Investigator Quantiplex HYres Kit

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1 These specifications are for the procurement of a validation study at GBI Division of Forensic Sciences facilities of two commercially available kits used in forensic DNA analysis. The two kits to be validated according to FBI Quality Assurance Standards for DNA Analysis Laboratories and ISO17025 standards are the Qiagen Investigator Quantiplex HYres DNA quantitation kit and the Life Technologies AmpFlSTR Globalfiler Kit. The vendor quote must include ALL necessary supplies (other than those specifically outlined in the specification below that will be provided by the GBI), travel expenses, and all other costs associated with the validation of these two kits. The validation studies must be completed and all associated documentation provided to the GBI Division of Forensic Sciences by December 31, The GBI Division of Forensic Sciences has three (3) locations that perform DNA casework: GBI Headquarters Laboratory, 3121 Panthersville Rd, Decatur GA GBI Coastal Regional Laboratory, 921 Mohawk St, Savannah, GA GBI Southwestern Regional Laboratory, 330 Veterans Parkway, Moultrie, GA, Vendors must respond to all requirements for each of the two validation studies outlined below and must provide separate pricing for validation of each individual kit type, along with total pricing for all items included in this Request for Quotations. Investigator Quantiplex HYres Kit 1. This validation is to be completed in conjunction with the validation of the AmpFlSTR Globalfiler Kit. The Contractor must be able to meet the validation specifications of both the Investigator Quantiplex HYres Kit validation and the AmpFlSTR Globalfiler Kit validation. 2. The Contractor must provide all consumable supplies and reagents in the volumes necessary for the completion of the validation project at each biology laboratory at the Georgia Bureau of Investigation Division of Forensic Sciences: Headquarters (Decatur, GA), Southwestern (Moultrie, GA) and Coastal (Savannah, GA). The Contractor must provide all consumable supplies and reagents in the volumes necessary for the completion of the subsequent performance checks at the following biology laboratories at the Georgia Bureau of Investigation Division of Forensic Sciences: Headquarters and Coastal lab. Upon completion of the validation projects and performance checks all purchased reagents and consumables will remain with the biology laboratories of the Georgia Bureau of Investigation Division of Forensic Sciences. 3. During the validation plan for the Investigator Quantiplex HYres Kit using the Applied Biosystems 7500 system and HID Real-Time PCR Analysis Software v1.2 conducted by the Contractor all samples will be quantified using the manufacturer s recommended conditions. Applied Biosystems 7500 and HID Real-Time PCR Analysis Software v1.2 are currently validated for casework use. All AmpFlSTR Globalfiler Kit and with AmpFlSTR Yfiler Kit amplifications will be performed by the 9700 thermal cycler.

2 4. The validation plan for the Investigator Quantiplex HYres Kit using the Applied Biosystems include a sensitivity study. The sensitivity study will be conducted using at minimum five (4 male and 1 female) DNA stock solutions which will be supplied by the Georgia Bureau of Investigation Division of Forensic Sciences Biology Units. Samples will be quantified. Serial dilutions will then be prepared from each DNA stock: 20, 5, 1.25, 0.313, 0.078, 0.019, , , , , , , , ng/µl. Serial dilutions will be quantified in triplicate. Evaluation of data will include establishing the limit of quantification and the limit of detection. The results must meet, at a minimum, the manufacturer s suggested values for sensitivity. All sample dilutions that quantify as undetected will be amplified with AmpFlSTR Globalfiler Kit and with AmpFlSTR Yfiler Kit using the maximum allowable template of DNA input. 