SWIFS FBU DNA Procedures For Multiplex STR Analysis v1.2 (06.24.08) PDF

Southwestern Institute of Forensic Sciences Criminal Investigation Laboratory
Forensic Biology Unit Procedures for Multiplex STR Analysis, Version 1.2 Effective Date: 6/24/2008
Approved by: Stacy McDonald, Ph.D., Deputy Section Chief Timothy J. Sliter, Ph.D., Section Chief Chris Heartsill, Acting Quality Manager
This is an uncontrolled copy of a controlled document
Not a Controlled Document

Corrections & Revisions SOP: Procedures for Multiplex STR Analysis, Version 1.X Date 5/28/2008 Description Authorized by Changes from Version 1.0 to Version 1.1 McDonald Revision: DNA Quantification using Quantifiler (revisions in wording to clarify the quality control process and documentation) Changes from Version 1.1 to Version 1.2 McDonald/Sliter Addition: Section 14.1- ABI Prism Procedures, Appendix 1 Increased injection times for low level samples
6/24/2008

ABI Prism 310 Procedures for STR Analysis Addendum 1. Increased injection time for low level samples 1. Principle This procedure describes a modification to the standard protocol for capillary electrophoresis 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. 2. Sample Requirements This procedure may be used under the following conditions: 1. Analysis of the sample has been completed using the standard capillary electrophoresis protocol, and all options for extract re-amplification and sample re-extraction have been exhausted. 2. The standard capillary electrophoresis analysis protocol gives no genetic information, and the possible presence of low level genetic information in the extract is indicated by one of the following: serology testing results, baseline fluctuations 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 genetic information, with no peaks exceeding 3000 RFUs. 3. Procedure 1. Following completion of the standard electrophoresis protocol, a follow-up electrophoresis is performed using an increased injection time. a. 15 second injection A 15 second injection time may be used when there are no peaks in the electropherogram exceeding 900 RFUs. b. 10 second injection A 10 second injection time may be used when there are no peaks in the electropherogram exceeding 3000 RFUs. 2. The increased injection time batch will include the following samples: a. Test extracts b. Corresponding reagent blanks (negative control) c. Corresponding amplification blanks (negative control) d. 9947A amplification positive control (injected for 5 seconds to avoid the possibility of off scale data) OR the positive extraction control. 3. Each of the test extracts and negative controls will be injected a minimum of two times to establish the reproducibility of any detected alleles.
ABI Prism 310 Procedures for STR Analysis Addendum 1. Increased injection time for low level samples Effective: 4/2/2008
4. Interpretation Guidelines 1. The data from the increased injection times will be interpreted using the standard interpretation guidelines (see SOP), with the following exceptions: a. All calls of homozygosity will be based upon the results of the standard 5 second injection times. Example 1: At a locus where a single allele is detected at 95 RFUs using the 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 85 RFUs. Example 2: At a locus where a single allele is detected at 80 RFUs using the standard 5 second injection time, the locus may not be called homozygous for the allele following the 10 second injection, because the standard results gave a peak height less than the 85 RFU threshold for calling homozygosity.
ABI Prism 310 Procedures for STR Analysis Addendum 1. Increased injection time for low level samples Effective: 4/2/2008

Procedures for Multiplex STR Analysis Appendix 4. Solutions, Kits and Materials: Preparation and Storage, Version 1.1 The following general instructions apply to the preparation of all solutions: 1. 2. 3. 4. Use graduated cylinder and pipettes close in capacity to the volume being measured. 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. Prepare all solutions with deionized water, and chemicals that are of reagent grade or better quality, unless otherwise noted. 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 correct final concentrations.
310 Buffer, 1X For 250 mL: 10X 310 Buffer Water 25 mL 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 by heating the buffer to 70oC with gentle agitation. Store at room temperature. QC test before use: QiaAmp DNA Micro Kit Reagents QC Test.
Procedures for Multiplex STR Analysis Appendix 4. Supplies, Kits and Reagents: Preparation and Storage, Version 1.1 Effective Date: 9/27/2007
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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) Ethanol, 100% 13 mL 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) Ethanol, 100% 60 mg 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
Procedures for Multiplex STR Analysis Appendix 4. Supplies, Kits and Reagents: Preparation and Storage, Version 1.1 Effective Date: 9/27/2007 -2-

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 in the QuantiBlot Human DNA Quantitation Kit. Final concentrations of DNA in the standards are as follows: Standard A Standard B Standard C Standard D Standard E Standard F Standard G 2 ng/L 1 ng/L 0.5 ng/L 0.25 ng/L 0.125 ng/L 0.0625 ng/L 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 the QuantiBlot Human DNA Quantitation Kit. Final concentrations of DNA in the standards are as follows:
Standard Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 Standard 8 Concentration (ng/L) 50.0 16.7 5.56 1.85 0.62 0.21 0.068 0.023 Amounts 50 L 200ng/L stock + 150 L TE-4 50 L Std 1 + 100 L TE-4 50 L Std 2 + 100 L TE-4 50 L Std 3 + 100 L TE-4 50 L Std 4 + 100 L TE-4 50 L Std 5 + 100 L TE-4 50 L Std 6 + 100 L TE-4 50 L Std 7 + 100 L TE-4 Dilution Factor 4X 3X 3X 3X 3X 3X 3X 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 Water 6.18 g 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 NaOH Water 186.1 g q.s. pH 8.0 0.2 q.s. 1 L
Add EDTA crystals to 800 mL water. While agitating on a magnet stirrer, add approximately 20 g NaOH. Adjust pH to 8.0 0.2 with 5 N NaOH. Adjust final volume to 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 5 M NaCI 0:5 M EDTA 20% SDS Water
10 ml, 20 mL 100 mL 100 mL 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 20% SDS Water 250 mL 25 mL 725 mL
Add 20X SSPE and 20% SDS to water. Mix thoroughly. Store at room temperature. Prior to use, 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 Water 10 mL 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 UVirradiate with 6 J/cm2 in a UV cross-linker prior to use. NaCl, 5 M For 1 L: NaCl Water 292 g q.s. 1 L
Dissolve NaCl in water. Autoclave solution. Store at room temperature. NaOH, 5 N For 1 L: NaOH Water 200 g q.s. 1 L
Dissolve NaOH in 800 mL water. Adjust volume to 1 L. Store at room temperature.

