Forensic Soil Science Final Exam

Soil Forensics

study of physical, chemical, and biological composition of soil as a part of criminal investigations

Soil microbial forensics

The study of how microbes in soil are used to forensic investigations

• 1 tbsp soil contains 10⁹ microbes

• vast majority microbes nonculturable

• Makes soil one of most complex ecosystems on earth

Microbiome

totality of microbes in a defined environment, especially their genomes and interactions with each other and surrounding environment

Soil microbiome

soils harbor the highest abundance and diversity of microorganisms

• bacterial, archaeal, fungal species

• bacteria dominate (mainly actinomycetota and pseudomonadota)

Why microbiome research is recently new

1. There was bias for culturable microbes, especially pathogens

2. Technology and tool availability has increased (PCR, sequencing, culture independent techniques)

Prokaryotes

contain no nucleus, no membrane bound organelles, single celled, single-circular chromosome for DNA, peptidoglycan cell walls, one origin of replication, coiled circular chromosomal DNA

• Ex: bacteria and archaea

Eukarytoes

have true nucleus, linear DNA in rod shaped chromosomes, membrane bound organelles, single cellular or multicellular

• Ex: fungi, protozoa, algae

Light microscopy vs Electron microscopy

Light: can visualize most plant and animal cells and mitochondria (100-1 micrometer)

Electron: visualize smallest bacteria, ribosomes, atoms (less than 1 micrometer)

Bacteria shapes

coccus= spherical

bacillus= rod-shaped

vibrio = curved

Types of bacterial classification

1. Phenetic —> physiology/morphology (under microscope and colonization)

2. Genotypic —> DNA composition

3. Phylogenetic —> evolutionary comparisons

Cell morphology

• size (bacteria around 1 micrometer)

• shape

• arrangement

• motility

Three cell wall features

1. Gram-negative (pink)

2. Gram-positive (purple)

3. Bacteria without cell walls

Gram-negative bacteria

contain one layer of peptidoglycan underneath outer membrane

• stain PINK because outer membrane is destroyed

• lack glycine peptide interbridge

Gram-positive bacteria

Thick peptidoglycan outer layer, then periplasmic space

• stain PURPLE because peptidoglycan maintained

• contain glycine peptide interbridge

Peptidoglycan features

glycan backbone and amino acid side chain

• NAG and NAM

• d form amino acids (unusuallll)

• diamino acids

Gram staining method

1. application of crystal violet (purple)

2. application of iodine

3. alcohol wash (decolorization)

4. Application of safranin (pink counterstain)

Biochemical properties observable in culture/colonies

• metabolism and enzymatic activity

• nutrient utilization

DNA-DNA hybridization assay

extent of hybridization indicates degree of similarity between samples

• more mismatches, more different organisms are from each other

GC content

G to C and A to T ratios are fixed in specific organisms

• these ratios can vary, indicating uniqueness

• increased GC content = increased withstanding of heat (curve shifted right)

DNA sequencing includes

sanger sequencing and whole genome sequencing

Average Amino Acid Identify (AAI) and Average Nucleotide Identity (ANI)

pairwise comparison between the whole or partial genome sequences of two microorganisms expressed as a percent identity

• 96% and up is good

Phylogeny

based on degree of DNA nucleotide similarity

• mutations can occur however

• high similarity = recent common ancestry w/ few mutations

• low similarity = distant common ancestry w/ more mutations

16S ribosomal RNA (or 18S in eukaryotes)

commonly used for measuring homology

• universally conserved

• suitable for identifying relationships (ideal length of 1.5 kb)

• conserved and variable regions (V4)

  • 515F and 801R flanking V4

Evolutionary distance (E_D)

number of nucleotide differences between sequences

• UNDERESTIMATES true genetic divergence

Corrected E_D

Adjusts for back and multiple mutations at the same site

Bacterial growth

Binary Fission

• grow exponentially

Growth characteristics

1. Lag phase = acquiring nutrients

2. Log phase = exponential growth (ONLY with resources)

3. Stationary phase = same amount replicating and dying

4. Decline phase = more death than growth

Chemotrophs

obtain energy through oxidation of chemical compounds

• Chemoautotrophs or Chemoheterotrophs

Chemoheterotrophs

use ORGANIC compounds for BOTH energy and carbon

• carbon source = organic compounds

• ex: Escherichia coli

Chemoautotrophs

Use chemical energy from INORGANIC compounds to FIX CO2 into organic molecules

• carbon source = CO2

• ex: nitrosomonas

Phototrophs

Obtain energy from light

Photoheterotrophs

use light for energy but require ORANIC CARBON

• carbon source= organic compounds

• ex: Rhodospirillum

Photoautotrophs

use sunlight energy to FIX CO2 into organic molecules

• carbon source= CO2

• ex: Cyanobacteria

Oligotrophs

thrive in LOW nutrient environments

• ex: Acidobacteria (low pH)

Copiotrophs

prefer nutrient rich environments

• ex: Pseudomonadota

Three physical factors that affect bacterial growth

1. Oxygen

2. Temperature

3. pH

Obligate aerobes

Lots of Oxygen required!

