GlobalFiler TM Extra Cycle Evaluation Report

Transcription

1 GlobalFiler TM Extra Cycle Evaluation Report Table of Content Chapter 1 Summary 1. Introduction Systems, Kits and Conditions Studies... 4 Chapter 2 Minimum Threshold and Background Study 1. Study Overview Experimental Design and Data Analysis Results Conclusions Chapter 3 Sensitivity and Stochastic Study 1. Study Overview Experimental Design and Data Analysis Results Conclusions Attachment Chapter 4 Inhibited Sample Study 1. Study Overview Experimental Design and Data Analysis Results Conclusion Attachment Chapter 5 Degraded Sample Study 1. Study Overview Experimental Design and Data Analysis Results Conclusions Attachment Page 1

2 Chapter 1 Summary Contents This chapter provides a summary for the evaluation of the GlobalFiler extra PCR cycle protocol, using GlobalFiler PCR Amplification Kit, Applied Biosystems 3500xl Genetic Analyzer, and GeneMapper ID-X Software v Introduction 2. Systems, Kits and Conditions 3. Studies 1. Introduction There can be challenges in getting full profiles with bone samples as this type of sample usually yields a low quantity of degraded DNA, the HID team decided to evaluate extra PCR cycles as a potential alternative workflow to improve the genotyping result in this specific situation where customers have low amount of degraded DNA samples. While the GlobalFiler PCR Amplification Kit was optimized at 29 cycles with 0.5-1ng input, the extra cycle validation study included 29, 30, and 31 cycles with different sample types and input amount. The goal of this study was to evaluate the outcome of GlobalFiler PCR amplification at 30 and 31 cycles with different sample types and inputs analyzed on Applied Biosystems 3500xl Genetic Analyzer and with GeneMapper ID-X v1.5 Software. Page 2

3 Chapter 1: Summary 2. Systems, Kits and Conditions The following components were used to perform the studies summarized in this report: Kits Quantification kit Amplification kit Real-time PCR system Thermal cycler Genetic analyzer Quantifiler TM Trio DNA Quantification Kit Part Number GlobalFiler PCR Amplification Kit Part Number Instruments Applied Biosystems 7500 Real-Time PCR System Serial no GeneAmp PCR System 9700 Thermal Cycler Serial no. G96S , G96S , G96S Applied Biosystems 3500xl Genetic Analyzer Serial no Software Quantification HID Real-Time PCR Analysis Software v1.2 Data collection 3500 Series Data Collection Software v3.1 Analysis GeneMapper ID-X Software v1.5 Amplification Injection 29, 30, 31 cycles 1.2 kv 24 seconds Parameters The following conditions were used to perform the studies summarized in this report: Process Quantification Amplification Genetic analysis Conditions 18 µl of Master Mix + 2 µl of DNA = 20 µl total The Master Mix consists of: 10 µl of PCR Reaction Mix 8 µl of Primer Mix 10 µl of Master Mix + 15 µl of DNA and/or TE -4 = 25 µl total The Master Mix consists of: 7.5 µl of Reaction Mix 2.5 µl of Primer Set 10 µl of Master Mix + 1 µl of PCR product or allelic ladder = 11 µl total The Master Mix consists of: 9.6 µl of Hi-Di Formamide 0.4 µl of GeneScan 600 LIZ Size Standard v2.0 Data analysis Threshold setting For Minimum Threshold and Contamination Study, the threshold was set at 1 RFU to analyze the negative controls. For Sensitivity and Stochastic Study, Mixture Study, Degraded and Inhibited Sample Study, the threshold was set at 175 RFU. Sizing calling method The Local Southern Sizing Method was used for all analysis Normalization Normalization was not applied to the data analysis in any of the studies. Page 3

