1
Q
what is a microbiome
A
community of micrrorganisms
2
Q
describe PCR amplification of 16s rRNA gene sequences
A
- extract DNA
- amplify and sequence 16s rRNA
- group
- group similar sequences and quantification of OTUs representation
3
Q
Use of microbiome assessments provides what in forensics
A
associative evidence between people and objects with places or with other people
4
Q
whats the diff between rRNA and rDNA
A
rDNA is the gene that CODES for the rRNA
5
Q
what is OTU
A
- operational taxonomical units
- group sequences by a fixed similarity threshold
6
Q
what is ASV
A
amplicon sequence variants*
7
Q
describe the goats that he looked at
A
- some were wild and some were domesticated
- ## some microbes were more abundant in wild ones, other more abundant in captive
8
Q
why might captive and wild goats have diff microbes
A
- diff diets captive will have the “ideal diet” as its controlled
- some microbes might need to be there to get as many nutrients as possible from the wild food sources, which arent needed in the captive
9
Q
describe the pandas being reintroduced to the wild
A
- 6/9 pandas survived in a 2018 reintroduction
- so they looked at the gut microbiome to see the differences in captive, training, reintroduced, and wild
- HUGE difference in proteobacteria (none there in captive but tons in wild)
- so maybe they should be trained to get better gut microbiomes pre-introduction so theyre best set up for survival
10
Q
Use of microbiome assessments provides _________ between people and objects with places or with other people
A
associative evidence
11
Q
microbiomes can be used similarly to what other DNA thing
A
microsatellites
12
Q
what does this mean “For post-mortem intervals, the focus becomes predictive modeling”
A
you give me a single unknown microbiome profile, I can tell you the PMI
13
Q
what are used to assist in finding PMI from an unknown microbiome profiles
A
- machine learning like elastic net regression
- cross-validation
14
Q
true/false linear regressions can be used to calculate PMI
A
true
15
Q
what are some of the pros to using linear regressions for PMI estimates
A
- automates variable selection
- addresses multicollinearity
16
Q
how do we do cross validation
A
- so we have training that we use to build the model
- and then have test data (withheld and known) in order to test to see that the linear regression is accurate enough
17
Q
what does MAE stand for
A
mean absolute error
18
Q
what does an R2 value of 0 mean
A
your regression explains NONE of the variation
19
Q
what does an R2 value of 1 mean
A
regression explains ALL of the vaiation
20
Q
how tools do we use to associate trace soil evidence to suspects
A
- gas chromatography
- electron microscopy
21
Q
Until _________ years ago, genetic/genomic analysis of soil microorganisms had largely been ignored by the forensic scientific community
A
~10-15
22
Q
why were genetic analyses of soil microorganisms ignored for so long
A
- limitations of traditional culturing techniques
- which allow only a small subset of organisms to be isolated and characterized
23
Q
true/false Techniques have been developed to circumvent the requirement to isolate and culture microorganisms as a prerequisite to identification
A
- true
- think of sanger vs now HTS
24
Q
how is soil microbial diversity now characterized
A
using simple molecular techniques (mostly) based on amplified rRNA (gene) DNA
25
Soil serves as powerful _______ because it is highly individualistic and has a high transfer and retention rate
contact trace evidence
26
why does soil serves as powerful contact trace evidence
because it is **highly individualistic** and has a **high transfer and retention rate**
27
what is involved in standard analyses of soil
- **look at intrinsic properties of soil** including
- mineralogy
- geophysics
- texture
- colour
28
Soils can support a vast number 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
29
describe the soil microbiome 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
- profiles were representative of the site of collection
- profiles were obtained using terminal-RFLP fingerprinting
- which uses fluorescent primer tech and semi-automated analysis techniques
30
describe the process of Terminal Restriction Fragment Length Polymorphism
- start w the environmental microbe sample
- extract the DNA
- PCR amplify the gene of interest
- and add a fluorescent primer
- so we add terminal label to make sure were only looking at the DNA of interest
- restriction enzyme will digest it
- making diff sizes of labeled fragments
- electrophoresis will happen and then detected w a laser, and an electropherogram will happen
31
how do the fragments differ in an electropherogram
- **peak intensity** y axis
- **fragment length in bp** x axis
32
what is the error fpr the peaks in an electropherogram
- **+/- 1 bp**
- due to incomplete adenylation
33
what is sorenson's similarity index
34
what must be done for forensic comparison of soils by bacterial community DNA profiling to be routinely applied to casework
- the methodological parameters that can significantly influence variability in profiles need to be determined
- *prob requires more sophisticated approaches than sorensons similarity index*
35
Differences in profiles with time _____ *(are/ are not)* unexpected
