© NYC OFFICE OF CHIEF MEDICAL EXAMINER FORENSIC BIOLOGY PROTOCOLS FOR FORENSIC STR ANALYSIS Approving Authority: Eugene Y. Lien, Technical Leader – Nuclear DNA Operations Working version as of 05/20/2016 Highlighted sections indicate a new revision to that procedure Table of Contents General Guidelines for DNA Casework ................................................................. 6 Laboratory organization .............................................................................................................. 6 Work Place Preparation .............................................................................................................. 6 Microcentrifuge tube and pipette handling ................................................................................. 6 Sample handling.......................................................................................................................... 7 Body fluid identification ............................................................................................................. 8 DNA Extraction Guidelines ........................................................................................................ 9 Extraction Negative Flow Charts .......................................................................................... 11 Controls for PCR analysis ......................................................................................................... 17 Concordant analyses and “duplicate rule” ................................................................................ 17 Exogenous DNA Policy ............................................................................................................ 20 Technical Deviations ................................................................................................................ 22 DNA storage ............................................................................................................................. 24 DNA Extraction ......................................................................................................25 Chelex Extraction from Blood and Buccal Swabs .................................................................... 25 Chelex Extraction from Soft Tissue (e.g. Fetus Samples) ........................................................ 27 Chelex DNA Extraction from Epithelial Cells ......................................................................... 28 Non-differential Chelex DNA Extraction from Semen Stains or Swabs .................................. 30 Differential Chelex DNA Extraction from Semen Stains or Swabs ......................................... 31 Controlled versions of Department of Forensic Biology Manuals only exist electronically on the Forensic Biology network. All printed versions are non-controlled copies. © NYC OFFICE OF CHIEF MEDICAL EXAMINER © NYC OFFICE OF CHIEF MEDICAL EXAMINER Differential Extraction from Semen Stains or Swabs utilizing the QIAcube and EZ1 ............ 35 Appendix A: QIAcube Loading Chart .................................................................................. 49 Appendix B: Retaining a substrate remains .......................................................................... 50 DNA Extraction from Hair ....................................................................................................... 51 Organic Extraction .................................................................................................................... 52 High Yield DNA Extraction ..................................................................................................... 65 Extraction of Exogenous DNA from Nails ............................................................................... 70 MagAttract DNA Extraction from Bloodstains and Exemplars ............................................... 75 A. Setting up M48 Test Batch and Saving Sample Name List ....................................... 75 B. Sample Preparation and Incubation ............................................................................ 75 C. BioRobot M48 Software and Platform Set-Up........................................................... 76 D. Importing Sample Names ........................................................................................... 80 E. Verifying Robot Set-Up and Starting the Purification ............................................... 80 F. Saving Extraction Report Page ................................................................................... 81 G. Post-Extraction Clean Up and UV Sterilization ......................................................... 82 H. BioRobot M48 Platform Diagram .............................................................................. 84 I. Troubleshooting .......................................................................................................... 85 Reduced Volume Magattract DNA Extraction from Bloodstains & Other Casework Samples86 A. Setting up M48 Test Batch and Saving Sample Name List ....................................... 86 B. Sample Preparation and Incubation ............................................................................ 86 C. BioRobot M48 Software and Platform Set-Up........................................................... 87 D. Importing Sample Names ........................................................................................... 92 E. Verifying Robot Set-Up and Starting the Purification ............................................... 93 F. Saving Extraction Report Page ................................................................................... 94 G. Post-Extraction ........................................................................................................... 