Genotyping Issues - The University of Vermontbiology/Classes/296D/15_Geno_Issues.pdf · Turns peaks...

Genotyping Issues

Chapter 15

Turns peaks into profiles

Overall goal is to determine a DNA profile:1. Extract and isolate DNA from sample2. PCR amplify DNA

• With fluorescent dye labels3. Separate PCR products4. Detect PCR products5. Analyze peaks and determine the

genotypes

Data Collection

Peak Identification

Data Review by Analyst/Examiner

Color Separation

Peak Sizing

Comparison to Allelic Ladder

Confirmation of Results by Second Analyst/Examiner

Genotype Assignment to Alleles

GeneScan or FMBIO Analysis

software

Genotyper or StaR Call

software

Internal sizing standard

(e.g., GS500-ROX)

Matrix file

Allelic ladder sample

GeneMapperIDsoftware

Expert Systems under Development

(e.g., True Allele)

Figure 15.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Data Collection Techniques:

Common Electrophoresis Machines ABI • Capillary machine• Currently most commonly used

– 96 capillaries currentlyFMBIO• Polyacrylamide gel• Then scan gel to read bands

Color Separation

• Matrixes files:– Assign the correct color spectra to the correct

fluorescent dye– Subtract overlap between dyes

• Run each dye separately– To determine the true spectra in given

conditions• Then run dyes together

– To determine amount of overlap

Blue (5FAM)

Green (JOE)

Yellow (NED)

Red (ROX)

Separate samples run for each dye colorEach sample contains multiple peaks All peaks labeled with the same dye color

ABI 310 Matrix Samples

ABI 3100 Matrix (Spectral Calibration)

Sample

Blue (5FAM)

Green (JOE)

Yellow (NED)

Red (ROX)

Single sample run containing all dye colorsOnly one peak per dye colorInjected into each capillary of the array

(A) (B)

A separate spectral calibration file is created for each capillary


Overlapping Spectra• Although each dye emits its maximum

fluorescence at a different wavelength they do overlap quite a bit

Wavelength (nm)

Fluo

resc

ence

Where laser reads emission

Peak Sizing• Internal size standards are run within each

lane/capillary• Size standard is a pre-determined ladder

– Every peak is a known sized product• Before you can determine the size of the

sample PCR products– Must go through and label every peak in the

size standard• Then computer uses size standard to

determine the size of sample peaks

3550

75 100 139 160 200 250300 340

350400 450 490

500150

DNA fragment peaks in sample

DNA Size

Data Point

147.32 bp147.32 bp

165.05 bp165.05 bp

100

139

150160

200

250

DNA fragment peaks are sized based on the sizing curve produced from the points on the internal size standard

(a)

(b)


Size Algorithms

Local Southern Method:• Take two size standard peaks above the

unknown sample• Two size standard peaks below• Therefore can only accurately call

unknown peaks that exist within the 50 bp to 490 bp range

• Any peaks outside this range cannot be called with standard size standard mix

Size Algorithms

Global Southern Method:• Fitting all the size standard peaks onto a

“best fit” line• Then using one line to determine all

unknown peaks• Seems to be more accurate method• Still need to keep within 50 to 490 bp

range

Comparison to Allelic Ladder

• First all peaks are sized– In terms of base pairs

• Then determine the actual allele of each peak

• Allele = the number of repeat units of the STR marker

• Need to be able to compare alleles between different labs with different technologies

Allelic Ladder

• Allelic ladder contains every single allele for the marker within one lane

• Determine the size of all peaks within the allelic ladder

• Then compare sample peaks to the allelic ladder – determine which alleles present

• Sample peaks need to be exactly the same as the allelic ladder to be called with confidence

D3S1358(8 alleles)

VWA(14 alleles)

D16S539(9 alleles) D2S1338

(14 alleles)

Blue panel

Green panel

Yellow panel

Orange panel

D21S11(24 alleles)

D8S1179(12 alleles)

D18S51(23 alleles)

TH01(10 alleles)

FGA low(19 alleles)

FGA high(9 alleles)

250 bp*139bp 200 bp160 bp 300 bp 340 bp 350 bp150 bp

LIZ-labeled GS500 DNA sizing standard

100 bp

Red panel

D19S433(15 alleles)

D5S818(10 alleles)

TPOX(8 alleles)

D13S317(8 alleles)

D7S820(10 alleles)

CSF1PO(10 alleles)

AMEL(2 alleles)


Off-ladder Alleles

• Accepted range of any allele– Within 0.5 bp of allelic ladder

• Any peaks that are outside this range are considered “Off-ladder”

• Two types of off ladder peaks:– Peaks that are bigger or smaller than ladder’s

complete range– Peaks that exist between the ladder’s peaks

Off-ladder Alleles

• If allele is smaller than smallest allele in ladder – genotype is written:– CSF1PO < 6

• If allele is larger than largest allele:– CSF1PO > 15

• Alleles between alleles written:– TH01 8.3 (8 repeat units plus 3 bases)– Or TH01 8.x

Off-ladder Alleles

• An expert may call a technically off-ladder allele as the closest allele– For example if the peak is only 0.7 bps off– Or if they know that certain colors always run

a certain amount high or low• Experts should compare any off ladder

alleles to databases of known microvariants– More than 220 microvariants are known for 13

core STR markers

Genotyping (Calling Alleles)

• If only one allele is present for a given STR marker = homozygous

• If two alleles are present = heterozygote• Genotype is usually reported as number of

repeats present– Number of time the repeat unit is present– Based on the size of PCR product

