swifs fbu dna procedures for multiplex str analysis v1.3 (03.12.2009) - re formatted

8/2/2019 SWIFS FBU DNA Procedures for Multiplex STR Analysis v1.3 (03.12.2009) - Re Formatted

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Southwestern Institute of Forensic Sciences

Criminal Investigation Laboratory

Forensic Biology Unit

Procedures for Multiplex STR Analysis,

Version 1.3

Effective Date: 3/12/2009

Approved by:

Stacy McDonald, Ph.D., Deputy Section Chief

Timothy J. Sliter, Ph.D., Section Chief

Karen Young, Quality Manager

This is an uncontrolled copy of a controlled document


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Corrections & Revisions

SOP: Procedures for Multiplex STR Analysis, Version 1.X

Date Description Authorized by

5/28/2008 Changes from Version 1.0 to Version 1.1 Revision: DNA Quantification using

Quantifiler (revisions in wording to clarify

the quality control process and

documentation)

McDonald

6/24/2008 Changes from Version 1.1 to Version 1.2

Addition: Section 14.1- ABI PrismProcedures, Appendix 1 Increased

injection times for low level samples

McDonald/Sliter

3.12.2009 Changes from Version 1.2 to Version 1.3

Deletion: Version 1.0 Approvals page(2000)

Deletion: Appendix 15 Approvals page(2003)

Deletion: SOP: DNA Quantitation byQuantiblot

Deletion: Table of Contents (hardcopyversion

Revision: SOP: Organic Extraction ofStains & Swabs. Revised to delete

specific references to the Quantiblotquantitation method.

Revision: SOP: Multiplex PCRAmplification. Revised to delete specific

references to the Quantiblot quantitationmethod; Revised to include reference to

ABI 9700 thermocycler.

Revision: Appendix 5. Critical Reagents.Revised to delete reagents and references

to the Quantiblot quantitation method.

Revision: Appendix 7. Quality ControlTests. Deleted QC test procedure for

Quantiblot test reagents deleted (test no

longer used). Revised QC test forQuantifiler Kits to include assessment ofthe Y-intercept for the standard curve in

the consideration for acceptability, and to

include communication with technicalleader in the response to failure of the QC

test.

Revision: ABI Prism 310 Procedures for

Sliter

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2

STR Analysis: Addendum 1. Increased

injection time for low level samples.

Revised of Principles and SampleRequirements sections to indicate that the

procedure is to be used only for samples

amplified using the Profiler Plus kit. Revision: SOP: DNA Quantitation by

Real-time PCR using the Quantifiler Kit.

Revision of the Evaluation of Standard

Curve section to include evaluation of theY-intercept.

Addition: SOP: Guidelines for EvaluatingArtifacts in STR Capillary Electrophoresis

Data.

Addition: SOP: Guidelines for ProcessingCODIS Candidate Matches

Revision: 30-Appendix 10. Guidelines forCalculating Statistical Weights for DNAMatches. Revised to consolidate and

organize various statistical proceduredocuments into a single set of guidelines.

Associated with this revision several

Version 1.2 documents were deleted (18-DNA Policy re Paternity Statistics; 31-

Appendix 10, Part 2; 33-Appendix 10,

Part 4; 38-Appendix 15, CalculatingLikelihood Ratios for Mixtures)

Deletion of sections related to equipmentmaintenance & calibration that arecovered in the Equipment Maintenance &Calibration manual (25-Weekly

Maintenance & Calibration Assignments

Log; 26- Weekly Maintenance &Calibration Assignments Log Sheet



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SOP: Organic Extraction of Stains & Swabs (rev. 3/12/2009)

Procedures for Multiplex STR Analysis

Organic Extraction of Stains & Swabs (rev. 3/12/2009)

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Organic Extraction of Stains & Swabs (rev. 3/12/2009)

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SOP: Differential Organic Extraction of DNA from Stains & Swabs (rev. 3/12/2009)

Procedures for Multiplex STR Analysis 1

Differential Organic Extraction of DNA from Stains & Swabs (rev. 3/12/2009)



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Differential Organic Extraction of DNA from Stains & Swabs (rev. 3/12/2009)

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SOP: Multiplex STR Amplification (rev. 3.12.2009)


Multiplex STR Amplification (rev. 3.12.2009)1



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Multiplex STR Amplification (rev. 3.12.2009)

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ABI Prism 310 Procedures for STR Analysis

Addendum 1. Increased injection time for low level samples (rev. 3.12.2009)

1. Principle

This procedure describes a modification to the standard protocol for capillaryelectrophoresis that may be used as a follow-up procedure (following analysis using the

standard method) for low quantity samples when options for extract re-amplification and

sample re-extraction have been exhausted. This procedure is for use with extracts

amplified using the Profiler Plus kit.

2. Sample Requirements

This procedure may be used under the following conditions:

1.

The sample for increased injection has been amplified using the Profiler Plusamplification kit.

2. Analysis of the sample has been completed using the standard capillaryelectrophoresis protocol, and all options for extract re-amplification and sample

re-extraction have been exhausted.

3. The standard capillary electrophoresis analysis protocol gives no geneticinformation, and the possible presence of low level genetic information in the

extract is indicated by one of the following: serology testing results, baselinefluctuations suggestive of trace level genetic markers below the RFU threshold

for allele calling in the data from the standard protocol;

OR

The standard capillary electrophoresis analysis protocol gives limited geneticinformation, with no peaks exceeding 3000 RFUs.

3. Procedure

1. Following completion of the standard electrophoresis protocol, a follow-upelectrophoresis is performed using an increased injection time.

a. 15 second injection A 15 second injection time may be used when thereare no peaks in the electropherogram exceeding 900 RFUs.

b. 10 second injection A 10 second injection time may be used when thereare no peaks in the electropherogram exceeding 3000 RFUs.