94 H. Clean Up and UV Sterilization ................................................................................... 94 I. BioRobot M48 Platform Diagram .................................................................................. 96 J. Purification and Concentration ................................................................................... 97 K. Troubleshooting .......................................................................................................... 99 DNA Extraction of Bone Samples .......................................................................................... 101 Bone Processing .................................................................................................................. 101 Large Volume Demineralization Extraction Procedure with Qiagen M48 Low Elution ... 107 Microcon DNA Fast Flow DNA Concentration and Purification ...................119 I. LIMS Pre-Processing ....................................................................................................... 120 II. Assay Preparation ......................................................................................................... 121 III. LIMS Post Processing I ................................................................................................ 127 Quantifiler® Trio DNA Quantification Kit ...............................................129 IV. LIMS Pre-Processing ................................................................................................... 129 V. Assay Preparation ......................................................................................................... 131 VI. Software Operations ..................................................................................................... 134 VII. Exporting Results ......................................................................................................... 135 VIII. LIMS Post Processing I ............................................................................................ 136 IX. Interpretation ................................................................................................................ 137 QC Summary Flagging Guide ............................................................................................ 140 X. LIMS Post Processing II .............................................................................................. 141 General Guidelines for Fluorescent STR Analysis ...........................................142 Controlled versions of Department of Forensic Biology Manuals only exist electronically on the Forensic Biology network. All printed versions are non-controlled copies. © NYC OFFICE OF CHIEF MEDICAL EXAMINER © NYC OFFICE OF CHIEF MEDICAL EXAMINER Batch processing ................................................................................................................. 142 Sample handling.................................................................................................................. 142 Instrument and computer maintenance ............................................................................... 143 Identifiler Kit ........................................................................................................144 Identifiler Sample Preparation for Amplification ................................................................... 144 Identifiler – Sample and Amplification Set-up ................................................................... 145 Samples and Controls ......................................................................................................... 146 Thermal Cycling – all amplification systems ..................................................................... 148 Amplification Troubleshooting ........................................................................................... 150 Identifiler Analysis on the ABI 3130xl Genetic Analyzer...................................................... 154 A. Setting Up A 3130xl Run.......................................................................................... 154 B. Creating a Test Batch ............................................................................................... 158 C. Foundation Data Collection (Importing Plate Record)............................................. 160 D. Preparing and Running the DNA Samples ............................................................... 161 E. Denature/Chill - For All Systems After Sample Addition ....................................... 168 F. Turning the Oven on and Setting the Temperature .................................................. 168 G. Placing the Plate onto the Autosampler (Linking and Unlinking Plate) .................. 170 H. Viewing the Run Schedule ....................................................................................... 172 I. Collecting Data ............................................................................................................ 175 J. Re-injecting Plates........................................................................................................ 175 K. Water Wash and POP Change .................................................................................. 176 TROUBLESHOOTING GUIDE ........................................................................................ 177 YFiler KitTM ..........................................................................................................181 Amplification using the YfilerTM System ............................................................................... 181 I. General Information for Amplification ........................................................................ 