Nuclear Fast Red Stain [0.05% Nuclear fast red, 2.5% Aluminum sulfate] For 600 mL: Aluminum sulfate Nuclear fast red Water 15 g 0.3 g 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 crosslinker prior to use. Picric Acid Solution, 1 % Purchase from commercial supplier (Aldrich p/n 31,928-7 or equivalent). Store at room temperature. Picroindigocarmine Stain [0.33% Indigo carmine, 1% Picric acid] For 600 mL: Indigo carmine 1 % Picric acid solution 2g 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 0.5 M EDTA Water 40 mL 25 mL 435 mL
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Add 5 N NaOH and 0.5 M EDTA to water. Mix thoroughly. Store at room temperature. QC test 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 before use: 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: Extraction Reagents 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 20 mg/mL Proteinase K 4 M DTT 1000 L 10 L 10 L
Add 20 mg/mL Proteinase K and 4 M DTT to EB. Mix thoroughly. Prepare fresh. Discard any 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 0.5 M EDTA 0.04% Bromothymol Blue Water 6 mL 3.75 mL 150 L 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 months following 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 at room 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 solution from a commercial supplier (Amresco p/n E112-100ML or equivalent). TE-4 Buffer [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 at room temperature. QC test lots before use: Amplification Reagents QC Test. Tris-Cl [Tris(hydroxymethyl)amminomethane], 1 M For 1 L: Tris base Water 121 g 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.

Wash Solution [1.SX SSPE, 0.5% SDS] For 2 L: 20X SSPE 20% SDS Water 150 mL 50 mL 1,800 mL
Add 20X SSPE and 20% SDS to water. Mix thoroughly. Store at room temperature. Prior to 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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Procedures for Multiplex STR Analysis Appendix 5. Critical Reagents, Kits and Materials, Version 1.1 Consult Appendix 4. Solutions, Kits and Materials: Preparation and Storage for information regarding required QC tests prior to use in casework. A. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. B.
1.
Listing of critical reagents, kits, and materials Biodyne B Membrane Buffer AL Buffer ATL Buffer AW1 Buffer AW2 Chromogen:TMB Citrate Buffer 4. Cofiler DNA Amplification Kit DNA Quantitation Standards (for Quantiblot and Quantifiler Kits) 4 M DTT EB Hybridization Solution 30% Hydrogen Peroxide PCIA Pre-Wetting Solution Profiler Plus DNA Amplification Kit Proteinase K Quantiblot Human DNA Quantitation Kit Quantifiler Human DNA Quantitation Kit Spotting Solution TE-4 Buffer Wash Solution Water Additional Notes QIAamp DNA Micro Kit, Quantiblot Human DNA Ouantitation 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 expiration date, 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
Procedures for Multiplex STR Analysis Appendix 5.Critical Reagents, Kits and Materials, Version 1.1 Effective date: 9/27/2007
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Addendum to Appendix 7, Quality Control Tests Quantifiler Reagents QC Test, Version 1.0 Quantifiler Kit Components: 1. 2. 3. Human DNA Standards 1-7 PCR Reaction Mix Human Primer Mix
Procedure 1. Perform the SOP: DNA Quantitation by Real-time PCR using the Quantifiler Kit 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. 2. 3. 4. 5. All DNA standards are detected. The R2 value 0.99. The slope ranges in value from -2.9 to -3.3. There is amplification of the Internal Positive Control (IPC). No DNA is detected in the Blank.
Procedures for Multiplex STR Analysis Addendum to Appendix 7. Quality Control Tests Quantifiler Reagents QC Test, Version 1.0 Effective Date: 9/27/2007
This is an uncontrolled copy of a controlled document TJS Initials
Date
1/4/2007
DNA049.2 ABI 310 Procedures for STR Analysis using Profiler Plus and Cofiler Windows XP Platform with 48 tray setup, Version 1/4/2006
1 Principle This protocol describes procedures used to perform fragment analysis for the purpose of STR typing using the ABI Prism 310 Genetic Analyzer and 310 Data Collection software run on a Windows NT platform and using a 48-sample tray. Fluorescently labeled PCR products produced using the Profiler Plus and Cofiler kits are detected following capillary electrophoresis. 2 Sample Requirements Samples for capillary electrophoresis using the ABI Prism 310 consist of amplified product produced using the Profiler Plus and Cofiler kits. 3 Procedures
3.1 Prepare Rox500 master mix 3.1.1 Prepare Rox500 master mix for the number of samples to be run, plus the allelic ladder(s). Prepare master mix for one or two more samples than you need so that you dont run short because of pipetting precision. The Rox500 master mix consists of the following components: Component Formamide, deionized Rox500 standard 3.1.2 3.1.3 Amount per sample 24 L 1 L
Label 310 sample tubes. Aliquot 25 L of the Rox500 master mix to each of the labeled 310 sample tubes.
3.2 Set-up 310 samples 3.2.1 Add 1.5 L of each test sample to the correspondingly labeled 310 sample tube. 3.2.2 Add 1.5 L of the ladder(s) to the correspondingly labeled 310 sample tube(s). 3.2.3 When all samples have been added, cap each tube with a rubber septum. 3.2.4 Place tubes in a 95C dry block for 3 min to denature the DNA. Chill tubes by placing in a cold block for at least 3 min. 3.2.5 Place sample tubes in a 310 sample tray (48 samples). 3.2.5.1 Place ladder samples as the last samples in the sample tray. 3.3 General Quality Considerations 3.3.1 All samples from a single amplification batch must be analyzed together in a single 310 batch, except when the size of the amplification batch exceeds the maximum 310 batch size of 48 samples. In setting up the 310 sample tray, samples must be placed consecutively, with no empty spaces, in the same order as the amplification batch. In the event that one or more samples must be rerun due to poor electrophoresis: 1
3.3.2 3.3.3