2. The increased injection time batch will include the following samples:a. Test extractsb. Corresponding reagent blanks (negative control)c. Corresponding amplification blanks (negative control)





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d. 9947A amplification positive control OR the positive extraction control(injected for 5 seconds to avoid the possibility of off scale data).

3. Each of the test extracts and negative controls will be injected a minimum of twotimes to establish the reproducibility of any detected alleles.

4. Interpretation Guidelines

1. The data from the increased injection times will be interpreted using the standardinterpretation guidelines (see SOP), with the following exceptions:

a. All calls of homozygosity will be based upon the results of the standard 5second injection times.

Example 1: At a locus where a single allele is detected at 105 RFUs usingthe standard 5 second injection time, the locus may be called homozygous

for the allele following the 10 second injection, because the standard

results gave a peak height greater than the 85 RFU threshold for callinghomozygosity in 5 second injections.

Example 2: At a locus where a single allele is detected at 80 RFUs usingthe standard 5 second injection time, the locus may not be called

homozygous for the allele following a 10 second injection, because the

standard results gave a peak height less than the 85 RFU threshold for

calling homozygosity in 5 second injections.



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Appendix 4. Solutions, Kits and Materials: Preparation and Storage, Version 1.1

The following general instructions apply to the preparation of all solutions:

1. Use graduated cylinder and pipettes close in capacity to the volume being measured.

2. Label solutions with the name of the solution, date prepared, and initials of preparer.If applicable, label solutions with the control/lot number and expiration date.

3. Prepare all solutions with deionized water, and chemicals that are of reagent grade orbetter quality, unless otherwise noted.

4. Solutions may be prepared to final volumes different from those specified here. The

amounts of the components should be adjusted accordingly, so as to obtain the correctfinal concentrations.

310 Buffer, 1X

For 250 mL:

10X 310 Buffer 25 mLWater 225 mL

Add QC'd 10X 310 Buffer to water. Filter through a 0.45 M filter. Store at 2-8C.

310 Buffer, 10X

Purchase 310 Genetic Analyzer Buffer with EDTA from Perkin Elmer (p/n 402824).

Store at 2-8C. QC test lots of 10X 310 Buffer before use: ABI 310 Reagents QC Test.

Biodyne B Membrane

Purchase from GibcoBRL (p/n 24800-013). Store at room temperature. QC test lots

of membrane before use: QuantiBlot Reagents QC Test.

Buffer ATL

Prior to use, check whether a precipitate has formed. Dissolve precipitate byheating the buffer to 70

oC with gentle agitation. Store at room temperature. QC test

before use: QiaAmp DNA Micro Kit Reagents QC Test.

Procedures for Multiplex STR Analysis - 1 -

Appendix 4. Supplies, Kits and Reagents: Preparation and Storage, Version 1.1




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Buffer AL

Prior to use, check whether a precipitate has formed. Dissolve precipitate by

heating the buffer to 70oC with gentle agitation. Store at room temperature. QC test

before use: Extraction Reagents - QiaAmp DNA Micro Kit QC Test.

Buffer AWI

Buffer AWI concentrate (Qiagen DNA Micro Kit) 19 mL

Ethanol, 100% 25 mL

Add ethanol to bottle containing concentrated Buffer AWI. Store at room temperature.

QC test before use: Extraction Reagents - QiaAmp DNA Micro Kit QC Test.

Reconstituted Buffer AWI is stable for up to one year.

Buffer AW2

Buffer AW2 concentrate (Qiagen DNA Micro Kit) 13 mL

Ethanol, 100% 30 mL

Add ethanol to bottle containing concentrated Buffer AW2. Store at room temperature.

QC test before use: Extraction Reagents - QiaAmp DNA Micro Kit QC Test.

Reconstituted Buffer AW2 is stable for up to one year.

Chromogen:TMB Solution [2% 3,3',5,5'-tetramethyl benzidine in ethanol]Chromogen:TMB (Perkin Elmer p/n N808-0092) 60 mg

Ethanol, 100% 30 mL

Allow Chromogen:TMB to warm to room temperature. Carefully remove rubber stopper

and add ethanol. Return stopper, and seal with parafilm. Shake on an orbital shaker for 30

min to dissolve. Store at 2-8 C. QC test before use: QuantiBlot Reagents QC Test.Solution is stable for at least 4 months.

Citrate Buffer [0.1 M Sodium Citrate, pH 5.0]

For 1 Liter:Trisodium citrate, dihydrate 18.4 g

Citric acid monohydrate q.s. pH 5.0

Water q.s. 1 L


Dissolve trisodium citrate in 800 mL water. Adjust to pH 5.0 t 0.2 by adding approximately

6 g of citric acid monohydrate. Adjust the final volume to 1 L with deionized water. Store at





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room temperature (glass or polypropylene container). QC test before use: QuantiBlot

Reagents QC Test.

Cofiler PCR Amplification Kit

Purchase from Perkin Elmer (p/n 4305246). Store at 2-8C. QC test lots of kit

before use: Amplification Reagents QC Test.

DNA Quantitation Standards (Quantiblot kit)

Prepare (using TE Buffer) a 2-fold serial dilution of the DNA Standard A supplied inthe QuantiBlot Human DNA Quantitation Kit. Final concentrations of DNA in the

standards are as follows:

Standard A 2 ng/L

Standard B 1 ng/L

Standard C 0.5 ng/L

Standard D 0.25 ng/LStandard E 0.125 ng/L

Standard F 0.0625 ng/LStandard G 0.03125 ng/L

Store at 2-8C. Diluted standards are stable for at least 3 months. QC lots of diluted

standard prior to use: QuantiBlot Reagents QC Test.