181 II. Generation of Amplification Test Batches ............................................................... 182 III. PCR Amplification – Sample Preparation ................................................................ 182 IV. Thermal Cycling ....................................................................................................... 185 YfilerTM – Capillary Electrophoresis ...................................................................................... 189 A. Preparation of 3130xl Batch ..................................................................................... 189 B. Mastermix and Sample Addition for Yfiler™ .......................................................... 190 C. Denature/Chill - For Yfiler™ After Sample Addition: ............................................ 191 D. 3130xl Settings ......................................................................................................... 191 Minifiler Kit ..........................................................................................................193 Amplification using the Minifiler System .............................................................................. 193 I. General Information for AmpFℓSTR® MiniFiler™ PCR Amplification ..................... 193 II. Generation of Amplification Sets ............................................................................. 194 III. PCR Amplification – Sample Preparation ................................................................ 195 IV. Thermal Cycling ....................................................................................................... 197 Minifiler – Capillary Electrophoresis ..................................................................................... 200 A. Preparation of 3130xl batch ...................................................................................... 200 B. Master Mix and Sample Addition for MiniFiler™ .................................................. 200 C. Adding Samples:....................................................................................................... 201 D. Denature/Chill – For MiniFiler™ After Sample Addition: ...................................... 201 E. 3130xl Settings ......................................................................................................... 202 Genemapper ID Analysis ....................................................................................204 Controlled versions of Department of Forensic Biology Manuals only exist electronically on the Forensic Biology network. All printed versions are non-controlled copies. © NYC OFFICE OF CHIEF MEDICAL EXAMINER © NYC OFFICE OF CHIEF MEDICAL EXAMINER A. CREATING A NEW PROJECT .................................................................................. 204 B. ANALYSIS SETTINGS .............................................................................................. 206 C. VIEWING ANALYZED DATA ................................................................................. 208 D. SIZING ......................................................................................................................... 209 E. PLOT VIEWS .............................................................................................................. 211 F. EDITING ......................................................................................................................... 213 G. EDITING - REVIEWER .............................................................................................. 219 H. PRINTING AND ELECTROPHEROGRAM GENERATION ........................................ 221 Quality Flags ........................................................................................................................... 225 Editing Codes .......................................................................................................................... 228 ReRun Codes .......................................................................................................................... 229 Genemapper ID Analysis Method Editor Settings .................................................................. 230 Identifiler Analysis Settings:............................................................................................... 230 MiniFiler Analysis Settings: ............................................................................................... 231 YFiler Analysis Settings: .................................................................................................... 232 Genemapper ID-Troubleshooting Guide ................................................................................ 233 1. REDEFINING THE SIZE STANDARD ................................................................. 233 2. ADJUSTING THE ANALYSIS DATA START POINT AND STOP POINT RANGE ............................................................................................................................... 237 3. Genotypes Plot – Locus Specific Quality Flags ....................................................... 239 4. PRINTING................................................................................................................ 242 5. ALLELIC LADDER ................................................................................................ 243 6. ALLELE HISTORY ................................................................................................. 244 7. SAMPLE HISTORY ................................................................................................ 245 8. TYPOGRAPHICAL ERROR IN SAMPLE............................................................. 