Degraded DNA can be successfully analyzed with STRs in many cases, thus removing a primary motivation for using SNPs

SNPs and Microsatellite Profiling:

• Probability of identity (PI)

  • Likelihood of two people having the same profile (genotype)

  • Different than RMP, which represents the estimated frequency of a profile occurring in a given population database

• Based on H-W principles

  • p is the frequency of allele A in a population

  • q is the frequency of allele a in a population

  • p^2 and q^2 are frequency of homozygotes

  • 2pq is the frequency of heterozygotes

• Why is there a difference between SNPs and microsatellites?

  • Example locus of 4 alleles with equal frequency

  • p^2 = aa,bb,cc,dd = 0.0625 for each homozygote

  • 2pq = ab, ac, ad, bc, bd, cd = 0.135 each heterozygote

  • P1 = sum of each genotype frequency squared

  • (0.0625)^2…+(0.135)^2… = 0.109375

H-W Example:

• Population contains: A, A, A, A, a

• p = ⅘ = 0.8

• q = ⅕ = 0.2

• Frequency of AA = (0.8)^2 = 0.64

• Frequency of aa = (0.4)^2 = 0.04

• Frequency of Aa = 1 - 0.64 - 0.04 = 0.32

SNP Sanger Sequencing:

• Chain termination with ddNTP (dideoxynucleotide)

  • 4’ OH group replaced with an H atom to terminate synthesis

• ABI 3730 series genetic analyzer is the largest fully automated system

• Other smaller models can be used to best suit lab needs

• Sanger sequencing = chain termination sequencing

• Automated sanger sequencing with dye termination:

  • Only one lane is needed

  • ddNTPs are labelled with different fluorescent dyes (one per base)

  • Hit with a laser and photograph

  • Shorter fragments migrate faster through the capillary tube

• Produces good quality sequences up to 1 kb (1000 bps) but normally 60-700 is good quality

• Somewhat costly and not high-throughput (each sample requires a sequencing reaction)

• Dominated the sequencing market until very recently and is still extensively used

• Best method for around 3 decades until Next Gen Sequencing

• SNPs are good for small data, high quality

• Provides the most complete set of genetic information but has until recently been to expensive for large-scale studies

Next-Generation Sequencing:

• Massively parallel sequencing, short-read sequencing, second-generation sequencing, high-throughput sequencing

• A suite of new sequencing technologies capable of producing a lot more sequence information, in much shorter time than Sanger sequencing

• Emerged from development in nano-technologies

• Generally (Illumina) produces shorter reads with lower quality at each base than Sanger sequencing

• The longest read chemistry available is 350 bp, the current norm is 125-150 bp

• Length and quality issues are outweighed by the massive number of sequences obtained (compared to Sanger)

• Huge implications for all commercial and research programs that rely on genetic information (medical genetics, evolutionary biology, population genetics, forensics)

• There are several new technologies:

  • Roche 454: Titanium, 500 bp

  • Illumina: NextSeq, 150-300 bp

  • ABI SOLiD sequencing: ligation based, 100 bp

  • Pacific Biosciences: Single Molecule (SMRT) Sequencing

  • Ion Torrent: ion semiconductor chips, 100-200 bp

• Different technologies have advantages for different applications

Illumina Technology:

• DNA is modified to make it compatible with the flow cell

• 1. DNA fragmentation and adapter ligation

• 2. Bridge amplification on solid support: Each fragment is amplified to generate a cluster

• 3. Sequence cluster in real time

Ion Torrent:

• Hydrogen ions are released when a nucleotide is added

• Similar concept to pyrosequencing

• The electrical current change which occurs when hydrogen is released is recorded

PacBio:

• Start with high-quality double-stranded DNA

• Ligate SMRTbell adapters and size select

• Anneal primers and bind DNA polymerase

• Circularized DNA is sequenced in repeated passes

• The polymerase reads are trimmed of adapters to yield subreads

• Concensus is called from subreads

RADseq:

• High-throughput sequencing of AFLPs

Genomic Data:

• Defined as high-throughput sampling of the genome

• Our genome is 3,000,000,000 basepairs

  • Most of the genome is not polymorphic

  • We are now getting 1000,000s of SNPs

  • Information on recombination

  • Information on methylation

• For now, genomics = 1000s to 1000,000s of SNPs

• Biggest shift in the past couple years has been to long-read single molecule sequencing

Massively Parallel Sequencing:

• The critical difference between Sanger sequencing and HTS is sequencing volume

• Sanger method only sequences a single DNA fragment at a time

• HTS is massively parallel, sequencing millions of fragments simultaneously per run

• This high-throughput process translate into sequencing hundred to thousands of genes at one time

• Poses a computational challenge in putting together millions of short reads into a meaningful sequence

• Easier is there is a reference genome sequence

• Tools are rapidly being improved and developed

• Bioinformatics is a necessary skill to have

• Applications to MPS technologies:

  • Genome sequencing

  • Expression analysis (RNA) including ID of noncoding RNAs

  • Analysis of methylation and chromatin packaging

  • Population genomics and genotyping

Epigenetic Assays:

• Most approaches use the above sequencing techniques

• PacBio simply detects methylation

• Bisulfite conversion involves the deamination of unmodified cytosines to uraci
  

Lecture 3: Non-Human Forensic Applications

Marker Terms and What they Identify:

• Genes: Any region in a genome

• Alleles: Variation in a gene

• Haplotype: Alleles inherited together

• SNPs: Phenotypes, individuals, populations

• Microsatellites: Individuals and populations

• Mitochondria: Populations and species

• Chloroplast: Species

Wildlife Forensics:

• Native species

• Domestrics

• Exotics

• Wildlife conservation: Enforcement of harvesting laws and agreements

• Largely based on microsatellites and mtDNA

• DNA applications:

  • Native species

  • Species ID

  • Sex ID

  • Individual ID

  • Population assignment

  • Parentage

• Novel Applications:

  • Environmental DNA

  • Models of illegal trade

The NRDPFC Forensic Laboratory:

• 1987: First case submitted

• HAs seen more than 1000 cases

• Provided evidence for every province and territory in Canada, plus some US states

• 1991: Provided the first DNA evidence for a wildlife infraction to be accepted into a North American court

• 2002: Involved in the first human homicide investigation with the OPP

• Lab involved in case against man found with Black Bear gallbladders

Why do we need Wildlife Forensics?

• Native species:

  • Has seen more than 1000 cases

  • Poaching (diminish natural resources)

  • Illegal movement of animals (introduction of non-native species)

• Ranched animals and domestics:

  • Track source and spread of disease

  • Animal cruelty cases

• Exotic species:

  • Illegal trade in animal parts: CITES and WAPPRIITA

  • Threats to species sensitive to overexploitation

Wildlife Investigations:

• Investigation by wildlife officers similar to police investigators

• Gather evidence regarding offences, verify accusations, carry out searches, testify in court

• The ID of tens of thousands of protected species, their parts, and derived products

  • Requires broad scientific knowledge: DNA largely bypasses that need

• The Convention on International Trade in Endangered Species (of wild fauna and flora)

  • Sets controls on the international trade and movement of animal and plant species that are/will be threatened

  • Appendix 1: Species are rare or endangered, and trade will not be permitted for primarily commercial purposes

  • Appendix 2: Species are not rare or endangered at present but could become so if trade is not regulated

  • Appendix 3: Species are not endangered but are managed within the listed nation

  • Wild Animal and Plant Protection and Regulation of Interprovincial and International Trade Act

    • Legislative vehicle by which Canada meets its obligations under CITES

    • Various national and taxa specific laws

  • World Wildlife Fund

    • Monitors wildlife trade

    • Founded to assist in the implementation of CITES

    • Illegal animal trade is a multi billion dollar industry

    • There are about 200-300 agents/officers in the US monitoring illegal trade

  • In Ontario:

    • Fish and Wildlife Conservation Act

    • Endangered Species Act

Pangolin Trade Case Study:

• Most common trafficked mammal

• 1,000,000 likely traded between 2006-2015

• Moved to CITES Appendix 1 in 2016

• 200,000 trafficked in 2019

• All 8 species at risk of extinction

• Trafficked for their scales which are powdered or made into paste for medicinal purposes

• There is no scientific basis for their use; the scales are made of keratin, the same material as our fingernails

• Fetuses are consumed in soup and are used to demonstrate social status

• Loss of pangolins is detrimental to ecosystems and communities, they eat termites which would otherwise eat agricultural crops

Narwhal Tusks Case Study:

• The Marine Mammal Protection Act in the United States

• Man smuggled 250 narwhal tusks worth up to 3 million dollars into US

• Marine mammals parts include any part of a marine mammal, both hard and soft, including parts derived from tissues, such as cell lines and DNA, but do not include urine or feces

Elephant Ivory Trade Case Study:

• 20,000 elephants illegally killed in 2015

• Legal sale of 107 tonnes of ivory in 2008 intended to flood market and crash the illegal trade market

• This backfired, illegal poaching increased

• Molecular markers (microsatellites) have been used to identify the networks of elephant trafficking

• Collection of elephant dung samples across Africa to create a DNA map of their genetics

• DNA extracted from ivory could then be cross-referenced to this database to figure out where the elephant was poached from

• Tracking ivory is linked to terrorist activity and has allowed for the identification of poaching hotspots and trafficking networks

• Armed guards posted in hotspots to protect wildlife

• More policing/.governance of animals has lead to a decrease in death

• The GDP of China increases as the number of poached elephants increases

Rhino Ivory Trade Case Study:

• Rhinoceros horns are worth up to 40,000$/kg

• Horns used in medicine, carvings, statues, and status symbols in Africa

• Blood and urine used for traditional Chinese medicine

• Poaching is projected to exceed natural growth rates in much of Africa

• DNA forensic tests track rhino poachers; daggers or powdered medicines are tracked back to source populations

