# Presentation: 571258

## 310 Data Collection Software

- Controls 310 run conditions
- Translates light on CCD camera into electropherogram (raw data)
- Sample sheets and injection lists are created

_**Macintosh**_
- 1.0.2
- 1.2.2
- 2.1 (5-dye)

_**Windows NT**_

**Just being released**

*ABI manual is P/N **904958B *

## Injection List in Data Collection Software

- Lists samples to be analyzed (repeats can be easily performed)
- Sets virtual filter on CCD camera
- Sets electrophoresis time and voltage
- Sets injection time and voltage
- Sets run temperature
- If desired, sample analysis can be set up for automatic matrix color separation and sizing with internal standards using defined analysis parameters

## Steps Performed in Standard Module

**Capillary fill** – polymer solution is forced into the capillary by applying a force to the syringe
**Pre-electrophoresis – **the separation voltage is raised to 10,000 volts and run for 5 minutes;
**Water wash of capillary – **capillary is dipped several times in deionized water to remove buffer salts that would interfere with the injection process** **
**Sample injection** – the autosampler moves to position A1 (or the next sample in the sample set) and is moved up onto the capillary to perform the injection; a voltage is applied to the sample and a few nanoliters of sample are pulled onto the end of the capillary; the default injection is 15 kV (kilovolts) for 5 seconds
**Water wash of capillary** – capillary is dipped several times in waste water to remove any contaminating solution adhering to the outside of the capillary
**Water dip** – capillary is dipped in clean water (position 2) several times
**Electrophoresis** – autosampler moves to inlet buffer vial (position 1) and separation voltage is applied across the capillary; the injected DNA molecules begin separating through the POP-4 polymer solution
**Detection** – data collection begins; raw data is collected with no spectral deconvolution of the different dye colors; the matrix is applied during Genescan analysis

## Raw Data from the ABI Prism 310

- (prior to separation of fluorescent dye colors)

## GeneScan® Software

- Calls peaks (based on threshold values)
- Separates colors with matrix file
- Sizes peaks with internal size standard

_**Macintosh**_
- 2.1
- 3.1
- 3.1.2 (5-dye)

_**Windows NT**_
- 3.7 (5-dye)

*ABI manual is P/N **4303189*

## Screens in GeneScan® Program

- Processed data
- Sizing data
- Electrophoresis history
- Sample Information
- Raw data
- Analysis log file

**Each screen can be used to aid in evaluation of samples and trouble shooting problem samples during data analysis**

## Matrix Standards (Raw Data)

- 6FAM

- TET

- HEX

- ROX

## Save 4 x 4 Matrix Created

## Process of Sizing DNA Fragments Using an Internal Standard

- 147.32 bp

- 165.05 bp

**100**

**139**

**150**

**160**

**200**

**250**

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

**Process of Sizing DNA Fragments Using an Internal Standard**

## Sizing Algorithm

- Local Southern is commonly used but may not be the best in all situations
- Local Southern involves using 2 peak above and 2 peaks below an unknown peak from the internal size standard to make a calculated DNA size

**Internal size standard**

**STR Allele**

## Internal Sizing Standards

**GS500 ROX (Applied Biosystems)**

**LTI 50-500 ROX (Life Technologies)**

**ILS600 CXR (Promega)**

## Thoughts on Size Standards

- Be consistent in use if you want to be able to compare data over time
- All size standards I have tested work
- Allele sizes are different with different internal sizing standards
- GS500 has a large “hole” in its sizing ability when using the local Southern algorithm for medium-sized STR alleles because of the 250 bp peak that cannot be used; also must be run out to 450 bp to be able to type large FGA alleles with ABI kits

## Genotyper Software

- Converts GeneScan sized peaks into genotype calls using macros
- Genotyping performed by comparison of allele sizes in allelic ladder to sample alleles

_**Macintosh**_
- 2.0
- 2.5
- 2.5.2 (5-dye)

_**Windows NT**_
- 3.7 (5-dye)