246 9. TABLE ERRORS ..................................................................................................... 246 References –Allelic Ladders, Controls, and Size Standards ................................................... 247 Identifiler Allelic Ladder .................................................................................................... 247 Identifiler Positive Control ................................................................................................. 248 LIZ-250-340 ........................................................................................................................ 249 MiniFiler Allelic Ladder ..................................................................................................... 250 MiniFiler Positive Control .................................................................................................. 251 YFiler Allelic Ladder .......................................................................................................... 252 YFiler Positive Control ....................................................................................................... 253 YFiler Size Standard (LIZ GS500) ..................................................................................... 254 Default Table and Plot Settings .............................................................................................. 255 STR Results Interpretation .................................................................................301 I. Allele Calling Criteria ...................................................................................................... 301 II. Manual Removal of Non Allelic Peaks ........................................................................ 302 III. Detection of Rare Alleles ............................................................................................. 306 IV. Interpretation of STR Data ........................................................................................... 307 V. Interpretation of controls .............................................................................................. 309 VI. Reporting Procedures ................................................................................................... 319 VII. Guidelines for Interpretation of Results ....................................................................... 321 VIII. Guidelines for reporting samples amplified with Identifiler for 31 cycles ............... 343 Additional Interpretations of Y-STR Results and Complex Y-STR Results .351 Controlled versions of Department of Forensic Biology Manuals only exist electronically on the Forensic Biology network. All printed versions are non-controlled copies. © NYC OFFICE OF CHIEF MEDICAL EXAMINER © NYC OFFICE OF CHIEF MEDICAL EXAMINER Population Frequencies for STR’s .....................................................................353 I. Random Match Probability for Autosomal STRs ............................................................ 353 II. Frequency for Y STRs.................................................................................................. 354 III. Combined Probability of Inclusion (CPI) for Mixtures ............................................... 356 Forensic Statistical Tool (FST) ...........................................................................358 I. A comparison profile must be available in order to use FST .......................................... 358 II. Sample Criteria for using the FST ............................................................................... 358 III. Hypothesis building...................................................................................................... 361 IV. User defined factors that affect the drop-out and drop-in rates.................................... 363 V. Instructions ................................................................................................................... 365 A. Creating Evidence, Comparison, and Known Contributor Files for FST ................ 365 B. FST Home Screen ..................................................................................................... 368 C. Uploading Files and Running FST ........................................................................... 373 D. Interpretation of Results ........................................................................................... 379 Sample Comparisons ...........................................................................................383 Autosomal STR Results .......................................................................................................... 383 Y-STR Results ........................................................................................................................ 389 Paternity Analysis ................................................................................................392 References .............................................................................................................394 DNA-View for Paternity and Kinship Analysis ................................................399 I. Creating a DNA-View Worksheet and Import Record .................................................... 399 II. Importing profiles into DNA-View .............................................................................. 401 III. Performing Paternity or Kinship Analysis ................................................................... 408 IV. Importing Raw Data ..................................................................................................... 423 V. Troubleshooting DNA-View ........................................................................................ 