3.3.3.1
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Negative controls from the original run need not be run again if they ran successfully the first time. 3.3.3.2 A 9947A positive amplification control sample should be included in each 310 run to verify proper run performance. In a rerun, if for some reason the 9947A sample can not be rerun, then with the Technical Managers approval, either the POS extraction control or the ladder sample may be utilized as a run control. 3.3.3.3 The allelic ladder sample must be run for sizing. 3.3.4 The allelic ladder samples should be reinjected several times during the 310 run to serve as an internal quality standard for the run. 3.3.5 Generally, when a completed amplification batch is analyzed, the order of samples in the 310 sample sheet and injection list will correspond to the order of samples in the STR Amplification Worksheet. Additional injections of some samples may be added to the injection list for confirmation purposes, or to reinject samples that electrophoresed poorly during the run. 3.4 Create a New Sample Sheet 3.4.1 Open the 310 Data Collection application. 3.4.2 Create a new 48-sample Sample Sheet. For reruns, the original sample sheet may be used. 3.4.3 Fill in the Sample Name fields following as a guide the examples shown below (Table 1) and using the information from the STR Amplification Worksheet. Include the amplification sample number, and the case and item identifiers. Indicate whether the sample is a Profiler (p) or Cofiler (c) amplification. 3.4.4 When Sample Name fields are correctly filled, copy the column and paste it into the Sample Information column. 3.4.5 When the sample sheet is complete, save it. The sample sheet will be named automatically according to the following format: SS_[Instrument Letter][Yr]_[Day Date Time Year]. For example: SS_F07_Tues Jan 01 15-45-23 CST 2007. Table 1. Guidelines for Sample IDs in the Sample Sheet Sample Type Sample ID Questioned evidence sample [Amp#]-[case#] [item/sample#] [sample name (OPTIONAL)] [p or c] e.g., 4-01P9999 #1T1 Stained swab p Standard [Amp#]-[case#] [item/sample#] [source name] [p or c] e.g., 4-01P9999 #23 J Doe p Amplification Blank [Amp#]-[AmpBlank] [p or c] e.g., A-AmpBlank c Reagent Blank [Amp#]-[RB] [p or c] e.g., 1-RB p Positive Control [Amp#]-[9947A] [p or c] e.g., B-9947A c Allelic Ladder [Profiler Ladder or Cofiler Ladder} e.g., Cofiler Ladder
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3.4.6
Alternately, sample sheet information may be imported from an electronic text file generated from an Excel workbook for DNA process worksheets.
3.5
Prepare the injection list 3.5.1 Create a new Injection List. 3.5.2 Select the appropriate sample sheet from the drop down menu. The Tube & Sample Name fields will be filled in automatically. 3.5.3 The Injection List should be automatically filled in with the following parameters. Field Length to Detector (cm) Module Inj secs Inj kV Run kV Run C Run Time Matrix File Auto Analyze Analysis Parameters Size Standard Auto Print 3.5.4 Value 30 GS STR POP4 (1 mL) F 5 15.0 15.0 60 24 <none> Off (leave blank) (leave blank) Off
Enter operator last name (do not use initials) in the operator field.
3.6 Add additional injections to the Injection List 3.6.1 Additional injections of ladder samples and 9947A samples should be inserted into the Injection List for Quality control purposes. These additional runs serve as quality control checks in the event of that fluctuations occur in run conditions. 3.6.1.1 The injection batch should begin with a ladder injection followed by a 9947A injection. 3.6.1.2 Paired injections of the ladder and 9947A should be inserted into the Injection List every 10-15 samples. 3.6.1.3 If the injection batch consists of both Profiler Plus and Cofiler samples, then the Profiler Plus injections should be flanked with corresponding ladder and 9947A injections, and the Cofiler injections should be flanked with Cofiler ladder and 9947A injections. 3.7 Start the 310 run 3.7.1 Open the doors of the 310. Press the Tray button to present the autosampler for loading. 3.7.2 Place the sample tray on the autosampler. 3.7.3 Press the Tray button to return the autosampler to its original position. 3.7.4 Close the doors of the 310.

3.7.5
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In the injection list window, click the Run button. The injection run will proceed automatically. 3.7.6 A run folder will be automatically created in the Runs folder and named according to the format: RF_[Instrument Letter][Yr]_[Day Date Time Year]. For example: RF_F07_Tues Jan 01 15-45-23 CST 2007. 3.7.6.1 NOTE. The default Run Folder name will be changed at the end of the run, before moving it to the Runs Archive Folder 3.7.7 The Injection List will be saved automatically to the run folder and named according to the following format: Inj_[Instrument Letter][Yr]_[Day Date Time Year]. For example: Inj_F07_Tues Jan 01 15-45-23 CST 2007. 3.7.7.1 The injection list is automatically saved to a newly created Run Folder when the 310 run is started. During the 310 run, the injection list can be modified (e.g., adding in repeat injections of a sample). 3.7.7.2 The modified list is not automatically saved, however. The modified injection list must be saved using the Save As option under the File menu, then navigating to the appropriate Run Folder. 3.7.7.3 Do not use the Save option under the File menu to save the modified injection list. If the modified Injection List is saved using the Save option under the File menu, then it will not be saved in the correct run folder; it will be saved with a different name in the parent directory containing the run folders. 3.8 Terminate the 310 run 3.8.1 When the 310 run is completed print out the Injection List. 3.8.2 Close the 310 Collection software, saving the project. 3.8.3 Locate the run folder in the Runs folder. Rename the run folder using the following convention: RF_[Instrument Letter]_[Date]_[Analyst Initials][Run Index Number]. The Run Index Number tracks the run batches that might be run on the same day. E.g., RF_F_2007Jan02_CF. 3.8.3.1 In the event that an analyst creates more than one run folder on a particular date, then the subsequent run folders (after the first one) are to be distinguished by an index number. The name of the run folders should conform to the following convention: RF_[Instrument Letter]_[Date]_[Analyst Initials]_[Run Index Number]. E.g., RF_F_2007Jan02_CF_2. 3.8.4 Immediately move the run folder to the Runs Archive Folder. 3.8.4.1 Note: Currently, each of the workstations that control the 310 devices has its own Runs Archive Folder. 3.8.4.2 In the future, the Runs Archive Folder will exist on a networked server. 3.9 Analyze the data in the run folder (located in the Runs Archive) folder using GeneMapper ID.