4 Chapter 1: Summary 3. Studies This section briefly describes each study in this evaluation report. For detailed information, see the subsequent evaluation study chapters. The following studies were performed in the evaluation of GlobalFiler extra PCR cycle protocol: Minimum Threshold and Contamination Study Sensitivity and Stochastic Study Degraded Sample Study Inhibited Sample Study Minimum Threshold and Contamination Study Goal Compare the levels of background noise detected from the 3500xl instrument under extra cycles (30 and 31 cycles) to that of the standard 29 cycles, to evaluate the impact of extra cycles (30 and 31 cycles) on the background noise and minimum threshold, if any. Determine the level of contamination, if any. In the minimum threshold and contamination study, fifteen negative samples (samples containing no DNA) from multiple amplification plates were analyzed for each of the cycle conditions (i.e. 29, 30 and 31 cycles) by setting 1 RFU as the detection threshold. Dye Average Peak Height of Background Calculated Minimum Threshold 29 cycles 30 cycles 31 cycles 29 cycles 30 cycles 31 cycles Blue (6-FAM ) Green (VIC ) Yellow (NED ) Red (TAZ ) Purple (SID ) The background noise level for the 3500xl instrument and GlobalFiler kit is very low when samples contain no human DNA (negative controls). At standard 29 cycles, the average peak heights of background ranged from 4 to 10 RFU, and the calculated minimum thresholds ranged from 35 to 60 RFU on blue, green, yellow, red and purple channels. The average peak heights of background were increased to the range of 5 to 11 RFU, and the minimum thresholds were increased to the range of 45 to 80 RFU by one extra cycle (30 cycles). Page 4

5 Chapter 1: Summary Two extra cycles (31 cycles) affected the background noise inconsistently on different channels. The average peak heights of background ranged from 4 to 8 RFU, and the minimum thresholds ranged from 35 to 75 RFU at 31 cycles. Sensitivity and Stochastic Study Establish a range of DNA quantities that can be successfully amplified with the GlobalFiler Kit and detected on the Applied Biosystems 3500xl Genetic Analyzer when amplifying under extra cycles. Goal Evaluate the advantages, and limitations of the extra PCR cycles with GlobalFiler kit for single source low input DNA. Provide a foundation for understanding the limitations of the extra cycles using with GlobalFiler Kit and the artifacts that are observed when very low or high amounts of DNA are amplified with standard and extra cycles male DNA and IMR-90 female DNA dilution series ranging from 16pg to 2ng were amplified in triplicates with GlobalFiler PCR Amplification kit at 29, 30, and 31 cycles respectively. Average peak heights, dropout of alleles, and extra non-allele peaks in electropherograms were analyzed with GeneMapper ID-X v1.5. One extra cycle (30 cycles) could improve the genotyping results in single source low-input DNA samples at 0.031ng and lower, in which one extra cycle could recover 20% to 44% of allele calls compared to standard 29 cycles while generating no or minimal artifacts. The extra cycle did not benefit the samples with input at 0.125ng or higher, because full profiles can be generated from these samples at 29 cycles; and extra cycle can only generate extra pull-up peaks, extra stutter peaks and extra artifacts. Two extra cycles (31 cycles) added less benefit to low input samples. Although two extra cycles could recover 2% to 14% more allele calls in samples with input at and lower compared to one extra cycle, they also added extra stutter peaks and other artifacts, which could cause problem in interpreting the genotype of samples. Two extra cycles did not add any benefit to samples with input at 0.063ng and above, because they did not add recovered alleles, but added off-scale peaks, pull-up peaks and extra stutter peaks. Inhibited Sample Study Goal Evaluate the advantages and limitations of the extra PCR cycles with GlobalFiler kit for single source inhibited DNA samples analyzed on the Applied Biosystems 3500xl Genetic Analyzer with GeneMapper ID-X Software v1.5. Provide a foundation for understanding the limitations of the extra cycles used with GlobalFiler Kit and the artifacts that are observed when extra cycles are used for PCR amplification. Two inhibitors (Hematin and Humic Acid) and three concentrations of each inhibitor were included in this study. Three replicates of each inhibitor and each concentration were amplified with GlobalFiler kit under three cycle conditions: 29, 30 and 31 cycles. Page 5