430 Appendix ...............................................................................................................445 Identifiler loci and approximate size range ......................................................................... 445 MiniFiler loci and approximate size range ......................................................................... 446 YFiler loci and approximate size range .............................................................................. 447 Macro Filter functions - Allele Filters ................................................................................ 448 Controlled versions of Department of Forensic Biology Manuals only exist electronically on the Forensic Biology network. All printed versions are non-controlled copies. © NYC OFFICE OF CHIEF MEDICAL EXAMINER © NYC OFFICE OF CHIEF MEDICAL EXAMINER FORENSIC BIOLOGY PROTOCOLS FOR FORENSIC STR ANALYSIS GENERAL GUIDELINES FOR DNA CASEWORK DATE EFFECTIVE APPROVING AUTHORITY PAGE 12-24-2015 NUCLEAR DNA TECHNICAL LEADER 6 OF 451 General Guidelines for DNA Casework Laboratory organization 1. To minimize the potential for carry-over contamination, the laboratory is organized so that the areas for DNA extraction, PCR set-up, and handling amplified DNA are physically isolated from each other. 2. Based on need, microcentrifuge tube racks have been placed in sample handling areas. These racks should only leave their designated area to transport samples to the next designated area. Immediately after transporting samples, the racks should be cleaned and returned to their designated area. 3. Dedicated equipment such as pipettors should not leave their designated areas. Only the samples in designated racks should move between areas. 4. Analysts in each work area must wear appropriate personal protective equipment (PPE). Contamination preventive equipment (CPE) must be worn where available. All PPE and CPE shall be donned in the bio-vestibules. Required PPE and CPE for each laboratory are posted conspicuously in each bio- vestibule. Work Place Preparation 1. Apply 10% bleach followed by water and/or 70% Ethanol to the entire work surface, cap opener, pipettes, and computer keyboard/mouse (when appropriate). 2. Obtain clean racks and cap openers, and irradiated microcentrifuge tubes, and UltraPure water from storage. Arrange work place to minimize crossover. Position gloves nearby with 10% Bleach/70% Ethanol/water in order to facilitate frequent glove changes and cleaning of equipment. Microcentrifuge tube and pipette handling 1. Microcentrifuge tubes, Microcon collection tubes, Dolphin tubes, and M48 tubes must be irradiated prior to use. Back to Table of contents Controlled versions of Department of Forensic Biology Manuals only exist electronically on the Forensic Biology network. All printed versions are non-controlled copies. © NYC OFFICE OF CHIEF MEDICAL EXAMINER © NYC OFFICE OF CHIEF MEDICAL EXAMINER FORENSIC BIOLOGY PROTOCOLS FOR FORENSIC STR ANALYSIS GENERAL GUIDELINES FOR DNA CASEWORK DATE EFFECTIVE APPROVING AUTHORITY PAGE 12-24-2015 NUCLEAR DNA TECHNICAL LEADER 7 OF 451 2. Avoid splashes and aerosols. Centrifuge all liquid to the bottom of a closed microcentrifuge tube before opening it. 3. Avoid touching the inside surface of the tube caps with pipettors, gloves, or lab coat sleeves. 4. Use the correct pipettor for the volume to be pipetted. For pipettors with a maximum volume of 20µL or over, the range begins at 10% of its maximum volume (i.e., a 100µL pipette can be used for volumes of 10-100µL). For pipettors with a maximum volume of 10µL or under, the range begins at 5% of its maximum volume (i.e., a 10µL pipette can be used for volumes of 0.5-10µL). 5. Filter pipette tips must be used when pipetting DNA and they should be used, whenever possible, for other reagents. Use the appropriate size filter tips for the different pipettors; the tip of the pipette should never touch the filter. 6. Always change pipette tips between handling each sample. 7. Never “blow out” the last bit of sample from a pipette. Blowing out increases the potential for aerosols, this may contaminate a sample with DNA from other samples. The accuracy of liquid volume delivered is not critical enough to justify blowing out. 8. Discard pipette tips if they accidentally touch the bench paper or any other surface. 9. Wipe the outside of the pipette with 10% bleach solution followed by a 70% ethanol solution if the barrel goes inside a tube. Sample handling 1. Samples that have not yet been amplified should never come in contact with equipment in the amplified DNA work area. Samples that have been amplified should never come in contact with equipment in the unamplified work area. Back to Table of contents Controlled versions of Department of Forensic Biology Manuals only exist electronically on the Forensic Biology network. All printed versions are non-controlled copies. © NYC OFFICE OF CHIEF MEDICAL EXAMINER © NYC OFFICE OF CHIEF MEDICAL EXAMINER FORENSIC BIOLOGY PROTOCOLS FOR FORENSIC STR ANALYSIS GENERAL GUIDELINES FOR DNA CASEWORK DATE EFFECTIVE APPROVING AUTHORITY PAGE 12-24-2015 NUCLEAR DNA TECHNICAL LEADER 8 OF 451 2. The DNA extraction and PCR setup of evidence samples should be performed at a separate time from the DNA extraction and PCR setup of exemplars. This precaution helps to prevent potential cross-contamination between evidence samples and exemplars. 3. Use disposable bench paper to prevent the accumulation of human DNA on permanent work surfaces. 10% bleach followed by 70% ethanol should always be used to decontaminate all work surfaces before and after each procedure. 4. Limit the quantity of samples handled in a single run to a manageable number. This precaution will reduce the risk of sample mix-up and the potential for sample-to-sample contamination. 