DNA Quantitation Standards (Quantifiler kit)

Prepare (using TE-4

Buffer) a serial dilution of the DNA Standard supplied in theQuantiBlot Human DNA Quantitation Kit. Final concentrations of DNA in the

standards are as follows:

Standard Concentration

(ng/L)

AmountsDilutionFactor

Standard 1 50.0 50 L 200ng/L stock + 150 L TE-4 4X

Standard 2 16.7 50 L Std 1 + 100 L TE-4 3X






Standard 8 0.023 50 L Std 7 + 100 L TE

-4

3X

Store at 2-8C. Diluted standards are stable for at least 3 months. QC lots of diluted

standard prior to use: Quantifiler Reagents QC Test.






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DTT (Dithiothreitol), 4 M

For 10 mL:

DTT 6.18 g

Water q.s. 10 mL

Dissolve DTT in sterile water. Aliquot to sterile 0.5 ml, tubes. Store at -20C. QC test before

use: Extraction Reagents QC Test.

EDTA (Ethylenediamine Tetraacetic Acid), 0.5 M

For 1 L:

Disodium EDTA dihydrate 186.1 g

NaOH q.s. pH 8.0 0.2Water q.s. 1 L

Add EDTA crystals to 800 mL water. While agitating on a magnet stirrer, addapproximately 20 g NaOH. Adjust pH to 8.0 0.2 with 5 N NaOH. Adjust final volumeto 1 L with water. Autoclave or filter sterilize. Store at room temperature. (Alternatively,

0.5 M EDTA, pH 8.0 solution may be purchased from a commercial vendor)

Extraction Buffer (EB) [10 mM Tris-Cl (pH 8.0), 100 mM NaCI, 50 mM EDTA, 2% SDS)

For 1 L:

1 M Tris-Cl, pH 8.0 10 ml,

5 M NaCI 20 mL0:5 M EDTA 100 mL

20% SDS 100 mL

Water q.s. 1 L

Add 1 M Tris-Cl, 5 M NaCI, 0.5 M EDTA and 20% SDS to sterile water. Transfer 10 mL

aliquots to sterile 15 mL polypropylene tubes. Irradiate tubes in a UV cross-linker

(30 J/cm2). Store at room temperature. QC test each lot before use: Extraction Reagents QC

Test.

Formamide, deionized

Purchase HiDi Formamide from Perkin Elmer (p/n 4311320). Store at 2-8C. QC test

lots of formamide before use: ABI 310 Reagents QC Test.






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Hybridization Solution [SX SSPE, 0.5% SDS]

For 1 L:

20X SSPE 250 mL

20% SDS 25 mL

Water 725 mL

Add 20X SSPE and 20% SDS to water. Mix thoroughly. Store at room temperature. Prior touse, warm if necessary to dissolve solids. QC test each lot before use: QuantiBlot Reagent

QC Test.

Hydrogen Peroxide, 30%

Purchase from commercial source (VWR p/n VW3742-1 or equivalent). Store at

2-8. Protect from light. QC test each lot before use: QuantiBlot Reagent QC

Test.

Hydrogen Peroxide, 3%

For 100 mL:

30% Hydrogen peroxide 10 mL

Water 90 mL

Add 30% Hydrogen Peroxide to water. Mix fresh daily from 30% Hydrogen Peroxide stock.

Microcon YM 100 Microconcentrators

Purchase from Amicon (p/n 42413). Assemble cups and collection tubes, and UV-irradiate with 6 J/cm

2in a UV cross-linker prior to use.

NaCl, 5 M

For 1 L:

NaCl 292 g

Water q.s. 1 L

Dissolve NaCl in water. Autoclave solution. Store at room temperature.

NaOH, 5 N

For 1 L:

NaOH 200 gWater q.s. 1 L

Dissolve NaOH in 800 mL water. Adjust volume to 1 L. Store at room temperature.






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Nuclear Fast Red Stain [0.05% Nuclear fast red, 2.5% Aluminum sulfate]

For 600 mL:

Aluminum sulfate 15 g

Nuclear fast red 0.3 gWater 600 mL

Heat water and add aluminum sulfate. Immediately add nuclear fast red stain and stir

with a glass rod. Allow solution to cool and filter through Whatman paper. Stable for

3-6 months. Store at room temperature.

PCIA Solution [25:24:1 Phenol: Chloroform:Isoamyl alcohol]

Purchase buffered PCIA Solution (pH 8.0) from a commercial supplier (Amresco p/n

0883100ML or equivalent). Store at 2-8C.

Phase Lock Gel Tubes, Heavy

Purchase from Eppendorf (p/n 0032 005.152). UV-irradiate with 6 J/cm2

in a UV cross-linker prior to use.

Picric Acid Solution, 1 %

Purchase from commercial supplier (Aldrich p/n 31,928-7 or equivalent). Store atroom temperature.

Picroindigocarmine Stain [0.33% Indigo carmine, 1% Picric acid]

For 600 mL:

Indigo carmine 2 g1 % Picric acid solution 600 mL

Dissolve ndigo carmine in 1 % picric acid solution. Store at room temperature in a brown

bottle. Solution is stable for approximately 4 months.

POP-4

Purchase Performance Optimized Polymer 4 (POP-4) from Perkin Elmer (p/n 402838). Store

at 2-8C. QC test lots of POP-4 before use: ABI 310 Reagents QC Test.

Pre-wetting Solution [0.4 N NaOH, 25 mM EDTA]

For 500 mL:

5 N NaOH 40 mL

0.5 M EDTA 25 mLWater 435 mL






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Add 5 N NaOH and 0.5 M EDTA to water. Mix thoroughly. Store at room temperature. QCtest before use: QuantiBlot Reagents QC Test.

Profiler Plus PCR Amplification Kit

Purchase from Perkin Elmer (p/n 4303326). Store at 2-8C. QC test lots of kit beforeuse: Amplification Reagents QC Test.

Proteinase K

Purchase from a commercial source separately (20 mg/mL) or as part of a kit

(Ameresco p/n E195-5mL, QIAamp DNA Micro Kit p/n 51306 or equivalent). Store

according to manufacturer's directions. QC test each lot before use: ExtractionReagents QC test or Extraction Reagents - QiaAmp DNA Micro Kit QC Test.