• A DNA database out of South Africa (RHODIS) has over 15,000 animals

  • 24 microsatellites, sex, and species markers

  • Over 2,000 forensic cases, multiple with >10 year jail terms

  • In these cases the horn is often linked to the carcass or tool

• Sexing rhinos: Zinc finger Gene found on the X and Y chromosome but there is a length polymorphism between X and Y

  • The male sex has two peaks (heterozygous)

Chilean Sea Bass Case Study:

• Also called Patagonian toothfish

• Long lived (50 years old)

• Unsustainably fished

• Became overharvested; Wholefoods stopped selling it

• Seafood Watch suggested avoiding this fish

• One population is certified sustainable (Marine Stewardship Council eco-label)

• Whole Foods started selling MSC eco-labeled fish

• Researchers looked at whether chilean sea bass carrying the MSC eco-label actually come from the certified fishery

  • Mitochondrial data was used in this study

  • This a haplotype that is inherited all at once

• 8% of eco-labeled fish were not Chilean Sea Bass to begin with

• 15% did not come from the certified population

• This is not restricted to Sea Bass, adulteration is widespread

• Now: Sea bass populations are rebounding and approximately 60% of stocks are considered sustainable

Monitoring Harvested Species:

• Harvested for sport, food, fur, and other derivatives (bones, urine, gallbladder for asian medicines)

• Not always clear how many animals and from what populations

• Use DNA profiling to define populations and non-invasively census

• Use DNA to enforce wildlife laws in the illegal harvest and trade of wildlife and better manage harvests

Goat Poaching Case Study:

• Hunter in possession of a goat claimed it was female

• The Alaska Dept. of Fish and Game and State Troopers believed the goat was male

• 5 DNA samples from different tissues were used for individual and species identification

  • DNA was extracted and amplified

  • Used gel electrophoresis for sex determination

  • All samples were from male goat

  • All samples were from the same individual

Wildlife Profiling Issues:

• Development of species-specific markers

• Low genetic variation and lower power of inclusion

• Need to establish databases for each species across their distributions

  • Need to identify species-specific markers

  • Markers for closely related species can be transferable

• No standardized loci among wildlife labs, needs to happen as the number of labs increases

• Calculations to establish PI need to be conservative when few individuals in database from region of crime scene

• Main difference to human forensics: Need to identify and distinguish between a variety of species (mtDNA or cpDNA)

• Might also include:

  • Sex determination (X/Y)

  • Individual identification (microsatellites)

  • Population identification (microsatellite/SNPs)

  • Parentage (microsatellites)

Microbial DNA Forensics:

• The properties that make microorganisms potentially harmful for cultivation and outbreaks:

  • Accessibility

  • Culturability

  • Capability for large-scale production

  • Stability during preparation

  • Ability to retain potency during transport and storage

  • Ability for dissemination

  • Stability and retention of potency after dissemination

  • Incubation period

  • Infectivity

  • Lethality

  • Pathogenicity

  • Toxicity

  • Transmissibility

  • Virulence

• 1,415 species known to be pathogenic to humans

  • 217 are viruses and prions

  • 538 are bacteria and rickettsia

  • 61% are zoonotic and 33% are transmissible among humans

• Human forensics relies on 20 microsatellite loci that are useful for the majority of scenarios encountered: This is not the case for microorganisms

• Unique genetic identification of a microorganism is likely never possible

• Clonal nature of many microorganisms means we cannot pin down the source

• Less than optimal population and phylogenetic data means it is difficult to develop genetic resources

• Limited historical and epidemiological information means limited databases and understanding of trends

• Widespread demand is somewhat limited but some well known examples are documented

  • Anthrax

Epidemiology Case Studies:

• Aq handful of nosocomial (originated from a hospital) outbreak studies have been performed in real-time with the goal of reducing the duration and impact of transmission

• Also being applied in the field (outside the hospital) as sequence data is useful to monitor outbreaks, but needs to be delivered in a timely fashion

Acinectobacter baumannii Case Study:

• Multi-drug resistant microorganism

• Opportunistic pathogen in humans, affecting people with compromised immune systems

• Outbreak lasted almost 2 years

• Genome sequencing revealed 57 potential transmission events that linked 55 patients by genomic (SNP) analysis

• Could identify likely transmission events and sites

• Burn-patient operating theatre and specialized burn-care bed were identified as contamination sources

• A total of 7 genotypes were identified over the course of the outbreak

  • Reproduce several thousand times a day

• The outbreak began with a contaminated bed; the patient left the hospital and the spread began three weeks later

• A subsequent incident occurred in a contaminated burn theatre

Carabapenem-Resistant K. pneumoniae:

• Bacterial pathogen Klebsiella pneumoniae is responsible for roughly 15% of Gram-negative infections in hospital ICUs

• Primarily affects immuno-compromised patients, and outside the hospital is manifested as pneumonia

• Infections caused by carabapenem-resistant strains; have few treatment options and are associated with mortality rates upwards of 50%

• Genome sequencing is used to track an outbreak of carbapenem-resistant K. pneumoniae in the U.S. National Institutes of Health (NIH) Clinical Center that colonized 18 patient, with 10 deaths

• Implementation of rigorous infection control procedures ultimately halted the outbreak