5. The validation plan for the Investigator Quantiplex HYres Kit using the Applied Biosystems include a single source non-probative/mock sample study. Samples for this study will be supplied by the Georgia Bureau of Investigation Division of Forensic Sciences Biology Units. 150 non-probative samples (50/laboratory) will be quantified using the Quantifiler Human DNA Quantification kit, the Quantifiler Y Human Male DNA Quantification Kit (male samples only), and the Investigator Quantiplex HYres Kit. Evaluation of data will be done by comparing quantification results of the three quantification kits. The following comparisons will be assessed: a. Percentage difference of quantification values of human and male targets. b. Average male/human DNA ratio from the Investigator Quantiplex HYres Kit as compared to the male/human DNA ratio developed from the combination of quantification values from the Quantifiler Human DNA Quantification kit and the Quantifiler Y Human Male DNA Quantification Kit. 6. The validation plan for the Investigator Quantiplex HYres Kit using the Applied Biosystems confirm the correlation of STR profile outcomes with DNA quantification results. At minimum three single-source DNA samples shall be created (previously extracted samples may be used). The samples will be quantified in triplicate prior creating the dilutions. Samples will be diluted in triplicate to the following dilutions: 1000, 500, 250, 187.5, 125, 93.8, 62.5, 46.9, 31.3, 23.4, 15.6 to 7.8 pg/µl. Samples will be quantified again to confirm the amount of DNA in each sample. After quantity confirmation, 2 µl of each dilution, amplification will be performed using AmpFlSTR Globalfiler Kit and using AmpFlSTR Yfiler Kit to enable testing from 2 ng to 15.6 pg of template DNA. All samples will be injected 1 µl for 15 seconds onto an Applied Biosystems 3500 Genetic Analyzer DC v2.0 and analyzed using GeneMapper ID-X v.1.4 software. Evaluation of the data will include number of detected alleles, peak heights, and average peak heights in correlation with the DNA template. 7. The validation plan for the Investigator Quantiplex HYres Kit using the Applied Biosystems

3 include a DNA mixture study. At minimum two male (M) and one female (F) donor DNA stocks (M1, M2, F) will be provided by the Georgia Bureau of Investigation Division of Forensic Sciences Biology Units. These DNA stocks will be quantified. Samples will be diluted and grouped into two different series sets. Series 1 will include the following six DNA ratios: 1:0, 1:1, 1:5, 1:10, 1:20 and 0:1 keeping the male concentration at ng/µl. Series 2 will include the following six DNA ratios: 1:0, 1:50, 1:100, 1:500, 1:1000 and 0:1 keeping the male concentration at ng/µl. These series will be comprised of the following samples: M1+F, M2+F. Series will be quantified in triplicate. Evaluation of data will be done by comparing the expected to the observed quantification values of the human and male targets for all series sets. 8. The validation plan for the Investigator Quantiplex HYres Kit using the Applied Biosystems include a study to assess the correlation between male DNA quantification results and male contribution in autosomal STR profiles. At minimum, twenty non-probative and mock mixture samples will be provided by the Georgia Bureau of Investigation Division of Forensic Sciences Biology Units. These samples will be quantified. The male to human (M/H) DNA ratios will be calculated by dividing the male DNA quantification results by the human DNA quantification results and will be expressed as percentages. Samples will be amplified using the AmpFlSTR Globalfiler Kit (using an optimal target range established in other studies within this validation), injected 1 µl for 15 seconds onto an Applied Biosystems 3500 Genetic Analyzer DC v2.0 and analyzed using GeneMapper ID-X v.1.4 software. The percentage of male DNA from Amelogenin will be calculated using the formula % Male = y/[(x y)/2 + y] where x is the peak height from Amelogenin X, and y the peak height from Ameloginin Y. The % Male and the M/H DNA ratios will be compared. Female and male alleles detected will be recorded. Samples will also be amplified using the AmpFlSTR Yfiler Kit (using an optimal target range established in another study within this validation), injected 1 µl for 15 seconds onto an Applied Biosystems 3500 Genetic Analyzer DC v2.0 and analyzed using GeneMapper ID-X v.1.4 software. This study will result in the establishment of an M/H DNA ratio cutoff to proceed to autosomal amplification vs YSTR amplification. 