6 Chapter 1: Summary Allele dropout was observed in samples with high concentration (210 ng/ul) of humic acid, but not in samples with low (90 ng/ul) or medium (150 ng/ul) concentration of humic acid, or in any samples with hematin regardless the concentration of hematin. One extra cycle (30 cycles) or two extra cycles (31 cycles) did not rescue the allele dropout presented at 29 cycles in humic acid inhibited samples, thus had no effect in improving the genotyping results. Moreover, two extra cycles (31 cycles) increased the numbers of off-scale peaks, pull-up peaks, extra stutter peaks, minus-a peaks and other artifacts, making the genotyping fail and data not usable. Degraded Sample Study Goal Evaluate the advantages, and limitations of the extra PCR cycles with GlobalFiler kit for single source degraded DNA Samples analyzed on the Applied Biosystems 3500xl Genetic Analyzer with GeneMapper ID-X Software v1.5. Provide a foundation for understanding the limitations of the extra cycles using with GlobalFiler Kit and the artifacts that are observed when different inputs of degraded DNA samples are amplified with standard and extra cycles. Artificially degraded male DNA samples were used in this study. Two levels of degraded (low-degraded and medium-degraded) DNA samples at two levels of input amount (1ng and 0.5ng) were amplified with GlobalFiler kit under three different PCR conditions: standard 29 cycles, 29 cycles with additional Taq Polymerase and BSA, and 30 cycles. One extra cycle (30 cycles) decreased the allele dropout rate in both low-degraded and medium-degraded samples and at both 1ng and 0.5ng DNA input, compared to standard 29 cycles. The effectiveness of one extra cycle on degraded sample is less significant compared to that observed in Sensitivity Study where non-degraded DNA sample was used. In the degraded samples, 9% to 14% of alleles were recovered by one extra cycle compared to 20% to 44% in non-degraded DNA sample. One extra cycle could improve the genotyping result of low-degraded sample at both 1ng and 0.5ng input by recovering dropped alleles while generating minimal artifacts. It also added benefit medium-degraded sample at 0.5ng input in the same way. However, extra cycle did not work well on 1ng of medium-degraded sample, because of the off-scale peaks, pull-up peaks, extra stutter peaks and other artifacts it generated. Additional Taq Polymerase and BSA did not rescue the dropped alleles in the degraded DNA samples at any input; it either did not change or even increased the allele dropout rate in most degraded samples. Page 6

7 Chapter 2 Minimum Threshold and Background Study Contents This chapter provides detailed information about Minimum Threshold and Background Study performed in the evaluation of the GlobalFiler extra PCR cycle protocol, using GlobalFiler Amplification Kit, Applied Biosystems 3500xl Genetic Analyzer, and GeneMapper ID-X Software v Study Overview 2. Experimental Design and Data Analysis 3. Results 4. Conclusions 1. Study Overview Goal Compare the levels of background noise detected from the 3500xl instrument under extra cycles (30 and 31 cycles) to that of the standard 29 cycles, to evaluate the impact of extra cycles (30 and 31 cycles) on the background noise and minimum threshold, if any. Determine the level of contamination, if any. This study was performed on an internal instrument; the goal of this study was not to determine the actual minimum threshold on this specific instrument, rather to evaluate the impact of extra cycles on the background noise and minimum threshold, if any. 2. Experimental Design and Data Analysis In the minimum threshold and contamination study, fifteen (15) negative samples (samples containing no DNA) from multiple amplification plates were analyzed for each of the cycle conditions (i.e. 29, 30 and 31 cycles) by setting 1 RFU as the detection threshold. After performing quantification, amplification, and capillary electrophoresis, the data were analyzed as follows: 1. Fifteen negative amplification controls (NTC) from Amplifications of Sensitivity Study, Degraded Sample Study and Inhibited Sample Study were analyzed. 2. Electropherograms were visually assessed. 3. Data from 60 bp to 460 bp were collected. Page 7

8 Chapter 2: Minimum Threshold and Background Study 4. The GeneMapper ID-X data was exported to txt file and then imported to the HPS excel macro tool for calculation. 5. The following statistics were calculated for the peak heights (RFU) observed in each dye channel: Maximum Peak Height Average Peak Height Standard Deviation Limit of Detection (LOD): average plus 3 standard deviations; the RFU value below which 99.7% of the background noise peaks should be observed Limit of Quantification (LOQ): average plus 10 standard deviations, provides an upper limit value below which all or nearly all background noise would generally be expected to fall Minimum Threshold: the minimum threshold values are calculated by rounding the LOQ of each dye channel to the nearest multiple of five. 6. Data from the three cycle conditions (29, 30 and 31 cycles) were analyzed respectively. 3. Results 3.1 Minimum thresholds Table 2-1. Minimum threshold data (29 cycles) Dye Channel Maximum Peak Height (RFU) Average Peak Height (RFU) Standard Deviation Average + 3 Standard Deviations (LOD) Average + 10 Standard Deviations (LOQ) Minimum Threshold (RFU) Blue (6-FAM) Green (VIC) Yellow (NED) Red (TAZ) Purple (SID) Table 2-2. Minimum threshold data (30 cycles) Dye Channel Maximum Peak Height (RFU) Average Peak Height (RFU) Standard Deviation Average + 3 Standard Deviations (LOD) Average + 10 Standard Deviations (LOQ) Minimum Threshold (RFU) Blue (6-FAM) Green (VIC) Yellow (NED) Red (TAZ) Purple (SID) Page 8