• Sought to better understand how the outbreak progressed to learn how to control future outbreaks

• Genome sequencing of isolates from the index (P1) patient taken from four body sites (urine, groin, throat, broncho-alveolar lavage)

• Sequenced isolates from each of the affected patients to find SNPs (6Mb genome) to infer transmission links

• Analysis of 41 SNPs to link patients

• Suggested three independent transmission events from this patient lead to hospital-wide dissemination of the outbreak strain

  • Used sequencing and patient overlap in hospital wards to predict the most probable transmission sequence

  • First transmission through patient 3

  • Second transmission though patient 4 by other patients as silent transmission vectors (five suspects)

  • Third transmission linked to a contaminated ventilator

SARS-CoV-2 Study:

• Coronaviruses (CoV) are a broad family of viruses named after the crown-like spikes on their surface

• These viruses can be found in people and animals

• CoV typically causes mild to moderate upper respiratory tract disease in humans, but can also cause more severe infections such as pneumonia

• In Nov/Dec 2019, an outbreak of a novel coronavirus in Wuhan China originally infected 40+ people, most of whom developed pneumonia and has contact with the same seafood market

• The N protein (end gene) in the SARS genome is used to identify different variants

• PCR and rapid antigen test results:

  • Positive (CoV detected)

  • Negative (CoV not detected)

  • Inconclusive/invalid

• Many mutations which lead to new variants occur at the end of the genome, where the membrane and spike proteins are encoded

  • Targeting these genes helps identify variants via sanger and Next Gen sequencing

• SARS-CoV-2 is zoonotic; a variant emerged in white-tailed deer with deer-to-human transmission

What is a Microbiome?

• A community of microorganisms

• Can be detected through PCR amplification of the 16S ribosomal DNA gene sequences followed by Illumina sequencing

• Measure aspects like diversity and species composition

• The 16S approach:

  • Extract DNA from microbial community sample

  • Amplify and sequence 16S rRNA

  • Group similar sequences and quantification of OTUs representation

• Complete sequencing technique:

  • Extract DNA from microbial community sample

  • Use shotgun and high-throughput metagenomics approaches

  • Sequence community DNA

  • Compare sequence to reference genomes and quantification

Microbiome Case Study Examples:

• Mountain goats:

  • Compared fecal microbiomes of captive vs wild mountain goats

  • Identified genus more abundant in wild and captive communities

  • Microbiome is influenced by diet and environment

• Pandas:

  • Captive pandas were reintroduced into the wild but only 6/9 survived

  • Wild pandas were found to have different gut/fecal microbiomes than captive pandas

  • Fecal microbiome transplants from wild to captive pandas to support reintroduction programs

The use of Microbiomes in Forensics:

• Use of microbiome assessments provides associative evidence between people and objects with places or other people

• Example: unknown soil on a suspect’s clothing or shoes could be compared with a known control soil sample from the crime scene

  • Soil contains a diverse community of microbes that can vary significantly in composition from one site to the next

• Post-mortem interval (necrobiome)

  • For most PMIs, the focus becomes predictive modeling

  • Given a single microbiome profile, the PMI can be estimated

• Machine learning algorithms and cross-validation are typically applied

• Linear regression for PMI:

  • Y = B0 + B1X1 + B2X2 +...+ E, where B1X1 represents microbiome metrics

  • Automates variable selection and addresses multicollinearity

  • Cross-validation using test and training samples from the complete data

  • R^2 value is a score from 0-1 to evaluate how the linear regression evaluates the variation

Trace Soil Evidence in Forensics:

• Traces of soil evidence may be associated with suspects and analysis can link the suspect to the crime:

  • Gas chromatography

  • Electron microscopy

• Analysis of soil microorganisms has to date largely been ignored by the forensic scientific community

  • Physically look at soil, properties, elements

• Mainly due to the limitations of traditional culturing techniques, which allow only a small subset of organisms to be isolated and characterized

• Techniques have been developed to circumvent the requirement to isolate and culture microorganisms as a prerequisite to identification

• Soil microbial diversity is now characterized using simple molecular techniques (mostly) based on amplified rRNA (gene) DNA

• The potential exists for such technologies to be used to analyze microbial populations in many diverse environments for forensic purposes

• Soil serves as powerful contact trace evidence because it is highly individualistic and has a high transfer and retention rate

• Standard analyses examine intrinsic properties of soil including mineralogy, geophysics, texture, and colour

• Soils can support a vast amount of organisms, which can be examined using DNA fingerprinting techniques:

  • Terminal restriction fragment length polymorphism (T-RFLP)

  • Denaturing gradient gel electrophoresis (DGGE)

  • Random whole metagenomic sequencing

  • High-throughput sequencing

• In practice:

  • Amplify 16S rRNA gene of the bacterial soil community DNA (1300 bp)

  • Each primer is fluorescently labelled at its 5’ end

  • PCR products are digested with restriction enzymes and separated using capillary electrophoresis