9. The validation plan for the Investigator Quantiplex HYres Kit using the Applied Biosystems include an inhibition study. The Contractor shall supply the following for this study: humic acid, tannic acid and hematin. Humic acid will be added to designated sample wells on the quantification plate in the following concentrations: 0, 5, 10, 12.5, 15, 17.5, 20, 25, 30, 40 and 50 ng/µl in the 20 µl reaction. Tannic acid will be added to designated sample wells on the quantification plate in the following concentrations: 0, 50, 100, 150, 175, 200, 225, 250, 275, 300, 325 and 350 ng/µl in the 20 µl reaction. Hematin will be added to designated sample wells on the quantification plate in the following concentrations: 0, 25, 50, 75, 80, 90, 100, 110, 120, 130, 140 and 150 µm in the 20 µl reaction. Plates will remain at room temperature for 24 h to permit the evaporation of the liquids pipetted in each well. The Investigator Quantiplex HYres Kit reagents will then be added to each well, allowed to sit for 10 minutes to resuspend the inhibitors before the addition of 2 µl of the control standard. Tolerance to the inhibitors will be assessed by evaluating the average cycle threshold values shift from normal (normal will be

4 established in other studies of this validation) for the human, male and internal control targets for each inhibitor concentration. 10. The validation plan for the Investigator Quantiplex HYres Kit using the Applied Biosystems include an accuracy study using the current NIST reference samples. National Institute of Standards & Technology Standard Reference Material sample will be purchased by the Contractor. The NIST samples will be diluted, at minimum, to 1/10, 1/100 and 1/1000 in TE. The neat NIST sample and the dilutions will be quantified in triplicate. Evaluation of data will include comparison (i.e. percentage difference) to the reported NIST sample DNA quantification value. Successful completion of this study will demonstrate the laboratory s compliance with standard of the Quality Assurance Standards for Forensic DNA Testing Laboratories. 11. The validation plan for the Investigator Quantiplex HYres Kit using the Applied Biosystems include a study on the reproducibility of the standard curve. This study may be obtained from data previously generated and collected in other studies within this validation. However, it will be the responsibility of the Contractor to organize and summarize the data in order to address the successful completion of the following reproducibility studies: a. Reproducibility of standard curve from different standard lot numbers. b. Reproducibility of standard curve cycle threshold (Ct) values for the human, male and internal control targets. i. Average of these Ct values ii. Standard deviation of these Ct values c. Reproducibility of the standard curve slope for the human and the male targets. i. Range of slopes produced ii. Range of amplification efficiency d. Reproducibility of the R 2 values for the human and the male targets. i. Average of R 2 values produced ii. Range of R 2 values produced e. Reproducibility of the Y-intercept for the human and the male targets. i. Average of Y-intercept values produced ii. Range of Y-intercept values produced f. Assessment of the linearity/reliability of the standard curve beyond the 20 ng/ µl data point. g. Reproducibility/stability of a set of standards over time. 12. The validation plan for the Investigator Quantiplex HYres Kit using the Applied Biosystems include a contamination study. This study may be obtained from data previously generated and collected in other studies listed above. However, it will be the responsibility of the Contractor to organize and summarize the data.