ROX-500

Purchase Genescan-500 (ROX) Size Standard from Perkin Elmer (p/n 401734). Store

at 28C. QC test lots of ROX-500 before use: ABI 310 Reagents QC Test.

Sample Extraction Buffer (SEB)

For 1 mL:

EB 1000 L

20 mg/mL Proteinase K 10 L4 M DTT 10 L

Add 20 mg/mL Proteinase K and 4 M DTT to EB. Mix thoroughly. Prepare fresh. Discardany unused solution. Do not store.

SDS (Sodium Dodecyl Sulfate), 20%

Purchase RNase-, DNase-, and Protease-Free solution from a commercial supplier (Fisher

p/n BP1311-200 or equivalent). Store at room temperature.

Spotting Solution [0.4 N NaOH, 25 mM EDTA, 0.00008% Bromothymol Blue]

For 75 mL:

5 N NaOH 6 mL0.5 M EDTA 3.75 mL0.04% Bromothymol Blue 150 L

Water 65 mL






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Add 5 N NaOH, 0.5 M EDTA and Bromothymol Blue solution (provided in QuantiBlot Kit)

to water. Store at room temperature in a glass bottle. Solution is stable for at least 3 monthsfollowing preparation. QC test before use: QuantiBlot Reagents QC Test.

SSPE Buffer, 20X [3 M NaCI, 200 mM NaHzP04, 20 mM EDTA, pH 7.4]

Purchase from commercial source (GibcoBRL pln 15591-027 or equivalent). Store atroom temperature.

SSPE Buffer, 5X [0.75 M NaCI, 50 mM NaH2P04, 5 mM EDTA, pH 7.4]

For 1 Liter:

20X SSPE 250 mL

Water 750 mL

Prepare fresh as needed. Store at room temperature.

TE Buffer [10 mM Tris-Cl (pH 8.0), 1 mM EDTA]

For 10 mL:

1 M Tris-Cl, pH 8.0 100 L

0.5 M EDTA 20 L

Water 9.88 mL

Add 1 M Tris-Cl, pH 8.0 and 0.5 M EDTA to sterile water. Autoclave. Store at

room temperature. Alternatively, may be purchased as a prepared sterile solutionfrom a commercial supplier (Amresco p/n E112-100ML or equivalent).

TE-4Buffer [10 mM Tris-Cl (pH 8.0), 0.1 mM EDTA]

For 100 mL:1 M Tris-Cl, pH 8.0 1 mL

0.5 mM EDTA 20 pL

Water 98.98 mL

Add 1 M Tris-Cl, pH 8.0 and 0.5 M EDTA to sterile water. Transfer 10 ml, aliquots to

sterile 15 ml, polypropylene tubes. Irradiate tubes in a UV cross-linker (30 J/cm). Store atroom temperature. QC test lots before use: Amplification Reagents QC Test .

Tris-Cl [Tris(hydroxymethyl)amminomethane], 1 M

For 1 L:Tris base 121 gWater q.s. 1 L

Dissolve Tris base in 800 mL water: Adjust to desired pH by adding concentrated HCI.Adjust final volume to 1 L. Autoclave. Store at room temperature.






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Wash Solution [1.SX SSPE, 0.5% SDS]

For 2 L:

20X SSPE 150 mL

20% SDS 50 mL

Water 1,800 mL

Add 20X SSPE and 20% SDS to water. Mix thoroughly. Store at room temperature. Priorto use, warm if necessary to dissolve solids. QC test before use: QuantiBlot Reagent QC

Test.

Water, Deionized

House Milli Q water, or deionized water from a commercial supplier. Prior to use in PCR

amplification set-up, transfer 10 mL aliquots of water to sterile 15 mL polypropylene

tubes, and irradiate in a UV cross-linker (30 J/cm2). Store at room temperature. QC test

lots of irradiated water before use: Amplification Reagents QC Test.






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Appendix 5. Critical Reagents, Kits and Materials (rev. 3.12.2009)

- 1 -

Appendix 5. Critical Reagents, Kits and Materials (rev. 3.12.2009)

Consult Appendix 4. Solutions, Kits and Materials: Preparation and Storage for information

regarding required QC tests prior to use in casework.

A. Listing of critical reagents, kits, and materials

1. Buffer AL2. Buffer ATL3. Buffer AW14. Buffer AW25. Cofiler DNA Amplification Kit6. DNA Quantitation Standards (for Quantifiler Kits)7. 4 M DTT8. EB9. PCIA10.Profiler Plus DNA Amplification Kit11.Proteinase K12.Quantifiler Human DNA Quantitation Kit13.TE-4 Buffer14.WaterB. Additional Notes

1. QIAamp DNA Micro Kit and Quantifiler Human DNA Quantitation Kit

Typically, multiple kits of the same lot will be ordered. It will be sufficient for a

single kit from a lot to pass the QC test in order for the lot as a whole to pass QC.Each lot of kits is marked by the manufacturer with an expiration date. In the event

that unused kits and/or unused portions of kits remain at the time of the expirationdate, then the unused kits and/or unused portions of kits of the same lot may be QC

checked at or about the time of the expiration date. If the lot passes the QC test then a

new expiration date will be assigned to the lot. This new expiration date will be two

months following the successful QC test. This two month recertification of the kitsmay be repeated as necessary provided that the kits continue to pass the QC test.