  • The output is an electropherogram that shows the digested PCR products

  • Fragments differ based on peak intensity and fragment length

  • Variations in the number and size (+/- 1 bp) of the peaks for each profile were compared and a Sorenson’s similarity index:

    • Cs = 2Nab/(Na + Nb)

    • Cs = sorenson’s similarity index

    • Nab = Number of matching peaks

    • Na = Total number of peaks in soil A

    • Nb = Total number of peaks in soil B

Bacterial Community DNA Profiling Case Study:

• A soil microbial community DNA profile was obtained from the small sample of soil recovered from the sole of a shoe and from soil stains on clothing

• The profiles were representative of the site of collection and could potentially be used as associative evidence to support a link between suspects and crime scenes

• Soil community profiles were obtained using the Terminal-RFLP fingerprinting method that uses fluorescent primer technology and semi-automated analysis techniques

  • 1. DNA is extracted from the microbial sample

  • 2. PCR is used to amplify the gene of interest

  • 3. Fluorescent primers copy and label the gene fragments

  • 4. Restriction digest cuts fragments

  • 5. Electrophoresis separates the labeled fragments by size

  • 6. Laser detection of colours representing OTUs                    

Forensic Comparison of Soils by Bacterial Community:

• Differences in profiles with time are not unexpected as seasonal fluctuations in parameters such as rainfall and temperature, may impact on the microbial community causing population shifts

• To be routinely applied to forensic casework the methodological parameters than can significantly influence variability in profiles need to be determined (optimization of DNA isolation, PCR conditions, PCR product digestion)

• Likely require more sophisticated approaches to the statistical interpretation of Sorenson’s similarity index

DGGE Analysis of 16S rDNA of Soil Bacterial Populations:

• The denaturing gradient gel electrophoresis uses gradient DNA denaturant to separate amplicons of roughly the same size based on sequence properties

• A GC camp within the last 5 nucleotides of primer is used in a special primer to anchor the PCR fragments together once they have denatured

• When the fragment reaches its melting point, it stops moving

• Partially melted dsDNA can no longer migrate through the gel

  • 1. Samples from different microbial communities

  • 2. Extract DNA from each community

  • 3. Add GC clamp (anchors DNA together once its denatured)

  • 4. Each lane in gel electrophoresis represents one microbial community

  • 5. Band alignments represent shared genes amongst communities while lone bands represent unique genes within the communities

• Limitations:

  • There is a strong bias for dominant populations

  • Biases generated by differential DNA extraction and PCR amplification, and bands can migrate to the same position (different genes of same size)

  • Lower specificity

• Case Study:

  • A blind test on soils from a crime an alibi scene and unrelated locations was conducted to evaluate the potential of environmental PCR and DGGE for use in forensic science

  • The primers for PCR were specific for conserved bacterial 16S rDNA sequences

  • Isolated PCR products of the expected size (550 bp) were subjected to DGGE

  • Digital image of gel and software performs cluster analysis

  • In most cases, soil patterns clustered according to soil type and location

  • Has potential but similar limitations to that of T-RFLP

Forensic Soil DNA Analysis using High-Throughput Sequencing:

• High-throughput sequencing offers a means to improve discrimination between forensic soil samples by identifying individual taxa and exploring non-culturable species

• Less than 1% of bacteria grow in a lab (no new class antibiotic has been discovered in the past 30 years)

• Technology that permits screening 10,000s of genes in many individuals simultaneously

• Previous genetic approaches relied on patterns of fragments length variation produced by amplification of unidentified microbial taxa in the soil extract and, these provide little resolution of spatial and temporal communities

• HTS technologies provide the ability to rapidly generate a detailed picture of soil microbial communities but require bioinformatics and significant computational abilities

• Reproducibility is a key area of concern when utilizing HTS in forensic casework

  • In DGGE, is it always the same genes that amplify? How is that impacted by sample quality? No, this is a limitation

• Validation standards must be met, which includes demonstrating:

  • Reproducibility

  • Robustness

  • Reliability

Forensic Soil DNA Analysis Limitations:

• Temporal effects could impact the DNA profile obtained since a time lapse often exists between the criminal activity and the recovery of reference samples during investigations

• Storage conditions of evidence samples could adversely affect the soil community profile

  • When soil is removed from the environment by transfer to an object, such as a shoe sole, conditions will be altered and in many cases such soil will dry-out

  • Soil moisture and sample drying will be critical in establishing the robustness of this method in practice, and further determining the most reliable target data

• Transfer of soil to objects also introduces further limitations:

  • Soil particles differ in persistence, with larger aggregates being primarily lost

  • Soil from the site of interest can be mixed with other soils, for example, a car tire may contain multiple layers of soils from different locations

• These alter soil composition and presumably the microbiota detected within samples, therefore careful consideration is required prior to analysis and interpretation of data

Microbiome and PMI:

• Current popular research topic but considerations:

  • Animal surrogates decompose differently and different microbiomes

  • Predictive models need to be developed for each region to account for factors like environment

  • Appreciate and understand the Mean Absolute Error of predictive models

Introduction to eDNA:

• Species and population monitoring has traditionally relied on physical identification by visual surveys and counting of individuals