4 Addendum to SOP - Screen Shots. 4.1 Windows - Users
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4.2
Data Collection Preferences File Names
Note: The Sample Sheet, Run Folder, and Injection List names include an identifier for both the instrument and the year (F07 in this example). The names will differ depending on the year and instrument used.
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4.3
Data Collection Preferences Folder Locations
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4.4
Data Collection Preferences General Settions
Note: the Global Serial Number is the beginning global serial number. The global serial number increments as it is used in naming folder, etc. The global serial number should not be reset except as part of yearly maintenance of the sample sheet, folders, and injection list names.
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4.5
Data Collection Preferences Genescan Injection List Defaults
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4.6
Data Collection Preferences Genescan Sample Sheet Defaults
10
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4.7
Data Collection Preferences Dye Indicators
11
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4.8
Data Collection Injection Sheet Values
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This is an uncontrolled copy of a controlled document TJS Initials Date
12/20/2006
DNA050.2 Analysis of Profiler Plus and Cofiler Data Collected with the ABI 310 and Analyzed using GeneMapper ID Software, Version 12/20/2006 1 Principle This protocol describes the steps taken following STR data collection with the ABI Prism 310 Genetic Analyzer to analyze the raw data using GeneMapper ID software, to evaluate the quality of the data, to perform automated allele calling, and to edit allele calls. 2 Sample Requirements Samples consist of raw data files collected with 310 Data Collection software and stored in the Runs folder. Data is analyzed using the GeneMapper ID (GMID) software. 3 Procedure
3.1 Create a new GMID project 3.1.1 From the desktop, open the GMID software by clicking on the icon. 3.1.2 Under the File menu, select Add Samples To Project. 3.1.3 Navigate to the Runs folder (D:\AppliedBio\310\Runs). 3.1.4 Select the appropriate run folder to analyze. 3.1.5 Click the <Add to List> button. The run folder will move to Samples to Add frame. 3.1.6 Press the <Add> button. The populated GMID sample table will open. 3.1.7 Save the GMID project. 3.1.7.1 The following format is to be used in naming GMID projects: [Analyst Initials]_[Date, format 2006JAN15]. Example: TJS_2006SEP28. 3.1.7.2 If more than one GMID project is created by the same analyst on the same day on the same machine, then the subsequent projects should be distinguished: TJS_2006SEP28_2, TJS_2006SEP28_3, etc. 3.1.7.3 Note: GMID project are temporary and will be discarded after a period of time. The only permanent record of the GMID analysis is the hardcopy printout and electronic copies of the printout. 3.2 Verify Data Analysis Parameters 3.2.1 In the GMID sample table, check to make sure that the correct parameters are selected. 3.2.2 Sample Type. Each sample should be designated one of the following sample types: Positive Control, Negative Control, Allelic Ladder, and Sample. The default sample type is Sample. 3.2.3 Analysis Method. The default Analysis Method is Profiler-Cofiler Advanced. The standards parameters for Profiler-Cofiler Advanced are shown in the Appendices. 3.2.4 Panel. Depending on the kit used, the Panel selection will be either Profiler_Plus_v1 or COfiler_v1. The default selection is Profiler_Plus_v1. 3.2.4.1 A single GMID project may contain both Profiler Plus and Cofiler data. The Panel parameter setting serves to distinguish these samples during
Analysis of Profiler Plus and Cofiler Data Collected with the ABI 310 and Analyzed using GeneMapper ID Software, Version 12/20/2006
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analysis so that Cofiler samples will be analyzed with the Cofiler Ladders, and Profiler Plus samples will be analyzed with the Profiler Ladders. 3.2.5 Size Standard. The default size standard is Rox500. 3.2.6 Matrix. The default matrix will be the most recent constructed matrix. 3.2.7 Allele labels. Default labeling for peaks is allele name only. Other labels (e.g., peak height, data point) may be added as appropriate for analysis and clarity. 3.3 Preliminary Analysis 3.3.1 After the sample table parameters are selected/confirmed, click the <Analyze> button. 3.3.2 After the analysis is complete, sort the table (Edit, Sort) first by Panel, then by Sample Type, then by Sample Name. 3.3.2.1 In sorting the table this way, Profiler Plus samples will be grouped together, and Cofiler samples will be grouped together. If there are only samples of one type in a project, then sorting by Panel is not necessary. 3.4 Evaluate the allelic ladders. 3.4.1 The allelic ladder (Profiler Plus or Cofiler) will be run several times during the course of each 310 injection batch. The function of the ladder is to provide empirical peak size data that will be used by the GMID program to define allele-specific size windows to identify peaks in the test samples. Each GMID project will define allele windows based on all ladder injections in the project that are designated the sample type allelic ladder. Therefore, it is important to review the ladder injections for quality. 3.4.2 In GMID sample sheet, select the ladder samples. 3.4.3 View the electropherograms. 3.4.4 In general, discrete, well-defined and well-separated peaks should be present in each ladder injection. The following features will generally characterize a good ladder injection: 3.4.4.1 Peak height of Rox500 standards. All defined Rox500 peaks must be present and labeled in the electropherogram (base pair sizes 75, 100, 139, 150, 160, 300, 340, 350, and 400). These peaks will typically exceed 500 RFU, and will be fairly even in height. Electropherograms with Rox500 peak heights significantly less than 500 RFU, or with significantly uneven peak heights may indicate a poor quality injection that should not be used for allele calling. Such samples should be evaluated carefully. 3.4.4.2 Peak height of ladder peaks. All defined ladder peaks should be called. Ladder peaks will typically exceed 500 RFUs with good peak height balance. Electropherograms with low or uneven peak heights may indicate a poor quality injection that should not be used for allele calling.