9 Chapter 2: Minimum Threshold and Background Study Table 2-3. Minimum threshold data (31 cycles) Dye Channel Maximum Peak Height (RFU) Average Peak Height (RFU) Standard Deviation Average + 3 Standard Deviations (LOD) Average + 10 Standard Deviations (LOQ) Minimum Threshold (RFU) Blue (6-FAM) Green (VIC) Yellow (NED) Red (TAZ) Purple (SID) Figure 2-1. Comparison of maximum peak height of back ground noise of three cycle conditions. 120 Maximum Peak Height of Background (RFU) cycles 30 cycles 31 cycles 20 0 Blue (6-FAM) Green (VIC) Yellow (NED) Red (TAZ) Purple (SID) The maximum peak heights of background were comparable among the three cycle conditions (29, 30 and 31 cycles). Extra cycles did not increase the maximum peak height of background, except for 31 cycles on blue channel. Page 9

10 Chapter 2: Minimum Threshold and Background Study Figure 2-2. Comparison of average peak height of back ground noise of three cycle conditions. 12 Average Peak Height (RFU) cycles 30 cycles 31 cycles 2 Blue (6-FAM) Green (VIC) Yellow (NED) Red (TAZ) Purple (SID) While 30 cycles slightly increased the average peak heights of background on all the channels, 31 cycles actually decreased the average peak heights of background. Figure 2-3. Comparison of LOD of the three cycle conditions LOD (RFU) cycles 30 cycles 31 cycles 5 Blue (6-FAM) Green (VIC) Yellow (NED) Red (TAZ) Purple (SID) 30 cycles increased the LOD on all the channels, with blue and green channels being more significantly affected. 31 cycles did not consistently increase the LOD on all the channels. Page 10

11 Chapter 2: Minimum Threshold and Background Study Figure 2-4. Comparison of LOQ of the three cycle conditions LOQ (RFU) 29 cycles 30 cycles 31 cycles Blue (6-FAM) Green (VIC) Yellow (NED) Red (TAZ) Purple (SID) Similar to the effect on LOD, 30 cycles increased the LOQ on all the channels, with blue and green channels being more significantly affected, while 31 cycles did not consistently increase the LOQ on all the channels. Figure 2-5. Comparison of calculated minimum threshold of the three cycle conditions Blue (6-FAM) Minimum Threshold (RFU) Green (VIC) Yellow (NED) Red (TAZ) Purple (SID) 29 cycles 30 cycles 31 cycles The minimum threshold of blue and green channels were increased from 35 to 55 RFU and from 60 to 80 RFU respectively at 30 cycles compared to 29 cycles. The minimum threshold of yellow, red and purple channels was slightly increased at 30 cycles compared to 29 cycles. 31 cycles did not consistently increase the minimum threshold on all the channels. Page 11

12 Chapter 2: Minimum Threshold and Background Study 3.2 Background noise and overlay of electropherograms Figure 2-6 through figure 2-8 show the overlay from all negative samples to demonstrate the level of background noise observed with the 3500xl instrument and GlobalFiler dye chemistry, as well as some of the observed spike peaks. To obtain a consistent view of the negative controls, the x-axis was scaled from 60 bp to 460 bp, and the y-axis was scaled from 0 to 100 RFU. Figure 2-6. All negative samples at 29 cycles overlaid in all channels except for Orange Figure 2-7. All negative samples at 30 cycles overlaid in all channels except for Orange Page 12