• Complex sightability models of one species

• In many cases these techniques fall short of performing efficient and standardized surveys

  • Phenotypic plasticity (same genotype, different phenotype)

  • Similar species

  • Sex and age class

  • Terrain and weather

• Some traditional methods are invasive/destructive on the species or ecosystem under study

  • Physical removal

  • By-catch and physical damage

• Morphological identification is heavily dependent on taxonomic expertise, which is often lacking or in rapid decline

eDNA:

• Environmental DNA can be defined as trace DNA released from skin, mucous, saliva, sperm, secretions, eggs, faeces, urine, blood, roots, leaves, fruit, pollen, and rotting bodies

• eDNA is a mixture of potentially degraded DNA from many different organisms

• This definition remains controversial due to the sampling of whole microorganisms that might appear in an environmental sample

• DNA based species identification or DNA barcoding is used in order to detect species through extracellular DNA or cellular debris, present in environmental samples, coming from cell lysis or living organism excretion or secretion

• Can detect species without catching, seeing, or hearing them

• eDNA methods are accelerating the rate of discovery, because no a priori information about the likely species found in a particular environment are required for ID of said species    

• Methodology overview:

  • 1. Environmental sampling

  • 2. DNA extraction

  • 3. Amplification of a gene fragment using universal primers

  • Next-Generation sequencing

  • Comparison to reference databases

• If a sample is not in a database, it is possibly a new species

• Absence of evidence is not evidence of absence

• Can to some degree give you trends

Who Works with eDNA:

• Those working with invasive species, biodiversity surveys and understanding functional diversity, wildlife and conservation biology

• Consulting companies now offer eDNA as a service

• Many government agencies

• US Army Corps of Engineers monitor Asian carp

• General focus on aquatic environments but changing

• The first application of the method in aquatic environments dates to 2008 where the invasive american bullfrog was successfully detected with an eDNA method in France

• Applications are increasing far and wide

What is eDNA?

• eDNA can be extracted from environmental samples such as soil, water and faeces without having to isolate the target organism

• Often a standard or modified DNA extraction protocol

• Composed of living and dead cells; intracellular DNA and extracellular DNA (from natural cell death and the subsequent destruction of its structure)

• To date, eDNA studies have predominantly focused on:

  • The method (improving, optimizing, comparing to standard field techniques)

  • Identifying species specifics (pathogenic, endangered, invasive, genetically modified, and game species)

  • Reconstruction of diets and ancient communities                          

eDNA in the Environment:

• DNA persistence in nature varies by environment (marine, freshwater or terrestrial) and substrates (water, soils, and sediments)

• In marine and freshwater environments, eDNA persistence varies considerably between studies, ranging from a few hours up to a month

• eDNA persistence may vary within the water column where higher levels of DNA degradation are observed at the top (warmer and exposed to UV radiation) compared to the bottom (colder) layer

• In general, cold and dry conditions severely slow down DNA degradation whereas, warm and wet conditions rapidly facilitate degradation

• In sediments and in terrestrial soils, a very high proportion of DNA can persist for long periods, adsorbed to organic or inorganic particles that protect it from several possible degradation agents

• In marine sediments it was demonstrated that extracellular DNA turnover is around 200 times slower than in sea water (93 days in sediments vs 10 hours in sea water)            

Factors Influencing eDNA:

• DNA is degraded by endonucleases (enzymes that cut the nucleic acids into smaller fragments), water, UV radiation, and microorganisms

• In aquatic ecosystems (or samples containing water), the main process that causes DNA damage is hydrolysis and the breakage of the DNA sugar backbone

• In order to stop this process, samples should be quickly dried, or put in a solution containing a preservative (alcohol)

• UV radiation can disrupt the DNA base-pair bonds

  • eDNA was not detectable in samples exposed to sunlight (8 days); detected in samples stored in the dark (up to 18 days)

• Microorganisms consume DNA as a source of nutrients (carbon, nitrogen, phosphorus) and to repair their own DNA

• The rupture of a cell releases DNA and cellular fluids into the environment, which stimulates the growth of microorganisms and leads to further DNA degradation

• Increased chlorophyll has shown increased faster rates of eDNA degradation

  • Increased chloroplast = increased decomposition rate

Advantages of eDNA:

• Studies have found higher detection probabilities using the eDNA method compared to traditional methods

• This does not apply to all species, but is true for rare and secretive/cryptic species, or invasive alien species at early stages of the invasion

• Once optimized, obtaining an environmental sample can be carried out in a very standardized manner across sites

• Traditional methods rely on the taxonomic knowledge and experience by personnel carrying out the surveys

• eDNA is a non-invasive method that inflicts little to no damage on the species or habitats under study

• For several species, traditional surveys are difficult outside particular seasons or certain conditions

• Several studies report shorter handling time and lower cost using eDNA compared to traditional monitoring techniques

• eDNA is often more cost-efficient than traditional methods because of its higher detection probability

  • 2.5 times cheaper, less time consuming than traditional methods for surveying invasive American bull frogs