3.4.4.3
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3.4.4.4 3.4.4.5
3.4.4.6
3.4.4.7
Peak resolution. A good injection will resolve ladder peaks that differ by 1 and 2 bp. For instance, in the Profiler Plus ladder, the D18S51 13, 13.2, 14, and 14.2 alleles must be resolved and called by the GMID software. In the Cofiler ladder data, the THO1 9, 9.3, and 10 alleles must be resolved and called by the GMID software. Baseline signal. In each color the baseline should be relatively flat, and generally <20 RFU. The 250 bp Rox500 peak. The 250 bp fragment in the Rox500 size standard is not assigned a standard size, and serves as an internal quality control standard. The 250 bp fragment will generally be sized from 244-248 bp, depending on the particular characteristics of the injection batch. Typically, in the first injection of a batch the 250 bp fragment will run a bit fast due to conditioning of the capillary. In a project, the empirical size of the 250 bp fragment in the injections will show a distribution. A ladder whose 250 bp ROX fragment lies significantly outside of the overall distribution may should not be used for allele calling. Other Rox500 peaks. For the local Southern method in GMID to accurately size peaks, the two Rox500 standard peaks smaller than Amelogenin (75 and 100 bp) must be included in the collected and analyzed data for each ladder. Similarly, the 400 bp Rox500 peak must be included in the analyzed data. When the ambient temperature of the lab is higher or lower than normal, this may affect the migration rate of fragments, resulting in failure to collect appropriate data for analysis. Under these conditions, it may be necessary to increase the data collection time and collect new data when the 400 bp Rox500 fragment is not collected. If the 75 bp Rox500 fragment is not included in the analyzed data, then data analysis parameters in GMID may be modified so as to include the peak. Artifacts. The presence of significant electrophoresis artifacts (e.g., dye blobs, spikes, high baseline, etc.) may compromise the quality of a ladder injection. Electropherograms that show such artifacts should be evaluated carefully.
3.5 Final Analysis 3.5.1 If there are any ladder injections that fail meet quality standards, change their sample types from Allelic Ladder to Sample. 3.5.2 Click the <Analyze> button in order to reanalyze the project using only ladder injections that meet quality standards. 4 Evaluate Negative Controls
4.1 Negative control samples 4.1.1 Injection batches will typically include Reagent Blanks (RBs) and Amplification Blanks (AmpBls).

4.1.2 4.1.3
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Injection batches that are reruns of samples that ran poorly in a previous run may not include negative controls.. Injection batches that are reamplifications of samples from a previously amplified batch may not include RBs.
4.2 General Quality Considerations 4.2.1 Each control must be successfully electrophoresed. 4.2.2 A successful electrophoresis is characterized by all defined Rox500 peaks being present and labeled in the electropherogram (base pair sizes 75, 100, 139, 150, 160, 300, 340, 350, and 400). Additionally, the 250 bp Rox500 fragment will typically be sized at 244-248 bp. 4.2.3 In most samples, the Rox500 peaks will typically exceed 500 RFU, and will be fairly even in height. 4.2.4 Samples in which the peaks are significantly less than 500 RFUs, which show uneven peak heights, or which show other abnormal characteristics, should be evaluated with care. Additional analysis (electrophoresis, amplification, etc.) may be performed. 4.2.5 Electrophoresis artifacts. The presence of significant electrophoresis artifacts (e.g., dye blobs, spikes, high baseline, etc.) may compromise the quality of an injection. Samples that show such artifacts should be evaluated carefully, and reinjected if needed. 4.3 Evaluate the AmpBls 4.3.1 The Amp Blank sample consists of the amplification mix without template DNA, and serves as a negative control for contamination of the PCR master mix with exogenous DNA. 4.3.2 The Amp Blank samples should not show a DNA profile. In the event that any Amp Blank sample shows a partial or complete DNA profile, then the analyst must consult with the Technical Leader to determine the proper course of action (e.g., trouble shooting, reanalysis, etc.). 4.4 Evaluate the RB samples 4.4.1 The RB samples serve as controls for contamination during the extraction setup, including contamination of the extraction solutions with exogenous DNA or biological material containing DNA. 4.4.2 The RB samples should not show a DNA profile. In the event that any RB sample shows a partial or complete DNA profile, then the analyst must consult with the Technical Leader to determine the proper course of action (e.g., trouble shooting, reanalysis, etc.). 5 Evaluate the positive control samples
5.1 Positive controls 5.1.1 Injection batches will typically include both an amplification positive control (9947A) and two extraction positive controls (POS). The positive control
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5.1.2
5.1.3 5.1.4
samples serve two purposes: 1) positive quality control samples for the extraction and amplification processes; and 2) electrophoresis run condition controls. Injection batches that are reruns of samples that ran poorly in a previous run may include only one of the positive controls. Typically this will be the 9947A control. However, the POS control may be used if the 9947A control was poorly amplified. Injections batches that are reamplifications of samples from a previously amplified batch will include only the 9947A positive control. In an injection batch, ladder samples may substitute for the positive controls as electrophoresis run condition controls.
5.2 General Quality Considerations 5.2.1 Each control must be successfully electrophoresed. 5.2.2 A successful electrophoresis is characterized by all defined Rox500 peaks being present and labeled in the electropherogram (base pair sizes 75, 100, 139, 150, 160, 300, 340, 350, and 400). Additionally, the 250 bp Rox500 fragment will typically be sized at 244-248 bp. 5.2.3 In most samples, the Rox500 peaks will typically exceed 500 RFU, and will be fairly even in height. 5.2.4 Samples in which the peaks are significantly less than 500 RFUs, which show uneven peak heights, or which show other abnormal characteristics, should be evaluated with care. Additional analysis (electrophoresis, amplification, etc.) may be performed. 5.2.5 Electrophoresis artifacts. The presence of significant electrophoresis artifacts (e.g., dye blobs, spikes, high baseline, etc.) may compromise the quality of an injection. Samples show such artifacts should be evaluated carefully, and reinjected if needed. 5.2.6 Amplification artifacts. The presence of significant amplification artifacts (off scale peaks, non-specifically amplified product, -A peaks, etc.) may compromise the quality of an injection. Samples show such artifacts should be evaluated carefully, and reinjected or reamplified if needed. 5.3 Evaluate the 9947A amplification positive control 5.3.1 The target profile of the 9947A amplification positive control is shown in the Appendix section. 5.3.2 Evaluate 9947A as an amplification positive control 5.3.2.1 The 9947A sample is the primary positive control for the amplification process. The POS extraction positive control serves as a redundant amplification positive control, which can be used in the case of a failure of 9947A control for trivial reasons (e.g., poor amplification of 9947A control DNA that has been stored for a long period of time). 5.3.2.2 Each of the 9947A injections should give a complete single source profile that matches the target profile.