13 Chapter 2: Minimum Threshold and Background Study Figure 2-8. All negative samples at 31 cycles overlaid in all channels except for Orange 4. Conclusions The background noise level for the 3500xl instrument and GlobalFiler kit is very low when samples contain no human DNA (negative controls). At standard 29 cycles, the average peak heights of background ranged from 4 to 10 RFU, and the minimum thresholds ranged from 35 to 60 RFU on blue, green, yellow, red and purple channels. The average peak heights of background were increased to the range of 5 to 11 RFU, and the minimum thresholds were increased to the range of 45 to 80 RFU by one extra cycle (30 cycles). Two extra cycles (31 cycles) did not increase the background noise consistently on all the channels. The average peak heights of background ranged from 4 to 8 RFU, and the minimum thresholds ranged from 35 to 75 RFU at 31 cycles. Page 13

14 Chapter 3 Sensitivity and Stochastic Study Contents This chapter provides detailed information about Sensitivity and Stochastic Study performed in the evaluation of the GlobalFiler extra PCR cycle protocol, using GlobalFiler Amplification Kit, Applied Biosystems 3500xl Genetic Analyzer, and GeneMapper ID-X Software v Study Overview 2. Experimental Design and Data Analysis 3. Results 4. Conclusions 1. Study Overview Establish a range of DNA quantities that can be successfully amplified with the GlobalFiler Kit and detected on the Applied Biosystems 3500xl Genetic Analyzer when amplifying under extra cycles. Goal Evaluate the advantages, and limitations of the extra PCR cycles with GlobalFiler kit for single source low input DNA. Provide a foundation for understanding the limitations of the extra cycles using with GlobalFiler Kit and the artifacts that are observed when very low or high amounts of DNA are amplified with standard and extra cycles. 2. Experimental Design and Data Analysis In this Sensitivity and Stochastic Study, male genomic DNA sample 7057 (Coriell Institute) and female genomic sample IMR-90 (New England Biolabs) were used. 2.1 Quantification 1. Human male genomic DNA 7057 and human female genomic DNA IMR-90 were quantified with the Quantifiler Trio DNA Quantification Kit on a 7500 Real-Time PCR System. 2. The result of 7057 DNA stock concentration (7.195 ng/µl) was then used to generate a serial dilution using TE buffer (10 mm Tris-HCl ph 8.0 and 0.1 mm EDTA) (Table 3-1). The serial dilution was quantified in duplicate. Page 14

15 Chapter 3: Sensitivity and Stochastic Study Table 3-1. Volumes of DNA and TE buffer added to generate dilution series. Samples were serially diluted from ng/µl 7057 stock. Final Target DNA volume (µl) TE -4 buffer Dilution tube concentration DNA from tube from stock or volume (µl) (ng/µl) previous tube M-A 2.0 stock M-B 1.0 A M-C 0.5 B M-D 0.25 C M-E D M-F E M-G F M-H G The result of IMR-90 stock concentration (8.417 ng/µl) was used to generate a serial dilution using TE buffer (10 mm Tris-HCl ph 8.0 and 0.1 mm EDTA) (Table 3-2). The serial dilution was quantified in duplicate. Table 3-2. Volumes of DNA and TE -4 buffer added to generate dilution series. Samples were serially diluted from ng/µl IMR-90 stock. Final Target DNA volume (µl) TE -4 buffer Dilution tube concentration DNA from tube from stock or volume (µl) (ng/µl) previous tube F-A 2.0 stock F-B 1.0 A F-C 0.5 B F-D 0.25 C F-E D F-F E F-G F F-H G Page 15