- **are not**
- seasonal fluctuations in parameters such as rainfall and temperature, may impact on the microbial community causing population shifts
36
what does DGGE stand for
denaturing gradient gel electrophoresis
37
how does DGGE work
- uses a gradient DNA denaturant *mix of urea and formamide*
- to separate amplicons of roughly the same size based on sequence properties
- GC clamp is used in a species primer to anchor the PCR fragments together once denatured *(the two strands cant separate cause theyre clamped together)*
- when the fragment reaches its melting point, itll stop moving
- partially melted dsDNA cant migrate through the gel
38
where are GC clamps found
typically last 5bp of a primer
39
what are the limitations of DGGE
- strong bias for dominant populations
- biases generated by differential DNA extraction and PCR amplification
- bands can migrate to the same position (diff genes, same size)
40
describe the case example of DGGE analysis of amplified 16s rDNA of soil bacterial populations
- a blind test on soil from a crime, alibi scene, and unrelated locations
- primers for PCR were specific for conserved bacterial 16s rDNA sequences
- isolated PCR products of the expected size (550bp) were subjected to DGGE
- in most cases, soil patterns clustered according to soil type n location
41
______% of bacteria grow in a lab
less than 1%
42
what are the benefits of using HTS in soil analyses
- improve discrimination between forensic soil samples by identifying individual taxa and exploring non-culturable species
- permits screening of 10,000s of genes in many individuals simultaneously
43
why do previous genetic approaches for soil have little resolution
cause they relied on patterns of **fragment length** variation produced by amplification of unidentified microbial taxa
44
whats the big downside of HTS for soil
- need bioinformatics and signifiant computational abilites
- also need reproducibility
45
what is included in validation standards that must be met for forensic soil DNA analysis w HTS
- reproducibility
- robustness
- reliability
46
what are the limitations of forensic soil DNA analysis
- temporal effects
- storage conditions of evidence could affect the soil profile
- transfer of soil to objects *soil particles diff in persistence, larger aggregates lost* *also things like car tires may have soils very various locations*
47
what considerations must be considered when researching microbiome and PMI
- Animal surrogates decompose differently AND have different microbiomes
- Predictive models need to be developed for each region *(i.e. environmental effects)*
- Appreciate and understand the MAE of predictive models
48
species/population monitoring has traditionally relied on what
- physical identification by visual surveys
- counting of individuals
49
whats the issue of traditional methods for species/population monitoring
- *fall short of performing efficient and standard surveys*
- phenotypic plasticity
- similar species
- sex and age class
- terrain/ weather
- *also some are invasive/ destructive on the species or ecosystem*
- physical removal
- by-catch and physical damage
50
what are some studies for PMI microbes done
- **6 mice sampled 4 times** orally, found an R2 of almost 1, really good but also HUGE error bars
- **65 mouse graves** in the soil, could see some noteable differences in the relative abundance of some microbes
- **21 cadavers** skin swabs at the tennessee body farm. only sampled them once (less than ideal) *cause even the location of the swab on the body can differ enough*
51
what the problem w morphological identification
- heavily dependent on taxonomic expertise
- which is often lacking o in rapid decline
52
what is eDNA
- **environmental DNA**
- trace evidence
- released from skin, mucous, saliva, sperm, secretions, eggs, faeces, urine, blood, roots, leaves, fruit, pollen, and rotting bodies
- mixture of DNA from many diff organisms
53
what is controversial about the eDNA definition
due to the sampling of *whole* microorganisms that might appear in an environmental sample
54
what is DNA barcoding for w eDNA
- to detect species through extracellular DNA or cellular debris
- present in environmetnal samples
- coming from cell lysis or living organism excretion or secretion
- **can detect species without catching, seeing, or hearing them**
55
eDNA methods are accelerating what and why
- **rate of discovery**
- no a priori information about the likely species found in a particular environment are required for identification of said species
56
**true/false** eDNA is a single source
FALSE its a mix
57
**true/false** if an eDNA sample is not in a database, it is *possibly* a new species
true
58
who works w eDNA
- **basically anyone**
- Those working with: invasive species, biodiversity surveys and understanding functional diversity, wildlife and conservation biology (researchers at Trent)
- 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
59
what is the first application of eDNA methods in aquatiic environments
- 2008
- invasive american bullfrog
- successfully detected w eDNA in france
60
where can eDNA be extacted from
- **environmental samples**
- like soil, water, and feces
- *without having to isolate the target organism*
61
**true/false** just cause we dont see it, doesnt mean its not there
true