5.3.2.3
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Occasionally, one or several 9947A injections may fail. A minimum of one of the 9947A injections in a 310 injection batch must give a complete profile in order for the amplification positive control to pass. 5.3.2.4 If none of the 9947A injections give a complete profile, then a POS extraction positive control sample may be used as an amplification positive control. 5.3.2.5 If neither the 9947A control or any of the POS control samples show a complete profile, then the analyst must consult with the Technical Leader to determine the proper course of action (e.g., trouble shooting, reanalysis, etc.). 5.3.2.6 If the 9947A sample amplifies but departs from the expected results (e.g., incorrect profile, mixed profile), , then the analyst must consult with the Technical Leader to determine the proper course of action (e.g., trouble shooting, reanalysis, etc.). 5.3.3 Evaluate the 9947A sample as an electrophoresis run control 5.3.3.1 On occasion, electrophoresis conditions will vary over the course of a 310 injection batch. 5.3.3.2 The 9947A sample is the primary control for evaluating the quality of the electrophoresis. Failure of a 9947A sample to correctly type may indicate a transient problem with the run conditions. 5.3.3.3 If a particular 9947A injection fails for a reason unrelated to the run conditions (e.g., no injection, poor injection), the ladder injection run adjacent to the 9947A injection may be used as a substitute for the corresponding 9947A injection. 5.3.3.4 If the GMID software fails to call correctly the alleles in a 9947A injection and in the corresponding ladder injection, then the affected test samples will be reinjected to determine the correct allele designations. 5.4 Evaluate the POS extraction positive control 5.4.1 The target profile of the POS extraction positive control is shown in the Appendices. 5.4.2 The POS control sample is the positive control for the extraction process. The POS control sample is expected to give a specific single source DNA profile. Failure of the POS control sample may indicate a failure in the DNA extraction process. Additionally, the POS control sample may be used as a supplemental QC sample (in addition to 9947A and ladder samples) to assess the quality of the electrophoresis process (see 5.3). 5.4.3 Evaluate the POS control sample as an extraction positive control 5.4.3.1 Each POS control sample should give a full single source DNA profile that matches the target profile. 5.4.3.2 If the profile obtained from the POS control sample departs from the expected profile (e.g., no profile, partial profile, incorrect profile, mixed profile, etc.) then the analyst must consult with the Technical Leader to determine the proper course of action (e.g., trouble shooting, reanalysis, etc.).
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Evaluate the test samples
6.1 Evaluate each injection for off-scale peaks. 6.1.1 Offscale data are indicated by a red lines/areas on the electropherogram. 6.1.2 Offscale data are typically a reflection of a high amount of DNA in the amplification reaction. 6.1.3 Peaks associated with off-scale data may exhibit characteristic features, including 6.1.3.1 Higher than normal pull-up 6.1.3.2 Higher than normal stutter percentage 6.1.4 Samples in which a high amount of DNA was used in the amplification may exhibit characteristic features, such as 6.1.4.1 The presence of -A peaks 6.1.4.2 The presence of +4 stutter peaks 6.1.4.3 Under-amplification of larger fragments 6.1.4.4 Non-specifically amplified products 6.1.5 Samples with offscale data should be evaluated carefully by the analyst to assess their suitability or unsuitability for analysis. 6.1.5.1 If a few of the homozygous peaks are offscale, and there is no significant occurrence of -A peaks, and there is no non-specifically amplified product, then the data may be analyzed routinely with appropriate editing of the allele calls. 6.1.5.2 If there are a large number of off-scale peaks, including both homozogous and heterozygous loci, but there is little or no occurrence of -A peaks, then the sample may be diluted and rerun to obtain onscale data. 6.1.5.3 If there is a significant occurrence of -A peaks, or if there are nonspecifically amplified products, then the sample should be reamplified if possible using less DNA. 6.2 Evaluate the sample for interfering artifacts. 6.2.1 Occasionally, run artifacts (4-color spikes, elevated baseline, dye-blobs, pullup, etc.) may be present in a sample. 6.2.2 Some artifacts are characteristic of the 310 run, and will not be observed when the 310 sample is rerun. (It may be necessary to change the buffers, water, and/or septa of the buffer and water tubes in order to eliminate dye-blob artifacts.) 6.2.3 If the artifact is clearly distinguishable from authentic allele peaks, and does not interfere with any authentic allele peaks, then the data may be analyzed routinely. A notation should be made on the GMID print-out for the sample. 6.2.4 If the artifact migrates closely with an authentic peak, or might potentially obscure an authentic peak, then the 310 sample should be rerun. 6.2.5 If rerunning the 310 sample does not eliminate an apparent artifact, then the sample should be reamplified if possible.
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6.3 Evaluate the baseline signal 6.3.1 Baseline signal is typically <20 RFU. 6.3.2 Higher baseline levels are often seen when high amounts of DNA are used in the amplification reaction. 6.3.3 If higher than normal baseline signal does not interfere with subsequent analysis, then the analysis may proceed routinely. 6.3.4 If higher than normal baseline signal might significantly interfere with subsequent analysis, then the sample may be rerun, diluted and rerun, or the extract may be reamplified. 6.4 Evaluate each sample for OL allele calls 6.4.1 All samples showing Off-Ladder (OL) allele calls should be evaluated carefully. 6.4.2 If the OL allele call is readily attributed to a spike, a dye blob, high baseline, or another type of routine electrophoresis artifact, then no further analysis is needed. 6.4.3 If the OL allele call is attributable to poor electrophoresis, then the sample should be re-electrophoresed. 6.4.4 If the OL allele call is attributable to nonspecifically amplified product, then it may be necessary to re-amplify the sample. 6.4.5 If the OL allele call is attributable to an authentic off-ladder allele, then the call must be confirmed by re-electrophoresis. 7 Prepare documentation for the report package
7.1 Print out the GMID sample table. 7.1.1 Notate on the hardcopy printout the success/failure of injections of positive and negative controls and the need for reinjections. 7.1.2 Note on the hardcopy printout the success/failure of injections of samples and the need for reinjections. 7.2 Print GMID plots showing allele calls. 7.2.1 In the GMID sample table, select all Alleleic Ladder, Positive Control, and Negative Control samples. Display the electropherograms in the plot window. Print these samples as a single batch at medium size. 7.2.1.1 In a project containing both Profiler Plus and Cofiler samples, print the two types of samples as separate projects. 7.2.1.2 If the project includes ladder samples that are of poor quality and are not used as allelic ladders in the analysis, do not print out these ladders 7.2.2 In the GMID sample table, print the sample plots individually (i.e., do not batch print). Print these samples as a single batch at medium size. 7.2.2.1 Print these plots using the small plot option. 7.3 Make hand notations on the electropherograms to edit allele calls for artifacts, etc.