Catalase, superoxide dismutase stops ROS species

Obligate Anaerobes

NO oxygen!

has no antioxidants to fight ROS species

Facultative Anaerobes

both oxygen and no oxygen! (prefers oxygen)

contains catalase and SOD for ROS species

Aerotolerant Anaerobes

No oxygen preferred (can still persist with O2, she dont care)

ONLY SOD, not catalase

Micro aerophiles

need LOW oxygen concentration

too much ROS bad

Psychrophiles

thrive in cold environments!

• <15 C

• ex: psychrobacter, Flavobacterium

Mesophiles

moderate temperatures

• 20-45 C

• most soil bacteria

• ex: Pseudomonas fluorescens, Bacillus subtilis

Thermophiles

Hot environments

• 45-80 C

• hot springs with high organic matter

• ex: Thermus aquaticus (Taq Polymerase!!!)

Hyperthermophiles

extreme heat

• above 80 C

• geothermal or deep sea vents

• ex: pyolobus fumarii, Thermococcus gammatolerans

pH naming systems for bacteria

range from:

extreme acidophiles (pH=1) to extreme alkalophiles (pH=14)

Agar

remains solid at incubation temperatures and indigestible to most microbes

• stable growth environment

Complex vs Defined Media

Complex: provides more than necessary nutrients

Defined: provide ONLY building blocks needed for growth

Three culture based techniques

1. Spread plate

2. Pour plate

3. Streak plate

Two culture independent techniques

Amplicon based —> 16S rRNA, 1 specific region

Shotgun metagenomic —> sequences all genes

PCR steps

1. Denaturation (95 C)

2. Annealing (60 C)

3. Extension (72 C)

PCR components

dNTP’s, Taq Polymerase, buffer solution, Mg2+, primers

Gel Electrophoresis

loading dye is added to the DNA, placed in wells, run from a negative to positive current, separating DNA by size and charge

• smaller DNA moves further

• UV visualization= based on fluorescence from dyes bound to aromatic nucleotides

• qualitative —> position + presence and absence

Prokaryotic ribosomes

50S (Large): contains peptidyl transferase center

30S (small): associates with mRNA during translation, mRNA decoding

How soils bacteria can be used in forensics

1. traced back to objects related to a crime

2. can estimate PMI

3 steps for investigating microbial communities

1. sample collection and storage

2. DNA extraction, purification, and quality control

3. 16S rRNA amplicon sequencing and analysis

Sample collection

use sterile tools, gloves, screw-top tubes

Sample storage

store samples at a low temperature (-20 to -80 C) to preserve DNA quality

Why is bead beating used?

to avoid biases in soil extraction so that gram negative and gram positive bacteria can both be lysed

Three steps for obtaining DNA samples

1. Lysis (extraction) [mechanical and non mechanical]

2. Isolation (purification)

3. Resuspension

Types of Lysis

Physical —> efficient, time consuming, costly

Chemical —> easy, fast, low cost, can cause disruption in sample

Enzymatic —> no equipment required, selective, expensive, time consuming

Three types DNA extraction

1. Phenol-Chloroform Extraction

2. Silica-Phase Extraction

3. Magnetic Bead Extraction

Phenol-Chloroform Extraction

uses 1. cell lysis, 2. phase separation (H2O= top, chloroform=bottom), 3. transference, 4. precipitation (ethanol), 5. resuspension

• more DNA obtained, collecting aqueous phase takes practice

Silica-Phase Extraction

uses 1. cell lysis, 2. DNA binding to column (high salt), 3. washing (ethanol), 4. elution (low salt aka water)

• fast and selective, amount DNA limited

Magnetic Bead extraction

uses 1. cell lysing 2. mix with beads, 3. put into separator, 4. wash, 5. DNA elution

• moneyyyy

Nucleic acid absorption values

around 260 nm

• proteins: 280

• salts and organic: 230

  • INCREASE TEMP INCREASE ABSORPTION

Qubit Flex (Fluorometry)

fluorescent dyes bind selectively to either DNA or RNA (specific)

• CANT see contaminants with this method

Three ways to assess DNA purity

1. Absorbance

2. Electrophoresis

3. Bioanalyzer

Absorbance (DNA purity)

A260/A280 = 1.9-2.1

• less than 1.9 —> protein contamination

• Greater than 2.1 —> wrong blank

A260/230 = 1.5-1.8

• less than 1.5 —> contamination with phenol, organic material, salts

Gel electrophoresis (DNA purity)

visualize DNA bands

• All at the top = larger DNA materials w/o contaminants

• Most in middle or bottom = contamination with fragments, RNA, etc.

Bioanalyzer (DNA purity)

electrophoretic separation on microchips!