• For species that are easy to observe or catch, eDNA will be less cost effective than traditional methods (context dependent)

• With eDNA methods, DNA-free materials are used between locations and many precautions are taken to prevent contamination with DNA

• This helps to reduce the risk of unintentional translocations of invasive species, or transmission of pathogens into new areas

• For example, the infectious amphibian disease chytridiomycosis, could be introduced into new areas by conventional field gear that is not disinfected

  • Use positive and negative controls to assess contamination
    

Limitations of eDNA:

• The majority of eDNA studies use a mitochondrial gene as a marker because there are hundreds to thousands of copies of mtDNA per cell (this is barcoding)

  • Smaller, circular, highly conserved, abundant

• This high copy number enhances the likelihood of detection of the DNA in degraded samples such as environmental samples; also think about its structure

• Some mitochondrial genes (Cox1, Cytb, control region) also have an evolutionary rate, which makes them more appropriate for species ID based on the genetic variation

• mtDNA is usually maternally inherited, which does not allow the ID of hybrid organisms, but only permits determination on the maternal species of the organism

• This is a major limitation in cases where an invasive species hybridizes with a native species since they do not get DNA from both parents

• Might also require conventional methods (morphological characteristics to identify hybrids)

• Assay development and bioinformatics are not straightforward

• eDNA is not universally entirely quantitative 

  • Current methods thus are only capable of detecting the presence or absence of a species

• Contrary to conventional methods, no information can be collected on life stages, demography, fecundity or health of the target species – all critical to management

• There is no collateral knowledge, it is gained from observations with traditional methods 

• eDNA is not homogeneously distributed throughout a water body (depth, shoreline) 

• Sampling locations should be selected according to the habitat preference of the target species

• Samples should be collected from areas where the target species is most likely to be detected

  • Weatherfish in the Netherlands were detected 2.5 times more often in habitats judged to be good

  • Random sampling or by non-specialists may not work

Lecture 5: Non-Human Models for Human Applications         

Questions of Interest to Researchers at Trent (Shafer Lab):

• Estimating time since deposition of blood

• Estimating rate of secondary DNA transfer from blood

• Clear applications to human forensics

• Use cow blood and semen for all experiments (pigs currently being used for decomposition)

Why Cow Blood?

• Easier to work with than human blood

•  Species specific attributes of the blood (viscosity, hematocrit levels, but useful (relevant) alternative and accessible

Time Since Deposition:

• No accurate or robust way to measure time since deposition of blood on crime scenes

• Important for establishing a time frame and we know blood undergoes a series of changes after being deposited ex vivo

• Can we determine the TSD of blood by pairing spectroscopy and high-resolution automated electrophoresis

• A tapestation machine is used for automated gel electrophoresis

  • Tapes containing gels with lanes fit into the machine

  • The standard ladder contains fragment sizes from 45000 bp to 250 bp

  • The top panel shows the size of any detectable DNA fragments

  • The bottom panel shows us the quantity of fragments at each size via quantification w intercalating dye detection

• Spectroscopy (Luminometer) shows blood degradation over time

  • The alpha and beta Soret bands reflect the stages of heme over time

  • “A Blue Spectral Shift of the Hemoglobin Soret Band Correlates with the Age (Time Since Deposition) of Dried Bloodstains

• Overall, DNA and RNA concentrations are expected to decline over time, but finer metrics such as shifts in DNA fragment length have not been quantified

• Fragments should become shorter over time, decrease on long fragments, increase in short fragments

Methodology of Hemoglobin Soret Band Correlates with TSD of Dried Bloodstains:

• 15 uL fresh pathogen free bovine blood

• 10 samples for each (5 TapeStation, 5 Luminometer)

• 11 time intervals (0-36 hours)

• Room temperature (Part 1)

• Warm and cool environments (Part 2)

• Look for a correlation to time and test predictability via cross-validation

• FTA paper: preserves blood samples

• Results:

  • Clear signature but not hourly; fresh vs old (days vs week)

  • Controlled environmental and high individual variation are current limitations

  • Difficult to implement in a court of law as we see environmental effects

  • Could RNA provide a finer resolution?

Automated Gel Electrophoresis (Tapestation) for RNA:

• The RNA integrity number equivalent (RINe) delivers an objective assessment of eukaryotic and prokaryotic total RNA degradation

• There is a strong inflection point at 28 hours for the ratio of RNA fragments above 200 bp (DV200)

• RNA decays drastically after 24 hours; could this be a diagnostic window?

• Could targeting specific RNA genes improve the model? qPCR techniques could be used to target specific genes

Time Since Deposition:

• High variation, often non-human

  • Could this be used in a court of law?

  • Non-human surrogates are often used

• Does our foundational understanding through human blood surrogates meet these standards

  • Often meet 3Rs but we must apply them to humans

  • If yes, how transferable are these patterns to human blood?

DNA Transfer:

• How common is it?

• What factors promote DNA transfer?

• Important for understanding potential links to evidence; has also been used as a defence

• Can determine the rate of secondary DNA transfer via qPCR and genotyping STRs

  • If a DNA profile is identified at a scene, does that mean the ID was involved in the crime?