7.4
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As necessary, print out additional electropherograms and raw data plots showing different views or additional labels as supporting documentation for artifact and OL allele calls.
7.5
In the event of an authentic off-ladder allele, print a plot window zoomed to the locus of interest. 7.5.1 Show the bin windows in the plot. 7.5.2 Include in the plot a panel showing the ladder so that the bins can be identified.

8 8.1 Addendum to SOP Screen Shots of Parameter Windows GeneMapper Logon
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Note: Database Host Name specifies the computer being used to host the database. Each computer will be specified uniquely.
10
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8.2
Analysis Method Editor - Allele
11
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8.3
Analysis Method Editor Peak Detector
Note: These are the default parameters. The Analysis range may be changed by the analyst to correct for ambient temperature fluctuations. Other parameter may not be changed, except as needed for troubleshooting purposes.
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8.4
Analysis Method Editor Peak Quality
13
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8.5
Analysis Method Editor Quality Flags
14
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8.6
GeneMapper Manager Analysis Methods
Note: Only the Profiler-Cofiler Advanced method is to be used for casework performed with the Profiler Plus and Cofiler kits. Other methods may be present for troubleshooting and validation purposes.
15

8.7 GeneMapper Manager Plot Settings
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16

8.8 GeneMapper Manager Size Standards
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17

8.9 GeneMapper Manager Table Settings
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18
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8.10 GeneMapper Options for adding samples to a new project. Shown is the set-up for a batch of Profiler Plus samples (panel Profiler_Plus_v1). For a batch of Cofiler samples, set panel to COFILER_v1. For a mixed batch of Profiler Plus and Cofiler samples, the panel for each sample may be set in the GeneMapper table. The matrix should be set to the most recent good matrix.
19

8.11 GeneMapper Options Analysis
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8.12 GeneMapper Options Startup
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8.13 GeneMapper Options Users
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Note: Only the gmid user is required. The gmid user is the standard user for casework analysis. Other users may be set up for troubleshooting, validation, research and other purposes.
22
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8.14 GeneMapper Panel Manager Cofiler_v1. Stutter percentages correspond to the upper bound of the 99% confidence interval as determined by SWIFS validation studies.
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8.15 GeneMapper Panel Manager Profiler_Plus_v1. Stutter percentages correspond to the upper bound of the 99% confidence interval as determined by SWIFS validation studies.
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DNA Quantitation by Real-time PCR using the Quantifiler Kit, Version 1.1 I. Principle This document describes procedures to estimate the amount of human DNA in extracts prior to PCR amplification. The procedure uses the Quantifiler Human DNA Quantification Kit (Applied Biosystems). II. Sample requirements DNA in water, TE, or TE-4. III. Procedure Note: Before beginning this protocol, assure that the kit has been QCd as appropriate, and has not reached its expiration date. Record lot information on the Reagent Log Form. A. 1. 2. 3. Sample Preparation (Note: The following steps will take place at the analysts workbench.) Thaw the primer mix completely, then vortex and centrifuge briefly. Briefly vortex and centrifuge the Quantifiler PCR reaction mix. Fill out Quantifiler Set-Up Sheet Determine the number of reactions to be run. Each run should include: 16 standards (for the standard curve), 1 blank containing only the primer and reaction mix, and between 1 and 78 samples. Calculate the volume of each component needed to prepare the reactions, using the following table: Component Quantifiler Human Primer Mix (stored at -20oC) Quantifiler PCR Reaction Mix (stored at 4oC) Volume Per Reaction (L) 10.5 12.5
Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during the reagent transfers. 4. 5. 6. 7. Pipette the required volumes of components into an appropriately sized microcentrifuge tube. Vortex the PCR master mix, then centrifuge briefly. Transfer 23 L of the PCR master mix into each reaction well. Transfer 2 L of sample or standard to the appropriate wells. 1
DNA Quantitation by Real-time PCR using the Quantifiler Kit, Version 1.1 Effective Date: 11/5/2007