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Appendix 7. Quality Control Tests (rev. 3.12.2009)

1. Organic Extraction Reagents QC Test2. QIAamp DNA Micro Kit QC Test3. Amplification Reagents QC Test4.

ABI 310 Reagents QC Test5. Quantifiler Reagents QC Test


Appendix 7. Quality Control Tests (rev. 3.12.2009)1



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Quality Control Test Procedure

Quantifiler Reagents QC Test

Quantifiler Kit Components:

1. Quantifiler Human DNA Standards 1-82. PCR Reaction Mix

3. Human Primer Mix

Procedure

1. Perform the SOP: DNA Quantitation by Real-time PCR using the QuantifilerKit for the following samples:

a. Standard curve (2 replicates)b. Blank (PCR Reaction Mix and Human Primer Mix only)

Specifications

Lots of kits are considered acceptable for casework if:

1. All DNA standards are detected.2. The R2 value 0.99.3. The slope falls between -2.9 to -3.3.4. The Y-intercept (CT value for 1 ng) falls between 28.0 and 30.0.5. There is amplification of the Internal Positive Control (IPC).6. No DNA is detected in the Blank.Note: If newly QCed kits fail QC, then this will be brought to the attention of the

technical leader for review and determination of the proper course of action

(troubleshooting, etc.).



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Guidelines for STR Interpretation and Statistical Weight Calculation for DNA Matches

(3.12.2009)

Principle

This document provides guidelines to be used by DNA analysts in calculating the statistical

weights of DNA matches. A statistical weight provides an estimate of the mathematicalsignificance of a DNA match.

General Considerations

A. Statistical weights will be calculated when a known individual is included as a possiblesource of, or as a possible contributor to, a DNA profile obtained from a questioned evidence

sample of probative value.

1. When a known individual is an expected contributor of biological material to an evidencesample for reasons unrelated to the incident under investigation, then no statistical weight

will be calculated or reported. E.g., if Suspect A is included as a source of a DNA profile

obtained from the door handle of his own car, then no statistical weight would becalculated because his profile is expected to be seen in this sample for reasons unrelated

to any criminal incident. Similarly, no statistical weight would be calculated to describe

the match between a victim and the DNA profile from the epithelial fraction of anintimate swab that matches her.

B. Statistical calculations are performed following completion of STR data collection, allelecalling, and interpretations of inclusion/exclusion of known individuals.

C. For the purposes of statistical calculations, the stochastic dropout threshold for Profiler Plusand Cofiler profiles is 85 RFU for 5 second injections on the ABI 310 Genetic Analyzer.

D. Calculations may be performed by hand, or with the assistance of computer software.Computer-based calculation methods include:

1. The current Popstats software distributed by the FBI as part of the CODIS softwarepackage. This package is used for the following calculations:

a. Single source random match probability - no dropout, with theta-correction.b. Mixture random match probability - no dropout, with theta-correction.c. Mixture likelihood ratio.d. Standard parentage involving a child, a known parent and an alleged parent

(Parentage index; Probability of parentage; Parentage probability of exclusion).

2. In-house methods using Microsoft Excel spreadsheets are used for the followingcalculations.

a. Single source random match probability with dropout, with theta-correction.b. Mixture random match probability with dropout, with theta-correction.c. Parentage cases involving a single alleged parent-child relationship.d. Missing person cases involving biological relatives of a missing person and alleged

remains of that person.


Guidelines for Calculating Statistical Weights for DNA Matches (3.12.2009)1



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E. The databases to be used for calculations of statistical weight will be appropriate to specificcase circumstances.

1. For routine casework involving incidents in Texas, the following databases will be used:a. Texas DPS - for Profiler Plus profiles when Cofiler testing was not performed (for

Texas Caucasians, Hispanics, & African Americans).

b. FBI - for combined Profiler Plus and Cofiler profiles (for Caucausians, SouthwestHispanics, & African Americans)

2. For routine casework involving incidents outside of Texas, the following databases willbe used:

a. FBI (Profiler Plus and Cofiler profiles; for Caucausians, Southwest Hispanics, &African Americans)

3. For casework that involves population groups other than the standard Caucasian,Southwest Hispanic and African-American population groups, in addition to the standarddatabases, databases for other population groups may be of value. This will be

determined on a case-by-case basis. In these circumstances the following databases willbe used;

a. FBI databases provided with Popstats (Southeast Hispanics, American Indians,Asians, Carribean)

b. Centre for Forensic Sciences (CFS) databases (Asians & East Indians)4. These guidelines are intended to cover the most commonly encountered categories of

DNA profiles. However, profiles may be encountered in casework that do not fall easilyinto the categories described in this document.

a. In the event that a profile does not correspond to one of the categories described inthis document, it will be referred to the Technical Leader for review and assessmentof the proper course of action to take.

5. In the following guidelines, it will be presumed that the questioned evidence profilematches a known individual whose profile is completely known at each locus tested.

a. Under circumstances where the DNA profile of the known individual originates fromdecomposed remains, the profile may be incomplete. In these cases, the statisticalcalculations will be limited to the loci at which the profile of the known individual is

known.

6. For the sake of brevity in the guidelines, known individuals who are included as possiblesources of or contributors to a sample may be referred to as Suspect 1, Suspect 2, etc.

However, the descriptions are equally applicable to other known individuals (e.g.,victims, consensuals, etc.).

7. Equations utilized in locus random match probabilities (Eq (1), (2), and (3)) and mixturelikelihood calculations are given in Section 2.

8. On occasion, a profile that shows the characteristics of a single source profile may alsoshow additional genetic markers at a low level. Under some circumstances, there may be

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a mixture of DNA from more than one person. However, under some circumstances the

low level genetic information may be insufficient to establish that the sample is amixture, as opposed to possible other explanations (such as somatic mutation when the

low level marker is in the +4 or -4 stutter positions). Under these circumstances, the low

level genetic markers will be reported as low or trace level alleles in the profile.

Section 1. Guidelines

Part A. Guidelines for Single Source Profiles

A. Simple single source profile (SSP). A simple SSP is a profile with one or two alleles at eachlocus. At each locus with one allele, the peak height of the observed allele is above the

stochastic dropout threshold (85 RFU), so dropout of alleles is not at issue for any locus.Therefore each locus can be categorized as homozygous (one observed allele) or

heterozygous (two observed alleles).