• All STR markers’ genotypes are put together forming a DNA profile

Genotyping Software

Currently software exists that can:• Size all peaks automatically given the size

standard’s known sizes• Then compare unknown peaks to allelic

ladders and determine the alleles automatically

• Convert the alleles into a genotype• Collect all alleles into a DNA profile

COfiler STR data

GeneScan view

Genotyperview

Allele call (repeat number)determined by comparison of peak size (bp) to allelic ladder allele peak sizes run under the same electrophoretic conditions

Peak height in relative fluorescence units (RFUs)

Data Review• All computer analyzed alleles must be

evaluated by a real life expert• Computer algorithms will call alleles based

on the information you feed into it:– The size standard’s expected sizes– The alleles present in the allelic ladder– The acceptable “bin” for each unknown peak

• Natural variations require that a human actually examine the data and verify the alleles make common sense

Allele Binning• Before computer can determine any

alleles• Give computer a base pair size range

– Known as a bin• Based on allelic ladder• Standard recommendation is 0.5 bps

below and above each peak in the ladder• However, the ladder’s alleles may show

variation – Not always at exact same position!

Allele Binning• Say the allelic ladder’s peaks vary by 0.5

bps to either side• Then you set your bin for that allele to be

another 0.5 bp to each side of that range• Therefore your bin will be 2 bps wide• Usually fine for tetranucleotide repeats

– May cause problems with microvariants

Bin

Partial DNA Profiles

• In some conditions only a partial Profile may be obtained

• Usually due to degradation of the DNA or the presence of PCR inhibitors

• Often the smaller PCR products may continue to show up when the larger products do not

• For this reason may decide to go back and re-genotype with mini-STRs

Partial DNA Profiles

• Only some of the genotypes can be obtained

• Significance of a match will go down• Because there are less markers

• Therefore the overall profile is less informative and specific

Mixture Interpretation

• While determining genotypes must also determine whether unknown sample is a mixture

Better chance of identifying that the sample is a mixture when examining:

• More markers; all highly polymorphic• Because more chance that both samples

will be heterozygous – see 3 or 4 peaks

Extra Peaks

• Sometimes extra peaks may not be caused by a mixture

• Possible reasons for extra peaks:– Stutter bands– Incomplete Adenylation of products– True rare anomalies causing extra

chromosomes or chromosomal regions– Mixtures

Extra Peaks

• Sometimes extra peaks caused by technology related artifacts

• Possible reasons for extra peaks:– Strong alleles bleeding through– Dye blobs– Air bubbles– Urea crystals– Sample contaminants that fluoresce

Dye blob

STR alleles

stutter

Pull-up (bleed-through)

spike

Blue channel

Green channel

Yellow channel

Red channel


D3S1358

Stutter products

6.0% 7.8%

Incomplete adenylation

D8S1179

-A

+A

-A

+A

Biological (PCR) artifacts

Deciphering Artifacts from the True Alleles

Contamination Possibilities

• Certain contaminants fluoresceWhen DNA is extracted along with any of

the following:• Dyes in fabrics• Chlorophyll from plants• People with certain pathological conditions

– Lead poisoning, Blood Porphyrins• People who took tetracycline antibiotics

Confirmation of Genotypes

• DNA profile is determined by:– Computer software– Expert review– Second expert

• Only when a genotype is confirmed by two experts is it accepted

• Both individuals need to be unaware of each other’s allele calls– Blind allele calls

Interpretation Strategy

Each laboratory must develop a strategy for calling alleles consistently:

• Conduct validation studies of lab equipment and personnel

• Gain experience examining peaks• Learn when to trust the computer and

when changing allele calls is appropriate• Reference literature and databases often

to keep up to date with CODIS markers

A Match?Three possible outcomes of comparing two

DNA profiles:1. Inclusion – if the DNA profiles match

– Probability of seeing this match at random is calculated

2. Exclusion – non-match– The profiles are too different to possibly be

the same individual3. Inconclusive - unknown

– There is not enough data to determine

A Match!Three possible explanations for a match:1. Suspect left DNA at crime scene

– Trial needs to determine whether that information proves suspect committed crime

2. Suspect’s profile matches by chance– This is why statistics of seeing this DNA

profile at random are calculated3. Match is a false positive result

– This is avoided at all times by validating technology, running controls and duplicates

Any Questions?

Read Chapter 16

Capillaries

Electrodes for Injection


Replace capillary

Refill syringe with polymer solution

Fill buffer vials

Prepare samples(denature, cool, and mix with size standard)

Prepare sample sheet and injection list

Automated Sample Injection, Electrophoresis and Data Collection

Automated Sample Injection, Electrophoresis and Data Collection

Genotype STR alleles

Size DNA Fragments

Perform Data Analysis

GeneScan Software

Genotyper Software

Manually inspect the data

Performed only once per batch of ~96 samples

Allelic ladder every tenth injection

ELECTROPHORESIS and DETECTION steps are simultaneous


FMBIO III Gel Imager System

PowerPlex 16 BIO

Penta E

D18S51

D21S11

TH01

D3S1358

Penta D

CSF1PO

D7S820

D13S317

D5S818

D16S539

TPOX

D8S1179

VWA

FGA

Amelogenin

SCAN GELSCAN GEL

Size DNA Fragments

Genotype STR alleles

Pour Gel

Load Samples(allelic ladder every third lane)

Electrophoresis

Pour Gel

Load Samples(allelic ladder every third lane)

Electrophoresis

STaR Call™Genotyping Software

FMBIO II Fluorescence Imaging System

Perform Data Analysis

FMBIO® Analysis Software

ReadImage Software

Manually inspect the data

Post-Electrophoresis

ELECTROPHORESIS and DETECTION steps are separate

Prepare samples: denature, cool, and mix with loading dye

Prepare samples: denature, cool, and mix with loading dye


GreenGreen

YellowYellow

BlueBlue


Genotyping Issues - The University of Vermontbiology/Classes/296D/15_Geno_Issues.pdf · Turns peaks...

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