62
**true/false** eDNA can give us info on trends
true
63
**true/false** eDNA is composed of living and dead cells
true
64
what have most eDNA studies focused on
- the method *improving, optimizing etc*
- identified species specifics *pathogenic, endangered, invasive, GMO etc*
- reconstruction of diets and ancient communities
65
DNA persistance in nature varies by what
- **environment** marine, freshwater, terrestrial
- **substates** water, soils, sedements
66
**true/false** eDNA persistance varies between studies in marine and freshwater environments
- **true**
- from hours to months
67
how does eDNA persistence differ between top and bottom layers of water
- **higher** more DNA degredation, cause UV radiation and warmer
- **lower** less degredation cause colder
68
what conditions slow down DNA degredation
cold and dry
69
how does DNA persist in sediments/ terrestrial soils
adsorbed to organic or inorganic particles that protect it from several possible degradation agents
70
extracellular DNA turnover is ______x _____ *(slower/faster)* in marine sediments than sea water
200x slower
71
what degrades DNA
- endonucleases
- water
- UV radiation
- microorganisms
72
in aquatic ecosystems, what is the main process that causes DNA damage, and how should we stop thi
- hydrolysis and breakage of the backbone
- samples should be quickly dried, or put in a solution w a preservative like alcohol
73
how does UV radiation affect eDNA
- **disrupts 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)
74
why do some microorganisms consume DNA
- as a source of nutrients (carbon, nitrogen and phosphorus)
- to repair their own DNA
75
what is the result of the rupture of a cell releases DNA and cellular fluids into the environment
- stimulates the growth of microorganisms
- leads to further DNA degradation
76
what is the result of increased chlorophyll w eDNA
faster rates of eDNA degredation
77
Studies have found _______ *(lower/higher)* detection probabilities using the eDNA method compared to traditional methods
- **higher**
- not for all species tho
- true for rare and secretive species OR invasive alien species at early stages of invasion
78
what are the advantages of eDNA
- once optimized, environmental samples can be done standardized across sites
- dont need to rely on taxonomic knowledge and experience
- non-invasive that inflicts little to no damage
- less difficult than traditional surveys (during certain conditions or seasons)
- higher detection probabilities compared to traditional
- shorter handling time and lower cost
- more cost efficient *most of the time*
79
why is eDNA more cost-efficient than traditional methods
- **because of its higher detection probability**
- (e.g. 2.5 times cheaper, and less time consuming, than traditional methods for surveying invasive American bull frogs)
- *doesnt apply for species that are easy to observe or catch*
80
what is done to reduce the risk of unintentional translocations of invasive species or transmission of pathogens into new areas
- DNA-free materials are used between locations and many precautions are taken to prevent contamination with DNA
- *rather than traditional methods where diseases might be spread using field gear that isnt disinfected*
81
what are the limitations of eDNA
- typically use mtDNA because there are more copies, which enhances the likelihood of detection, but also some genes have an evolutionary rate so theyre good for species identification
- but also mtDNA doesnt allow identification of hybrid organisms, just the maternal species
- this is a HUGE deal w invasive species when they hybridize w a native species
- sp we might need conventional methods to assist (like morphological things)
- so assay development and bioinformatics arent straightfoward
- w a blast search, youre told to sort by % similarity, but itll acc only give you the top 50 results, but since it wasnt at the top of that initial list pre-sorting, it might just not be there
- eDNA isnt entirely quantitative yet, just looks at presence or absence (not amount of each)
- no info on age, demography, fecundity, or health *which are all critical to management... so theres no collaterol knowledge*
- its not homogenously distributed throughout a water body (think waves move things around), so sampling locations should be selected based on preferences
82
whats an example of collateral knowledge
this fungus that was a deadly disease for fire salamanders, was detected because volunteers found them dead and reported it
83
describe the mediterranean monk seal case study
- in 2017, there were only 700 of them
- they were split apart 4 subpopulations
- they looked at fecal samples
- were able to look at markers to investigate diet
- they saw a shift in diets between diff populations, so even tho theyre the same species, they were eating diff things
84
Applied Molecular Genetics
flashcards
Decks in class (12)
# Cards
-
Intro + Overview80
-
DNA markers and platforms99
-
Wildlife frsc, conservation, non-human application98
-
Microbial genomics84
-
Non-human models for human applications63
-
Readings19
-
Human Genome Project79
-
New Sequencing + FRSC Applications54
-
Comparative genomics52
-
RNA expression and Forensic applications49
-
Methylation & Gene Editing50
-
Investigative Genetic Genealogy8