Locus probabililties:

Homozygous loci: Eq (1) Section 2

Heterozygous loci: Eq (2) Section 2

Combined multilocus random match probability (F):

F = f, for all loci

1. Variation 1. Low level/degraded SSP. This profile shows the characteristics of a simpleSSP except that one or more loci shows a single allele with a peak height less than the

stochastic dropout threshold (85 RFU). Additionally, there may be loci at which no

alleles are detected.


Homozygous loci (>85 RFU): Eq (1) Section 2


Single allele loci (


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Heterozygous loci: Eq (2)

Triallelic locus: Eq (2), using the two rarest alleles of the threeobserved


F = f, for all loci

3. Variation 3. SSP with a triallelic locus consistent with somatic mutation. This profileshows the characteristics of a simple SSP except that at one a locus three alleles are

detected. Two of the alleles have peak heights characteristic of a heterozygous locuscompared to other loci in the profile. The third allele is a low level peak one repeat,

either smaller or larger than one of the large peaks (a position that would be expected of

most somatic mutations). Unlike aneuploidy, a somatic mutation observed in aquestioned evidence sample would not necessarily be observed in the standard of a

matching known individual, which may not originate from the same tissue type.




Triallelic locus: Eq (2) Section 2, using the two major alleles of thethree observed


F = f, for all loci

4. Variation 4. SSP with an apparent somatic mutation at a homozygous locus. This profileshows the characteristics of a simple SSP except that at one locus there is a single allelewith a peak height consistent with homozygote when compared to other loci in the

profile, and a low level peak one repeat smaller or larger than the large peak. The small

peak is therefore explainable as a somatic mutation of the main allele. Unlikeaneuploidy, a somatic mutation observed in a questioned evidence sample would not

necessarily be observed in the standard of a matching known individual, which may not

originate from the same tissue type. Locus probabililties:



Triallelic locus: Eq (1) Section 2, using the major allele


F = f, for all loci

5. Variation 5. SSP with an apparent primer site mutation at one locus. This profile showsthe characteristics of a simple SSP, except that at one locus there is a single allele whosepeak height when compared to other loci is not consistent with homozygosity but is



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consistent with heterozygosity. This locus is therefore explainable as a primer site

mutation that prevents amplification of one of the locus on one sister chromosome. Therecognition of a locus affected by a primer site mutation is complicated by the effects of

degradation in questioned evidence samples. A locus affected by a primer site mutation

in a questioned evidence sample would be expected to be similarly affected in the

standard of the individual who was the source of the sample. If possible, putative primersite mutations should be confirmed by amplification with a kit that uses a different primer

site. However, this is often not feasible.




Apparent primer site mutation locus:f= 1


F = f, for all loci

Part B. Guidelines for Simple Mixtures

A. Simple mixture profile (SMP). This profile shows three or more alleles at multiple loci.The alleles are not distinguishable into major and minor contributions. The alleles detectedare 85 RFU so stochastic dropout is not at issue at any locus. Based upon the maximum

number of alleles at any one locus, it is possible to determine the minimum number of

contributors to the mixture (N). For instance, if the maximum number of alleles at any locusis 5 or 6, then N = 3.

When all of the alleles observed in Suspect A are observed in the mixture, Suspect Ais included as a possible contributor to the mixture. A Mixture Likelihood Ratio(Section 2) will be calculated with Popstats that evaluates the following competing

and mutually exclusive hypotheses:

o Hypothesis 1. Suspect 1 and (N-1) unknown individuals are contributors to themixture.

o Hypothesis 2. N unknown individuals are contributors to the mixture When more than one suspect is included as a possible contributor to the mixture, then

an appropriate Mixture Likelihood Ratio (Section 2) will be calculated using Popstats.

For instance, if all of the alleles observed in the profiles of Suspect A and Suspect Bare observed in the mixture and N=2, then one possible set of competing hypotheses

would be:

o Hypothesis 1. Suspect A and Suspect B are contributors to the mixtureo Hypothesis 2. Two unknown individuals are contributors to the mixture.

1. Variation 1. 2-contributor SMP with a known contributor (subtraction). This profile is a2-contributor SMP. However, because of the nature of the evidence sample, one of the

contributors is a known individual. (E.g., a 2-contributor mixture seen in the sperm





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fraction of a vaginal swab when all of the genetic markers of the victim are observed so a

contribution by the victim is indicated.) With this type of profile, the contribution of theknown contributor will be taken into account in the interpretation so as to deduce

additional information regarding the unknown contributor. For instance, if a victim is

AA at a locus and the mixture is ABC then the non-victim contributor is deduced to be

BC. In this analysis, relative peak height information may be important. For instance, ifthe victim is AB and the mixture is ABC with A and B of equal peak heights and C is

approximately twice the peak height of A or B, then the non-victim contributor can bededuced to be CC. Alternatively, if the victim is AB and the mixture is ABC with A and

C of equal peak heights and B is approximately twice the height of A or C, then the non-

victim contributor would be deduced to be BC. Depending upon the profile, the result of

subtraction may be a single source profile at each locus in the profile. However,depending upon the profile, subtraction may not be possible at every locus. For instance,

if the victim is AA and the mixture is AB, then depending upon the mixture it might not

be possible to distinguish if the non-victim contributor is AB or BB.

If the unknown contributors profile is fully deduced as a result of the subtractionprocess, and if Suspect A fully matches the non-victim contribution, then a randommatch probability will be calculated as described above for a simple Single Source

Profile.





F = f, for all loci

If the unknown contributors profile is not fully deduced as a result of the subtractionprocess (so at some indeterminate loci there are multiple not-excluded genotypes),

and if Suspect A fully matches the non-victim contribution (and is included in the set

of not-excluded genotypes at the indeterminate loci), then a random match probabilitywill be calculated:




Indeterminate loci: Sum of the probabilities associated with the not-

excluded genotypes, using Eq (1) and Eq (2)


F = f, for all loci

2. Variation 2. Low level SMP. This profile shows the characteristics of the SMP, exceptthat at some loci alleles are observed that are less than the stochastic dropout threshold

(


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loci with the smallest amplicons. In the event that slight degradation indicated (e.g., by a

general decrease in peak height with increasing amplicon size) then the profile will betreated as a SMP (see following section).