• DNA transfer examples:

  • It was not possible to say whether the DNA had been deposited directly (primary transfer) or indirectly (secondary transfer)

DNA Transfer on Denim Methodology:

• Passive, pressure, friction, and control transfers

• qPCR was used to amplify the cytochrome b sequence (mtDNA)

• Results:

  • The results showed that DNA is transferred between materials and can be amplified

  • The pressure samples showed the best measure of variance

  • DNA does transfer, varies by substrate but differs in concentration between primary and secondary transfer

  • Often the transfer substrate did not have a blood stain (we knew where to look), whereas there is no visual of DNA transfer in real world cases

• Can you amplify STRs? This is the primary marker used in human applications

• Can we generate a profile from the DNA present?

Lab Studies to Date:

• Incremental , multi-pronged assessment of real forensically relevant phenomenon with molecular tools

• High variation, does make application challenging but lots of options

• Large enough database might allow for diagnostic patterns to be identified and validated

• A meta-analysis has been done on ex-vivo whole blood degradation

  • This is a statistical analysis of the data from independent primary studies and is not a literature review paper

• This paper calls for:

  • Considering interdisciplinary research that integrates multiple analytical techniques

  • Use of the most readily available mammalian blood source for proof of concept TSD studies

  • Validation with human blood prior to crime scene implementation

  • Focus on research on non-porous substrates due to their larger effect size (appears most promising)

  • Report exact environmental conditions for room or ambient temperature, and humidity

  • Application of multiple analytical techniques where possible

The Role of Cats in Human DNA Transfer:

• Background samples from the cat’s head, back, right, and left

• Transfer from a cat to a gloved hand after petting of the neck

• Transfer from an ungloved hand to a cat

• 20 cats

• Quantifiler trio (specific to human DNA) to not amplify other sample type, gives female/male ratios as well

• PowerPlex 21 (STR assay kit)

• Assessed the background amount of DNA on the cats at the four locations

• Detectable DNA was collected from:

  • 95% of the back samples (19/20)

  • 90% of the head samples (18/20)

  • 80% of the right samples (16/20)

  • 10% of the left samples (2/10)

• The researcher patted and scratched a cat’s back of neck 8 times while wearing new gloves

• The same, foreign to the household, researcher patted and scratched the cat’s chest and neck area 8 times using their bare dominant hand

Integrating Microbial Profiling and Machine Learning for Drowning Site Inference:

• Drowning locations are typically difficult to pinpoint

• Can we leverage our understanding of microbial communities to assist with this process?

• Used mice and rabbit surrogates

• Alpha diversity indices of eight sampling locations

  • Highlights variations in species richness and diversity across different sampling points

• Results suggest that when inferring drowning sites across different animal species, relatively good accuracy can still be achieved, even when there are significant differences between water samples from the target location and those from other locations

Changes in Oral Health During Aging in Primate Model:

• Marmoset system, comparing 3 stages of life (young, middle, old)

• Some work in humans, largely gut microbiome

• Looked at morphological changes in oral health and in the oral microbiome

• Trying to establish marmoset model recapitulating the changes in oral health associated with human aging

• Results:

  • Despite some differences in abundances, no significant findings associated with the microbiome and ageing

  • The changes in oral manifestations (tooth loss) and alpha diversity of the oral microbiome that failed to show any statistically significant differences between age groups

  • Could be due to the limited statistical power of our sample size (6)

Detecting Human DNA in Aquatic Environments – Potential of eDNA in Forensics:

• Human eDNA is largely untouched in the area of investigation

• Looked at the rate of DNA decay and profile recovery of temporal samples

• Results demonstrated that human eDNA remains detectable for up to 36 hours in freshwater samples and up to 84 hours in saltwater samples

• nuDNA profiles are less stable and reliable

• Degradation index = ratio of small nuDNA fragments to large nuDNA fragments

  • Ratios below 1 have little to no degradation and those greater than 10 have severe degradation

• After 24 hours in both conditions, no viable DNA remained for analysis

Comparison of NGS (SNPs) and Capillary Electrophoresis (STRs) in the Genetic Analysis of Human Remains:

• Two laboratories

• 3 different methods; 2 CE-STR and 1 NGS-SNP approach

  • Powerplex ESX 17 Fast System (17 autosomal STRs)

  • Powerplex Y23 system (23 Y-STRs)

  • ForenSeq Kintelligence Kit (NGS; 10,230 SNP markers curated for forensic kinship)

• Applied to 40-83 year old remains of war victims

  • Only obtained sufficient DNA from 16 samples to proceed with all workflows

  • The data support that high throughput SNPs could be the method of choice for missing persons cases

  • The current STR systems would not yield viable data or would not be able to be associated with family members

  • SNP data is best for smaller and lower quality strand samples

  • This is best for degraded samples and missing persons cases

Fourth Generation Sequencing:

• In 2nd and 3rd generation sequencing, we do not know where DNA/RNA is coming from within samples

• With 4th generation sequencing, we can track where DNA, RNA, and single cell samples are coming from