*ABI manual is P/N **904648*

## Profiler Plus Allelic Ladders

**D3S1358**

**FGA**

**VWA**

**AMEL**

**D8S1179**

**D21S11**

**D18S51**

**D5S818**

**D13S317**

**D7S820**

## COfiler Allelic Ladders

**D3S1358**

**AMEL**

**D7S820**

**D16S539**

**TH01**

**TPOX**

**CSF1PO**

## SGM Plus Allelic Ladders

**D3S1358**

**D16S539**

**VWA**

**AMEL**

**D8S1179**

**D21S11**

**D18S51**

**D19S433**

**TH01**

**FGA**

**D2S1338**

## Identifiler Allelic Ladders

**D3S1358**

**D16S539**

**VWA**

**AMEL**

**D8S1179**

**D21S11**

**D18S51**

**D19S433**

**D5S818**

**FGA**

**D2S1338**

**TPOX**

**TH01**

**D13S317**

**CSF1PO**

**D7S820**

## PowerPlex® 16 Ladders

- PowerPlex® 16 Ladders

**D3S1358**

**D16S539**

**VWA**

**D8S1179**

**D21S11**

**D18S51**

**FGA**

**Penta D**

**TPOX**

**D13S317**

**CSF1PO**

**D7S820**

**AMEL**

**Penta E**

**TH01**

**D5S818**

## Steps in STR Genotyping Process

**GeneScan**
**software**

**Genotyper**
**software**

**Internal sizing standard **
**(e.g., GS500-ROX)**

**Matrix file**

**Allelic ladder sample**

**Data Collection**
**software**

***Expert Systems under Development (e.g., True Allele)***

## Three Possible Outcomes

**Match** – Peaks between the compared STR profiles have the same genotypes and no unexplainable differences exist between the samples.  Statistical evaluation of the significance of the match is usually reported with the match report.
**Exclusion** – The genotype comparison shows profile differences that can only be explained by the two samples originating from different sources.
**Inconclusive** – The data does not support a conclusion as to whether the profiles match.  This finding might be reported if two analysts remain in disagreement after review and discussion of the data and it is felt that insufficient information exists to support any conclusion.

## ABI 3100          16-capillary array

**ABI 3100          16-capillary array**

**ABI 310       single capillary**

**Increasing Sample Throughput with Parallel Processing**

**Subtle differences in matrix formation and sizing algorithms – NOT directly equivalent to 310**

## ABI 3100 Array Detection

## Identifiler Data from a Bad Capillary in 3100 Array

## Identifiler Data from a Good Capillary in 3100 Array

## Biological “Artifacts” of STR Markers

- Stutter Products
- Non-template nucleotide addition
- Microvariants
- Null alleles
- Mutations

## Stutter Products

- Peaks that show up primarily one repeat less than the true allele as a result of strand slippage during DNA synthesis
- Stutter is less pronounced with larger repeat unit sizes
- (dinucleotides > tri- > tetra- > penta-)
- Longer repeat regions generate more stutter
- Each successive stutter product is less intense
- (allele > repeat-1 > repeat-2)
- Stutter peaks make mixture analysis more difficult

## STR Alleles with Stutter Products

**D21S11**

**D18S51**

**D8S1179**

- DNA Size (bp)

*Stutter Product*

*Allele*

- Relative Fluorescence Units

## Schematic of Stutter Product Formation Process

- Walsh *et al* (1996) *Nucleic Acids Res.* 24: 2807-2812

**Slipped Strand Mispairing Model**

**Normal STR Allele Replication**

## Non-template Addition

- Taq polymerase will often add an extra nucleotide to the end of a PCR product; most often an “A”
- Dependent on 5’-end of the reverse primer
- Can be enhanced with extension soak at the end of the PCR cycle (e.g., 15-45 min @ 60 or 72 oC)
- Can be reduced with new polymerase
- Best if there is NOT a mixture of “+/- A” peaks

## Impact of the 5’ nucleotide on Non-Template Addition

## Null Alleles

- Allele is present in the DNA sample but fails to be amplified due to a nucleotide change in a primer binding site
- Allele dropout is a problem because a heterozygous sample appears falsely as a homozygote
- Two PCR primer sets can yield different results on samples originating from the same source
- This phenomenon impacts DNA databases
- Large concordance studies are typically performed prior to use of new STR kits