If Suspect A is included as a possible contributor to the mixture because all of hisalleles are detected in the mixture, then a Mixture Random Match Probability will be

calculated using Popstats:


f= (pA + pB + pC + )2, with correction


F = f, for all loci

If Suspect A is included as a possible contributor but at some loci alleles seen inSuspect A are not observed and the absence of these alleles can be reasonably

accounted for by stochastic dropout model, then a Mixture Random MatchProbability will be calculated:


Loci at which Suspect As alleles are all observed:


Loci at which some or all of Suspect As alleles are not observed:

f= 1


F = f, for all loci

3. Variation 3. Degraded SMP. This profile shows the characteristics of the SMP exceptthat it is degraded. Peak heights at larger amplicons are reduced due to degradation, and

the loci with larger amplicons include peaks


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not exclusionary), then the mixture will be carefully evaluated to determine which

loci to include in the statistical weight calculation. The smallest locus at which adegradation/dropout model must be invoked for Suspect A will define the cutoff for

loci that will be used in the statistical weight calculation. Loci with amplicons

smaller than the cuttoff locus will be included in the calculation. The cutoff locus and

loci with amplicons larger than the cutoff locus will not be used in the calculation(since degradation/dropout would be expected to affect these loci to a greater degree

than the cutoff locus). For loci that are included in the calculation, a Mixure RandomMatch Probability will be calculated using Popstats:




F = f, for all loci in the calculation

Part C. Guidelines for Major-Minor Mixtures

A. Simple major-minor mixture profile (MMMP). This profile is a mixture profile with clearlydistinguishable sets of major alleles and minor alleles (at lower peak heights). The set ofmajor alleles consists of one or two alleles at each locus indicating a single major contributor.

The set of minor alleles consists of one or two minor alleles at each locus consistent with a

single minor contributor; all of the observed alleles are 85 RFU so dropout of major orminor alleles is not at issue at any locus.

If the Suspect matches the major contributor, then a random match probability will becalculated as described above for a simple Single Source Profile.





F = f, for all loci

If the Suspect matches the minor contribution (all minor alleles in the mixturecorrespond to alleles observed in the Suspect; all other alleles observed in the suspectcorrespond to alleles that would be masked by the major contributor), then at each

locus the set of genotypes that would not be excluded as the minor contributor will be

defined. For instance, if the major contributor is AB and there is a minor C, then theset of not-excluded minor genotypes would be: AB, AC, CC. Alternatively, if the

major contributor is AB and there are minor CD alleles, then the set of not excludedminor genotypes would be CD only. At each locus a random match probability willbe calculated for the minor contribution based upon the set of not-excluded

genotypes:


All loci: Sum of the probabilities associated with the set of not-excluded

genotypes, using Eq (1) and Eq (2) Section 2



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F = f, for all loci

1. Variation 1. MMMP with one major contributor and more than one minor contributor.This profile shows the characteristics of a simple MMMP except that there are more than

two minor alleles at some loci, indicating that there is a mixture of individuals in the

minor contribution. However, all minor alleles are 85 RFU so dropout of minor allelesis not at issue.

If the Suspect matches the major contributor, then a random match probability will becalculated as described above for a simple Single Source Profile.





F = f, for all loci

If the Suspect is included as a possible minor contributor (all alleles observed in theSuspect are observed as either minor alleles or in positions that would be masked bythe major contributor), then a Mixture Random Match Probability will be calculated

using Popstats for the combined set of major and minor alleles:




F = f, for all loci

2. Variation 2. MMMP with more than one major contributor. This profile shows thecharacteristics of a simple MMMP, except that the major contribution is a mixture of

DNA from two (or possibly more) people.

The statistics for possible contributors to the major contribution (a mixture) will behandle in the same way as a SMP.

If a Suspect is included as a possible minor contributor (all alleles observed in theSuspect are observed as either minor alleles or in positions that would be masked by

the major contributor), then a Mixture Random Match Probability will be calculatedusing Popstats for the combined set of major and minor alleles:




F = f, for all loci

3. Variation 3. MMMP with one or several loci where the major & minor contributions cannot be distinguished. This profile shows the characteristics of a simple MMMP.





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However, at one or more loci the observed alleles cannot be clearly assigned to the major

or minor contributions. This may be seen in situations where there is differentialdegradation of either the major or minor contribution. For instance, in the case a small

blood stain on clothing where the major contribution is from the blood and the minor

contribution is from epithelial cells on the clothing, the DNA from the epithelial cells

may be highly degraded at larger amplicons and less degraded at smaller amplicons.Under these circumstances, the major and minor contributions may be distinguishable at

all loci except the smallest loci, which would be indeterminate/inconclusive in regard tothe major and minor contributions except inasmuch as defining a combined set of alleles

for the contributors.

If Suspect 1 is included as a possible source of the major contribution, then a randommatch probability will be calculated based upon the set of not-excluded genotypes for

the major contributor at each locus:




Indeterminate loci: Sum of the probabilities associated with the set of

not-excluded genotypes, using Eq (1) and Eq (2)

Section 2


F = f, for all loci

If Suspect 1 is included as a possible source of the minor contribution, then a randommatch probability will be calculated based upon the set of not-excluded genotypes for

the minor contributor at each locus:


For loci where the major and minor contributions are distinguishable:

f= Sum of the probabilities associated with the set of not-excludedgenotypes, using Eq (1) and Eq (2) Section 2.