5 13. The Contractor must provide on-site training for 41 analysts at the Headquarters Biology section, 9 DNA analysts at the Coastal Regional Biology section, and 4 analysts at the Moultrie Regional Biology section to cover all aspects of the validation including but not limited to; all results and analysis involved in the validation study and the chemistry of the Investigator Quantiplex HYres Kit. This training must consist of a combination of bench work and lecture material. 14. The validation will be completed using one Applied Biosystems 7500 system at each of the following biology laboratories of the Georgia Bureau of Investigation Division of Forensic Sciences: Headquarters, Southwestern, and Coastal labs. The Contractor will be responsible for conducting a performance check using the Investigator Quantiplex HYres Kit on two additional Applied Biosystems 7500 systems at the Headquarters Biology section and one additional Applied Biosystems 7500 system at the Coastal Biology section. The performance checks must include, at a minimum, a sensitivity study, single source non-probative/mock sample study, reproducibility study and contamination study. The performance check may be composed of samples previously used in the validation study. 15. The Contractor must provide a Standard Operating Procedure (SOP) based on the validation study results. This document should include results, data, charts and information obtained from the validation study which addresses the following aspects: a. Introduction to the chemistry, instrument and analysis software involved. b. QA/QC recommendations for the chemistry. c. Protocol for DNA quantification with the Investigator Quantiplex HYres Kit that includes quantification set up and Applied Biosystems 7500 parameters. d. Protocol includes parameters for acceptable quantification results such as the following: standard curve slope ranges, standard curve R 2 ranges, standard curve Y-intercept ranges. 16. Contractor should provide estimated time of completion of laboratory portion of work 17. Contractor should provide estimated time of completion of analysis of data and submission of final results. 18. The Contractor must provide electronic copies of all data, summaries, charts, tables, worksheets and other written documents involved with the validation study. Also included must be electronic copies of the validation report, summary of the results and literature references. A CD of the data files and any other electronic data must also be provided. 19. Contractor must submit resume of the personnel whom will perform the validation. 20. Contractor must be an ISO ASCLD-LAB accredited vendor. 21. Travel, lodging and food expenses for vendor personnel conducting the validation studies will be included in the bid amount.

6 AmpFlSTR Globalfiler Kit Validation 1. This validation is to be completed in conjunction with the validation of the Investigator Quantiplex HYres Kit. The Contractor must be able to meet the validation specifications of both the Investigator Quantiplex HYres Kit validation and the AmpFlSTR Globalfiler Kit validation. 2. The Contractor must provide all consumable supplies and reagents in the volumes necessary for the completion of the validation project at each biology laboratory at the Georgia Bureau of Investigation Division of Forensic Sciences: Headquarters (Decatur, GA), Southwestern (Moultrie, GA) and Coastal (Savannah, GA). The Contractor must provide all consumable supplies and reagents in the volumes necessary for the completion of the subsequent performance checks at the Headquarters biology section at the Georgia Bureau of Investigation Division of Forensic Sciences. Upon completion of the validation projects and performance checks all purchased reagents and consumables will remain with the biology laboratories of the Georgia Bureau of Investigation Division of Forensic Sciences. 3. During the validation plan for the AmpFlSTR Globalfiler Kit using the Applied Biosystems 3500 Genetic Analyzer DC v2.0 and GeneMapper ID-X v.1.4 software conducted by the Contractor all samples will be amplified with the 9700 thermal cycler using the manufacturer s recommended conditions. Applied Biosystems 3500 Genetic Analyzer DC v2.0 and GeneMapper ID-X v.1.4 software are currently validated for casework use. 4. The validation plan for the AmpFlSTR Globalfiler Kit using the Applied Biosystems 3500 Genetic Analyzer DC v2.0 Genetic Analyzer and GeneMapper ID-X v.1.4 software conducted by the Contractor must include an allelic ladder precision study. The allelic ladder precision study must be performed using, at a minimum, five injections of the allelic ladder in four different wells of a 96 well optical plate to assess the variation observed in the base pair size of all alleles for each locus of the ladder. The completed analysis must include the following generated statistics: minimum base pair size, maximum base pair size, average base pair size, and standard deviation of base pair size. The results must meet, at a minimum, the manufacturer s suggested values for precision. 5. The validation plan for the AmpFlSTR Globalfiler Kit using the Applied Biosystems 3500 must include a minimum threshold study in order to determine the level of background noise detected. This study must consist of, at a minimum, nine negative amplification control samples injected at 15 seconds analyzed using a Peak Threshold value of 1 RFU for each of the four dye channels. The final analysis will consist of establishment of the maximum RFU, the average RFU, the standard deviation, limit of detection, and the limit of quantization for each dye. The successful completion of this study will establish the minimum threshold value to use in casework analysis and must meet, at a minimum, the manufacturer s recommended threshold.