## Impact of DNA Sequence Variation in the PCR Primer Binding Site

**Impact of DNA Sequence Variation in the PCR Primer Binding Site**

**No mutation**

**Mutation at 3’-end of primer binding site ****(allele dropout)**

**Mutation in middle of primer binding site**

## Apparent Null Alleles Observed During Concordance Studies

**Apparent Null Alleles Observed During Concordance Studies**

**7/13 CODIS loci affected so far**

## Microvariants

- Defined as alleles that are not exact multiples of the basic repeat motif or sequence variants of the repeat motif or both
- May exist as insertion, deletion, or base change
- Sequence variation can occur within repeat, in the flanking region, or in a primer binding site
- Can cause PCR failure due to polymorphism in the primer site -- “null alleles”

## Detection  of a Microvariant Allele at the STR locus FGA

- 1 = S25-L25 = 244.34 - 244.46 = -0.12 bp
- 2 = SOL - L28 = 257.51-256.64  = +0.87 bp
- c = |1 -2| = |-0.12-0.87|  = 0.99 bp

## Caution with Sizing Extreme “Off-Ladder” Alleles

_**FGA ladder allele 30**_

_**Unknown FGA allele**_

_****__** **__**bp**_

_****__** **__**repeat**_

**264.79****	****	****	****330.60****	****	****65.81****	****16.45   (****46.2****)**
**264.96****	****	****	****330.63****	****	****65.67****	****16.42   (****46.2****)**
**264.66****	****	****	****330.50****	****	****65.84****	****16.46   (****46.2****)**

**267.88****	****	****	****329.44****	****	****61.76****	****15.44   (****45.2****)**
**267.13****	****	****	****329.89****	****	****61.76****	****15.44   (****45.2****)**
**267.56****	****	****	****329.23****	****	****61.67****	****15.41   (****45.2****)**

**310 Data**

**377 Data**

- Data courtesy of Melissa Fiebelkorn (Maine State Police Crime Lab)

## Three-Peak Patterns

**D21S11**

**“****Type 1”**

**“****Type 2”**

**Balanced peak heights**

**Sum of heights of two of the peaks is equal to the third**

**D18S51**

*Most common in TPOX and D21S11*

*Most common in D18S51 and .....*

## Database of Variant Alleles

**Database of Variant Alleles**

- 15 total D7S820 variants

- CSF1PO: 9
- D3S1358: 13
**D7S820: 15**
- D8S1179: 2

- FGA: **45**
- TH01: 3
- TPOX: 5
- VWA: 4

- D13S317: 6
- D16S539: 10
- D18S51: 20
- D21S11: 15

**Number of variants reported**       *(as of Sept 2001)*

- 150 New Variants

- D5S818: 3

**AND 33 unique 3-banded patterns**

**STRBase**** ****	****http://www.cstl.nist.gov/biotech/strbase**

## Mutation Observed in Family Trio

- Normal Transmission of Alleles (No Mutation)

- Paternal Mutation

## Measured Mutation Rates

- http://www.cstl.nist.gov/biotech/strbase/mutation.htm
- *Data used with permission from American Association of Blood Banks (AABB) 1999 Annual Report.

## Summary of STR Mutations

- Mutations happen and need to be considered
- Usually 1 in ~1000 meioses
- Paternal normally higher than maternal
- VWA, FGA, and D18S51 have highest levels
- TH01, TPOX, and D16S539 have lowest levels

## STRBase

- Intro to STRs (downloadable PowerPoint)
- STR Fact Sheets
- Sequence Information
- Multiplex STR Kits
- Variant Allele Reports

- _Forensic Interest Data_
- FBI CODIS Core Loci
- DAB Standards
- NIST SRM 2391
- Published PCR Primers
- Y-Chromosome STRs
- Population Data
- Validation Studies

- _Supplemental Info_
- Reference List
- Technology Review
- Addresses for Scientists
- Links to Other Web Sites

**http://www.cstl.nist.gov/biotech/strbase**

**Short Tandem Repeat DNA Internet Database**

## Degraded DNA Results

**D19**

**AMEL**

**D3**

**D8**

**VWA**

**TH01**

**D21**

**FGA**

**D16**

**D18**

**D2**

**D19**

**AMEL**

**D3**

**D8**

**VWA**

**TH01**

**D21**

**FGA**

**D16**

**D18**

**D2**

**15 years old (room temp storage)**

**6 years old (-20 ****o****C storage)**

***Results with SGM Plus STR kit (Applied Biosystems)***

## Sample Mixture ExampleProfiler Plus data

**Sample Mixture Example**Profiler Plus data