For loci where the major and minor contributions are not distinguishable:

f= Sum of the probabilities associated with the set of not-excluded

genotypes, using Eq (1) and Eq (2) Section 2


F = f, for all loci

4. Variation 4. MMMP with minor alleles at some loci in the stochastic dropout range. Thisprofile shows the characteristics of the simple MMMP, except with minor alleles


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excluded genotypes for the minor contributor (who contributed C) would be {CC and

CX}, where X is any allele other than C (AC and BC are included in CX). If a suspecthas a C allele, then he would be included as a possible contributor of the C allele in the

profile. The locus random match probability associated with the inclusion would be the

sum of the probabilities associated with the CC and CX genotypes, where p X = (1-pC).

Alternatively, if major alleles AB and minor alleles CD are observed with C and D both


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1. The following policies will be followed in DNA testing for the purpose of standardpaternity testing:

a. Testing for the purpose of determining parentage in a standard paternity case willrequire the following samples for analysis:

i. Known maternal standard (MO)ii. Known child/fetal standard (CH)iii. Known standard of alleged father (AF)

b. Initial testing will be performed using the Profiler Plus kit. The result for this testingwill be considered sufficient under the following circumstances:

i. The alleged father is excluded as being the biological father, based uponexclusion at three or more STR loci.

ii. The alleged father is not excluded as being the biological father and the mostconservative values for both the Probability of Exclusion (PrE) and the

Probability of Parentage (W) are

99.99%.c. When the result of the Profile Plus testing is a failure to exclude, but the most

conservative PE and W statistics are


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e. Paternity Index (PI)

f. Probability of Parentage (W)

g. Probability of Exclusion (PrE)

2. Variation 1. Paternity with an apparent germline mutation.





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Part E. Missing Person Calculations

A. Missing Child, Two Parents Case. A typical missing person case will consist of parents of amissing person, and remains alleged to be from the missing person.

B. Missing Child, One Parent Case. Some missing person cases will involve a single parent of amissing person, and remains alleged to be from the missing person.





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C. Missing Parent, with one child and its other biological parent. Some missing person caseswill involve a biological child of a missing person, the other biological parent of the child,and remains alleged to be from the missing person.





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Section 2. Calculation methods and equations

Part A. Locus Random Match Probability (RMP) Calculations

Note: For calculations of the locus probability (f) for homozygous genotypes that include theta

() correction, = 0.01.

Eq (1) Calculation for homozygote AA

f= pA2

+ pA (1- pA), = 0.01

Eq (2) Calculation for heterozygote AB

f= 2pApB

Eq (3) Calculation for single allele (A) that is detected at a level less than the stochastic dropout

threshold (85 RFU). This is calculated based upon the set of not-excluded genotypes, G, where

G = {AA and AX}, where X is any allele other than A. Therefore, the px = 1 - pA. AX istherefore the set of all heterozygotes that include A, and the probability associated with AX is

2(pA)(1- pA).

f= [pA2

+ pA (1- pA)] + [2 (pA)(1- pA)], = 0.01



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Part B. Mixture Likelihood Ratio Calculation and Reporting Guidelines





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Section 3. Additional Reporting Guidelines

Part A. Reporting guidelines for parentage tests.

1. For standard parentage tests, statistical results will be reported for the Parentage(Paternity) Index, the Probability of Parentage (Paternity) and the Probability of

Exclusion in the Statistical Analysis section of the report.

2. The Statistical Analysis section of the report will structured as illustrated in the followingexample:

Statistical Analysis:

The Paternity Index, Probability of Paternity and Probability of Exclusion were calculated

for the Caucasian, African-American, and Southwest Hispanic population groups using the

Federal Bureau of Investigation database of STR population frequencies.

Based upon the genetic results described above, the combined probabilities related to John

Smith being the biological father of Jane Doe are:

Population Group Probability of

Exclusion

Paternity Index Probability of

Paternity *

Caucasian >99.99% 11.9 million >99.99%

African-American >99.99% 14.3 million >99.99%

Southwest Hispanic >99.99% 5.94 million >99.99%

* Assuming a prior probability of 0.5.

Based upon these calculations, the following statistical statements can be made regarding

the parentage of Jane Doe:

1. John Smith cannot be excluded as being the biological father of Jane Doe. Basedupon the most conservative Probability of Exclusion statistic, the probability that a

randomly selected man would be excluded as being the biological father of Jane Doe

is greater than 99.99%.

2. Based upon the most conservative Paternity Index statistic, the DNA profile of JaneDoe is 5.94 million times more likely if John Smith is the biological father than if a

randomly selected, unrelated male is the biological father.

3. Based upon the most conservative Probability of Paternity statistic, and ignoring allnon-genetic evidence, the probability that John Smith is the biological father of Jane

Doe is greater than 99.99%.

Part B. Reporting guidelines for random match statistics for single source samples and mixtures.

1. For single source samples (including major contributors to major-minor mixtures), theStatistical Analysis statement will be generally structured as shown in the following

example:

The probability of selecting at random an unrelated individual with the same DNA

profile as item 1A (stained fabric from shirt, major contributor), which matches John





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Does DNA profile, was calculated for the Caucasian, African-American, and Hispanic

population groups using the Texas Department of Public Safety database of STR

population frequencies. The combined probabilities for STR systems D3S1358, vWA,

FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, THO1, TPOX

and CSF1PO:

Population Group Probability

Caucasian 1 in 14.6 billion

African-American 1 in 45.3 billion

Hispanic 1 in 74.9 billion

2. For mixtures, the Statistical Analysis statement will be generally structured as shown inthe following example:

The probability of selecting at random an unrelated individual who would be included asa possible contributor to the mixture of DNA obtained from item 1A (stained fabric from

shirt), as John Doe is included, was calculated for the Caucasian, African-American, and

Hispanic population groups using the Texas Department of Public Safety database of

STR population frequencies. The combined probabilities for STR systems D3S1358,

vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, THO1,

TPOX and CSF1PO:

Population Group Probability

Caucasian 1 in 5.33 million African-American 1 in 7.85 million

Hispanic 1 in 3.26 million



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