7 6. The validation plan for the AmpFlSTR Globalfiler Kit using the Applied Biosystems 3500 must include a sensitivity study. This sensitivity study will be conducted using Human Genomic DNA. This DNA must be diluted to a final concentration ranging from of ng/μl to 2.5 ng/μl and re-quantified (using the Investigator Quantiplex HYres Kit) to verify the concentration. The final concentration of the input DNA in the amplification will be the following: 5.0ng, 2.5ng, 1.0ng, 0.5ng, 0.25ng, 0.1ng, 0.05ng, 0.025ng, 0.010ng, and 0.005ng. Each of these will be amplified in triplicate and injected 1µL for 15 seconds. Evaluation of the data will include peak height imbalance, off-scale peaks, pull-up, minus stutter, plus stutter, minus A, background and artifacts. The successful completion of the study will provide information used to establish a target range of amplification, a suggested optimal amplification target (OAT), interpretation guidelines including stochastic threshold and allele calling thresholds, minus stutter percentages for each loci, and plus stutter percentages for each loci. 7. The validation plan for the AmpFlSTR Globalfiler Kit using the Applied Biosystems 3500 must include a mixture study. The Contractor will supply the male and female Human Genomic DNA used to generate the mixed samples in the following ratios: 19:1, 9:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:9, and 1:19. For each of these ratios, the OAT will be amplified and injected 1µL for 15 seconds. Analysis of the data will include which ratios produced profiles where all expected alleles were detected and which ratios allow for the distinction between the major and minor DNA contributors. The results should be concordant with the results obtained in the AmpFlSTR Globalfiler Kit mixture developmental validation studies. 8. The validation plan for the AmpFlSTR Globalfiler Kit using the Applied Biosystems 3500 must include a complex mixture study. The Contractor will supply the male and female Human Genomic DNA used to generate mixed samples of 3 and 4 persons in the following ratios: 1:1:1, 3:1:1, 5:1:1, 3:2:1, 4:2:1, 8:2:1, 1:1:1:1, 6:2:1:1, 6:2:2:1, 8:2:2:1, 10:1:1:1, and 10:2:1:1. For each of these ratios, the OAT will be amplified and injected 1µL for 15 seconds. Analysis of the data will include which ratios produced profiles where all expected alleles were detected and which ratios allow for the distinction between the major and minor DNA contributors. 9. The validation plan for the AmpFlSTR Globalfiler Kit using the Applied Biosystems 3500 must include a concordance study using the current NIST reference samples. A minimum of 10 National Institute of Standards & Technology Standard Reference Material samples will be purchased by the Contractor. Each one of these samples will be quantified (using the Investigator Quantiplex HYres Kit) and amplified using the OAT. The correct genotypes must be obtained from all NIST reference samples tested. In addition, the results should demonstrate well balanced alleles as well as similar peak heights observed across all profiles. Successful completion of this study will demonstrate the laboratory s compliance with Standard of the Quality Assurance Standards for Forensic DNA Testing Laboratories.