8. Seal the reaction plate with the Optical Adhesive Cover. (Note: The following steps will take place in the post-amplification instrument room) 9. B. 1. 2. Place the compression pad over the Optical Adhesive Cover with the foam side down and with the holes positioned directly over the reaction wells. Running the Reactions Open the SDS software by clicking on the icon. Under the File menu open a new file. A new document window should open on the screen. In the new document window, select: i. Assay Absolute Quantification ii. Container 96 wells clear plate iii. Template Quantifiler.sdt Click the OK button to accept the parameters. 3. 4. With the template open, verify that the plate grid has the correct number of wells (96) selected. Verify that the IPC and Quantifiler Human detectors are selected. Fill out the sample sheet. a. To Manually fill out the sample sheet: i. Select well ii. Enter name of sample iii. Repeat for all samples iv. Select all samples and click IPC and Quantifiler Human boxes v. In the task column, verify that Unknown is selected for all samples (the standards will already be set to Standard from the template) b. To Import samples from a text file: i. Under the File menu select Import ii. Navigate to the appropriate file iii. Click Import 5. Under the Instrument tab, open the Real-time tab and click Open/Close to rotate the instrument tray to the Out position. (Note: If the instrument is not already connected to the computer, then connect it by clicking the connect button.) Load the plate in the instrument tray. Click Start to rotate the instrument tray to the IN position and to start the run. Save the name files using the following convention: TP_2007Sep25.
6. 7.

C. 1. 2. 3. D. 1. 2. Data Analysis Once the run is complete, select Analysis >Analyze for the software to convert the raw data to analyzed data. In the Results tab, select the Standard Curve tab. In the Detector drop-down list, select the Quantifiler Human detector. Print out the Standard Curve and Amplification Plots. Evaluation of Standard Curve In the Grid Panel, select the wells for all standards. View the Standard Plot (Quantifiler Human setting) and Amplification Plot (Detector = IPC; Plot = Ct vs Well Position) for the standards to evaluate the performance of the standards. View the IPC CT values for the standard reactions and the calculated regression line slope and R2 values. a. CT values the expected CT value range for the IPC of the standards is between 20 and 30 cycles. i. If the IPC CT values for any of the standards fall outside of the recommended range, see the Technical Manager regarding appropriate remedial steps. b. R2 value 0.99 indicates a close fit between the standard curve regression line and the individual CT data points of quantification standard reactions. If the R2 value is <0.98 check the following: i. If the R2 value is <0.98, then consult with the Technical Manager regarding appropriate remedial steps. ii. An R2 value of <0.98 may be the result of one of the following: 1. Incorrect quantity values entered for quantification standards in the Well Inspector tab during plate document setup 2. Serial dilutions of quantification standards were made improperly 3. Operator error in the addition of samples to the reaction plate 4. Failure of the reactions containing the quantification standards c. Slope - Indicates the PCR amplification efficiency for the assay. A slope of -3.3 indicates 100% amplification. For the Quantifiler Human kit, the typical slope range is -2.9 to -3.3 with -3.1 being the average slope. i. If the slope falls outside of the recommended range, consult with the Technical Manager regarding appropriate remedial steps.
3.

E. 1. 2. Print Results In the Grid Pane, select all wells. In the Amplification Plot (under the Results tab), set the parameters as follows: a. Detector = IPC b. Plot = Ct vs Well Position Under the File menu, select Print Report. In the Print Report dialog box, click the Uncheck All button. following reports to print: a. Quantifiler Human b. Amplification Plot c. Standard Curve Plot Evaluation of Sample Results Evaluate the Internal Positive Control (IPC) data for all samples (including the blank sample) by selecting samples in the Grid Pane and viewing the IPC detector data in the Amplification Plot. If the IPC amplifies, use the following table to interpret the quantitation results: IPC (VIC Dye)
Amplification
3. 4.
Then select the
F. 1.
2.
Quantifiler Human (FAM Dye)

Amplification
Interpretation
DNA extract sufficient for STR analysis no apparent PCR inhibition
Recommended Action
Perform STR amplification with target amount of approximately 2ng of DNA Option for dilute extracts with a sufficient volume of extract: Concentrate sample extract (requantify when appropriate) Concentrate sample extract (re-quantify when appropriate) or Perform STR amplification with 20l of the sample extract
No amplification
Amplification
Amplifiable DNA may or may not be present in sample
3.
For samples where the IPC fails to amplify: 4

a. Select the well containing the sample b. Select the Multicomponent Plot icon and observe the amplification of the FAM dye (Quantifiler Human detector) and the VIC dye (IPC detector) c. Use the following table to interpret the quantitation results: Quantifiler Human (FAM Dye)
No amplification
IPC (VIC Dye)

No amplification
Interpretation
Failure of quantification reaction or complete inhibition
Recommended Action
Depending on the circumstances: Re-quantify 2l of extract or Make a 1:10 dilution of sample extract and requantify 2l of extract or Re-cleanup sample extract and re-quantify 2l of extract Perform STR amplification with target amount of 2ng of DNA Depending on the circumstances: Make a 1:10 dilution of sample extract and requantify 2l of extract or Perform STR amplification with less than 2ng of DNA (targeted amount of DNA) or Re-cleanup sample extract and re-quantify 2l of extract
Amplification (low CT and high Rn) Amplification (high CT and low Rn
No amplification No amplification
DNA extract sufficient for STR analysis no apparent PCR inhibition Partial PCR inhibition

Revisions and Corrections Log SOP: DNA Quantitation by Real-time PCR using the Quantifiler Kit Effective Date 11/5/2007 Description Version 1.0 replaced by Version 1.1. Changes incorporated into Version 1.1: Minor revisions of wording to eliminated redundancies and improve clarity F. Evaluation of Sample Results. In table, recommended actions for dilute samples revised to make re-quantification optional following concentration. Authored by McDonald / TJS

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This is an uncontrolled copy of a controlled document

This is an uncontrolled copy of a controlled document

This is an uncontrolled copy of a controlled document

This is an uncontrolled copy of a controlled document

This is an uncontrolled copy of a controlled document

This is an uncontrolled copy of a controlled document

This is an uncontrolled copy of a controlled document

This is an uncontrolled copy of a controlled document

This is an uncontrolled copy of a controlled document