• smaller molecular weights measured first, larger later

Library Preparation

two step PCR method in which

A. target specific PCR occurs

B. Addition of barcodes and sequencing adaptors by 2nd PCR

C. Final amplicon ready to be sequenced

Parts of Library Preparation

Primer —> bind to conserved regions around variable regions

Linker —> overhang that barcodes bind to

Barcodes —> specific DNA tags to identify DNA after multiplexing

Sequencing adaptors —> bind fragments to flow cells

Specific variable amino acids

Y or W

• include many specific primers to account for that

PCR product sizes

V4 region = 252 bp

PCR 1 product = 319 bp

PCR 2 product = 388 bp

Multiplexing

Samples are sequenced together for efficiency, barcodes identify specific samples

Normalization

make libraries equally represented in concentration

First vs Second vs Third Generation sequencing

1st - Sanger (dNTP’s and electrophoresis, 500-1000 bp fragments)

2nd - Illumina (each nucleotide fluorescing, 50-500 bp fragments)

3rd - Nanopore (tens of kb fragments)

Cluster generation

amplification of library with primers fixed to flow cell surface

• each unique baterial 16S V4 sequence forms its own cluster

Illumina sequencing by synthesis

one cycle generates one base

• add first base, image, deblock

• fluorescent dye added to 3’ -OH, Polymerase cant add next nucleotide till this is removed

Steps of bioinformatic analysis

1. Demultiplexing

2. Quality Control

3. Denoising reads

4. Taxonomy assignment

5. Microbial diversity analysis

1st : Demultiplexing

assigning reads to corresponding sample based on barcodes

• we only used forward reads because our sample was too long

2nd : Quality Control

FastQC —> forward and reverse reads separated, quality decrease towards end of sample

MultiQC —> Overall reads together, gives PHRED score

• PHRED greater than 20 is good

• PHRED less than 20 is bad

PHRED scores

corresponds to probability P for nucleotide to be wrong

• Q = -10log₁₀ x P

• P = 10^-Q/10

• Q= 20 means P=0.01, meaning probability of 1% to be incorrect

3rd : Denoising reads

DADA2 = Divisive Amplicon Denoising Algorithm

• uses abundance data and error model to remove sequencing errors and detect ASV’s

ASV’s = Amplicon Sequence Variants

• highly resolved DNA sequences that represent distinct biological entities in microbiome studies

Operational Taxonomic Units (OTU’s)

Before DADA2

• comparison of divergence

• 94% = genus , 97% = species

4th : Taxonomy assignment

comparison of sequences against database

• be careful with dominating taxas that require deep sequencing

• universal primers might not amplify all taxa evenly

5th : Microbial diversity analysis

Rarefaction = standardize unequal sampling sizes

Measuring Diversity

gamma diversity = TOTAL species diversity

alpha diversity = the mean species diversity at sites (higher = more diverse, lower = less)

beta diversity = differentiation among those habitats (higher = less diverse, lower = more)

Qualitative

presence/absence of taxa in samples

Quantitative

considers presence/absence and relative abundance

Non-phylogenetic diversity metrics

Observed features: calculates # ASV’s (qualitative)

Chao1: measures richness based on rare classes (qualitative)

• Chao1= N + S²/(2D)

• S= singletons, D=doubletons

Shannons H: Measures based on richness AND evenness (quantitative)

• 0 = 1 species, 1.5-3.5= normal range, 5= very diverse

Phylogenetic diversity metrics

Faiths phylogeneti diversity (PD): measures diversity by incorporating phylogenetic difference between species (qualitative)

• greater PD = greater branch length = more divergent

Rarefaction

standardizes sample sizes by randomly subsampling data

• multiple rarefactions give curves that show where to subsample

• Plateaus = # observed species dont change (SAMPLE HERE)

Jaccard (dis)similarity

quantifies based on presence/absence

• J(A,B) = shared taxa/total unique taxa

• Similarity of 1 = perfectly similar

• Dissimilarity of 0 = no dissimilarity

Bray Curtis dissimilarity

presence and abundance of taxa

• BC (A,B) = sum of subtracted similar samples / sum of added similar samples

• BC = 0, identical

• BC = 1, no species in common

Unifrac (Phylogenetic)

Unweighted: abundance not taken into account

Weighted: abundance taken into account

• Unifrac = 0, identical

• Unifrac = 0.5, half shared

• Unifrac = 1, no overlap

Principal coordinate analysis (PCoA)

uses disssimilarity matrices and creates plot

• dots closer = more similar

Key functions of soil microbiomes

a. climate regulation —> methylomonas take in CH4, methanosarcina release CH4

b. nutrient cycling —> Rhizobium take in N2, Nitrosomonas take in NH4 and release NO3, pseudomonas take in NO3, release N2O

c. plant growth promotion and stress tolerance —> Rhizophagus take in H2O, arthrobacter release phosphate

d. pest and disease control

e. degradation of pollutants

Soil structure drives the microbiome

clay, silt, and sand have different pores and aggregates

Root influenced soil - Rhizosphere

most organic matter

higher microbial biomass an activity

lower diversity

high rates of organic matter flow