8 10. The validation plan for the AmpFlSTR Globalfiler Kit using the Applied Biosystems 3500 must include a concordance study using approximately 90 non-probative samples (30/laboratory) which will be supplied by the Georgia Bureau of Investigation Division of Forensic Sciences Biology Units. These samples will be a range of type and quality. The DNA extracts provided will be quantified (using the Investigator Quantiplex HYres Kit) and amplified using the OAT with the AmpFlSTR Globalfiler Kit. The samples will be injected on the Applied Biosystems 3500 Genetic Analyzer DC v2.0 Genetic Analyzer 1µL for 15 seconds. Successful completion of this study will demonstrate that concordant results were obtained from these samples amplified with the AmpFlSTR Globalfiler Kit, run on the Applied Biosystems 3500 Genetic Analyzer DC v2.0 Genetic Analyzer and analyzed with the GeneMapper ID-X v.1.4 software when compared to results previously obtained from these samples amplified with the AmpFlSTR Identifiler Amplification Kit, run on the Applied Biosystems 3500 Genetic Analyzer DC v2.0 Genetic Analyzer and analyzed with GeneMapper ID-X v.1.4 software. 11. The validation plan for the AmpFlSTR Globalfiler Kit using the Applied Biosystems 3500 must include a reproducibility study. This study may be obtained from data previously generated and collected in other studies listed above. However, it will be the responsibility of the Contractor to organize and summarize the data in order to address the successful completion of the following reproducibility studies: a. Reproducibility of the genotype obtained when aliquots of the same DNA sample are amplified repeatedly. b. Reproducibility of the genotype obtained when the same lot of positive control is amplified repeatedly. c. Reproducibility of the genotype obtained for samples prepared and typed in another laboratory. d. Reproducibility of genotype obtained for mixed samples. e. Reproducibility of injection of different aliquots of the same sample in different capillaries. f. Reproducibility of the base pair sizes estimated for all alleles of all loci from the allelic ladder of the AmpFlSTR Globalfiler Kit run in different wells. 12. The validation plan for the AmpFlSTR Globalfiler Kit using the Applied Biosystems 3500 must include a contamination study. This study may be obtained from data previously generated and collected in other studies listed above. However, it will be the responsibility of the Contractor to organize and summarize the data. 13. The Contractor must provide on-site training for 35 DNA analysts at the Headquarters Biology section, 7 DNA analysts at the Coastal Regional Biology section, and 4 analysts at the Moultrie Regional Biology section to cover all aspects of the validation including but not limited to; all results and analysis involved in the validation study and the chemistry of the AmpFlSTR

9 Globalfiler PCR Amplification Kit. This training must consist of a combination of bench work and lecture material. 14. The validation will be completed using one Applied Biosystems 3500 Genetic Analyzer DC v2.0 at each of the following biology laboratories of the Georgia Bureau of Investigation Division of Forensic Sciences: Headquarters, Southwestern, and Coastal labs. The Contractor will be responsible for conducting a performance check using the AmpFlSTR Globalfiler PCR Amplification Kit on four additional Applied Biosystems 3500 Genetic Analyzers at the Headquarters Biology section. The performance check must include, at a minimum, a sensitivity study, concordance study, reproducibility study and contamination study. The performance check may be composed of samples previously used in the validation study. 15. The Contractor must provide a Standard Operating Procedure (SOP) based on the validation study results. This document should include results, data, charts and information obtained from the validation study which addresses the following aspects: a. Introduction to the chemistry, instrument and analysis software involved. b. QA/QC recommendations for the chemistry. c. Protocol for DNA amplification with the AmpFlSTR Globalfiler Kit that includes amplification set up and thermal cycler parameters. d. Protocol for detection of the PCR product on the Applied Biosystems 3500 Genetic Analyzer DC v2.0 genetic analyzer including matrix standardization, instrument preparation, sample preparation and detection. 16. Contractor should provide estimated time of completion of laboratory portion of work 17. Contractor should provide estimated time of completion of analysis of data and submission of final results. 18. The Contractor must provide electronic copies of all data, summaries, charts, tables, worksheets and other written documents involved with the validation study. Also included must be electronic copies of the validation report, summary of the results and literature references. A CD of the data files and any other electronic data must also be provided. 19. Contractor must submit resume of the personnel whom will perform the validation. 20. Contractor must be an ISO ASCLD-LAB accredited vendor. 21. Travel, lodging and food expenses for vendor personnel conducting the validation studies will be included in the bid amount.

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