5. Change gloves frequently to avoid sample-to-sample contamination. Change them whenever they might have been contaminated with DNA and whenever exiting a sample handling area. 6. Make sure the necessary documentation is completely filled out, and that the analyst’s ID is properly associated with the notations. Body fluid identification 1. The general laboratory policy is to identify the stain type (i.e., blood, semen, or saliva) before individualization is attempted on serious cases such as sexual assaults, homicides, robberies, and assaults. However, circumstances may exist when this will not be possible. For example, on most property crime cases when a swab of an item is submitted for testing, the analyst will cut the swab directly for individualization rather than testing the swab for body fluid identification. 2. A positive screening test for blood followed by the detection of DNA in a real-time PCR assay is indicative of the presence of human blood. Back to Table of contents Controlled versions of Department of Forensic Biology Manuals only exist electronically on the Forensic Biology network. All printed versions are non-controlled copies. © NYC OFFICE OF CHIEF MEDICAL EXAMINER © NYC OFFICE OF CHIEF MEDICAL EXAMINER FORENSIC BIOLOGY PROTOCOLS FOR FORENSIC STR ANALYSIS GENERAL GUIDELINES FOR DNA CASEWORK DATE EFFECTIVE APPROVING AUTHORITY PAGE 12-24-2015 NUCLEAR DNA TECHNICAL LEADER 9 OF 451 3. High Copy Number (HCN) testing is performed when the samples have a quantitation value ≥10.0 pg/uL for YFiler (at least 100 pg per amp), ≥20 pg/µL for Identifiler 28 cycles (at least 100 pg per amp) or ≥10 pg/uL for Minifiler (at least 100pg per amp). High Sensitivity DNA testing (Identifiler 31 cycles) can be performed if samples have a quantitation value of less than 7.5 pg/µL (or 20 pg/µL) and greater than 1 pg/µL. DNA Extraction Guidelines Slightly different extraction procedures may be required for each type of specimen. Due to the varied nature of evidence samples, the user may need to modify procedures. 1. All tube set-ups must be witnessed/ confirmed prior to starting the extraction. 2. Use lint free wipes or a tube opener to open tubes containing samples; only one tube should be uncapped at a time. 3. When pouring or pipetting Chelex solutions, the resin beads must be distributed evenly in solution. This can be achieved by shaking or vortexing the tubes containing the Chelex stock solution before aliquoting. 4. For pipetting Chelex, the pipette tip used must have a relatively large bore – 1 mL pipette tips are adequate. 5. Be aware of small particles of fabric, which may cling to the outside of tubes. 6. With the exception of the Mitochondrial DNA Team, two extraction negative controls (E- neg) must be included with each batch of extractions to demonstrate extraction integrity. The first E-Neg will typically be subjected to a micro-con and will be consumed to ensure that an E-neg associated with each extraction set will be extracted concurrently with the samples, and run using the same instrument model and under the same or more sensitive injection conditions as the samples. The second E-Neg will ensure that the samples in that extraction set can be sent on for further testing in another team or in a future kit. In the Mitochondrial DNA Team, only one extraction negative control is needed. Refer to the end of this section for flow charts. Back to Table of contents Controlled versions of Department of Forensic Biology Manuals only exist electronically on the Forensic Biology network. All printed versions are non-controlled copies. © NYC OFFICE OF CHIEF MEDICAL EXAMINER © NYC OFFICE OF CHIEF MEDICAL EXAMINER FORENSIC BIOLOGY PROTOCOLS FOR FORENSIC STR ANALYSIS GENERAL GUIDELINES FOR DNA CASEWORK DATE EFFECTIVE APPROVING AUTHORITY PAGE 12-24-2015 NUCLEAR DNA TECHNICAL LEADER 10 OF 451 The extraction negative control contains all solutions used in the extraction process but no biological fluid or sample. For samples that will be amplified in Identifiler (28 or 31 cycles), YFiler or MiniFiler, the associated extraction negative should be re-quantified to confirm any quantitation value of 0.2 pg/µL or greater. 7. If a sample is found to contain less than 20 pg/µL of DNA, then the sample should not be amplified in Identifiler (28 cycles); if a sample is found to contain less than 10 pg/µL of DNA, then the sample should not be amplified in YFiler; if a sample is found to contain less than 10 pg/µL of DNA, then the sample should not be amplified in MiniFiler. Samples that cannot be amplified may be re-extracted, reported as containing insufficient DNA, concentrated using a Microcon (see Section 3 of the STR manual), or possibly submitted for High Sensitivity testing. The interpreting analyst shall consult with a supervisor to determine how to proceed. Other DNA samples may also be concentrated and purified using a Microconif the DNA is suspected of being degraded or shows inhibition or background fluorescence during quantitation. Samples that are 1 pg/µL to 20pg/µL may be submitted for High Sensitivity testing with a supervisor’s permission. 8. After extraction, the tubes containing the unamplified DNA should be transferred to a box and stored in the appropriate refrigerator or freezer. The tubes should not be stored in the extraction racks. 9. All tubes must have a LIMS label and/or the complete case number, sample identifier and IA initials on the side of the tube. This includes aliquots submitted for quantitation. Back to Table of contents Controlled versions of Department of Forensic Biology Manuals only exist electronically on the Forensic Biology network. All printed versions are non-controlled copies. © NYC OFFICE OF CHIEF MEDICAL EXAMINER