16 Chapter 3: Sensitivity and Stochastic Study 2.2 Amplification 7057 male DNA and IMR-90 female DNA dilution series ranging from 16pg to 2ng were amplified in triplicates with GlobalFiler PCR Amplification kit at 29, 30, and 31 cycles respectively on 9700 Thermo Cycler with golden block. Input DNA amount included 2, 1, 0.5, 0.25, 0.125, , , and ng per reaction, as shown in Table 3-3. Table 3-3. Male and Female DNA input, replicates and cycle numbers used in the sensitivity and stochastic study. Male Number of replicates of Female Number of replicates of 7057 Male DNA 7057 IMR-90 Female DNA IMR90 DNA input 29 cycles 30 cycles 31 cycles DNA input 29 cycles 30 cycles 31 cycles 2ng ng ng ng ng ng ng ng ng ng ng ng ng ng ng ng The reaction mix was prepared with GlobalFiler Master Mix and Primer Set. 2. Input DNA and TE buffer were added to the 1.5-mL tubes. Based on the Quantifiler Trio results, GlobalFiler amplification reactions were prepared using the volumes listed in Table 3- (for male 7057 DNA) and Table 3- (for female IMR-90 DNA) to cover enough reaction mix for twelve (12) replicates of each dilution series. Table 3-4. Quantities of DNA and TE buffer added for Male DNA 7057 amplification. Concentration measured by the Quantifiler Trio DNA Quantification Kit DNA Quant Trio Final Master tubes for 12 reactions Volume dilutions from Results Input (µl)/rxn Master step 2 (ng/µl) (ng)/rxn TE (µl) DNA (µl) Mix (µl) M-Neg M-Neg M-A_2.0ng M-B_1.0ng M-C_0.5ng M-D_0.25ng M-E_0.125ng M-F_0.063ng M-G_0.031ng M-H_0.016ng M-POS Page 16

17 Chapter 3: Sensitivity and Stochastic Study Table 3-5. Quantities of DNA and TE buffer added for Female DNA IMR-90 amplification. Concentration measured by the Quantifiler Trio DNA Quantification Kit. IMR-90 DNA Quant Trio Final Master tubes for 12 reactions Volume dilutions from Results Input (µl)/rxn Master step 3 (ng/µl) (ng)/rxn TE (µl) DNA (µl) Mix (µl) F-Neg F-Neg F-A_2.0ng F-B_1.0ng F-C_0.5ng F-D_0.25ng F-E_0.125ng F-F_0.063ng F-G_0.031ng F-H_0.016ng F-POS Amplification controls were prepared: Control DNA 007 was added to the positive amplification control (POS) TE buffer was added to the negative control (NTC) 4. Each 1.5-mL tube of reaction mix was distributed into three replicate wells on a 96-well plate and three identical plates (for 29, 30 and 31 cycles) were prepared. 2.3 Capillary Electrophoresis The Amplified PCR products were run on 3500xl Genetic Analyzer instrument with standard injection parameters specified in the GlobalFiler PCR Amplification kit User Guide. Data Collection Software v3.1 was used for data collection. 2.4 Data analysis 1. GeneMapper ID-X v1.5 was used for data analysis. Average peak heights, dropout of alleles, and extra non-allele peaks in electropherograms were analyzed by using the threshold at 175 RFU. 2. The profiles were visually assessed, and extraneous peaks were reviewed and relabeled manually with allele edit comments. The known genotype was used to assess the electropherograms. 3. The GeneMapper ID-X data was exported as txt files and imported to HPS excel macro tool for calculation. Page 17

18 Average Heterozygous Peak Height (RFU) Average Heterozygous Peak Height (RFU) Chapter 3: Sensitivity and Stochastic Study 3. Results 3.1 Peak heights Figure 3-1. Average heterozygous peak heights (RFU) for the 7057 dilution series -29 cycles 7057 GlobalFiler (29c - 24s) Sensitivity , , ,085 2,771 1, ng 0.031ng 0.063ng 0.125ng 0.25ng 0.5ng 1ng 2ng Input DNA Figure 3-2. Average heterozygous peak heights (RFU) for the IMR-90 dilution series -29 cycles IMR-90 GlobalFiler (29c - 24s) Sensitivity ,564 12,276 6,101 3,479 1, ng 0.031ng 0.063ng 0.125ng 0.25ng 0.5ng 1ng 2ng Input DNA Page 18

19 Average Heterozygous Peak Height (RFU) Average Heterozygous Peak Height (RFU) Chapter 3: Sensitivity and Stochastic Study Figure 3-3. Average heterozygous peak heights (RFU) for the 7057 dilution series -30 cycles GlobalFiler (30c - 24s) Sensitivity 18,737 9,678 5,221 2, ng 0.031ng 0.063ng 0.125ng 0.25ng 0.5ng 1ng Input DNA Figure 3-4. Average heterozygous peak heights (RFU) for the IMR-90 dilution series -30 cycles IMR-90 GlobalFiler (30c - 24s) Sensitivity , , , , , ng 0.031ng 0.063ng 0.125ng 0.25ng 0.5ng 1ng Input DNA Page 19

20 Average Heterozygous Peak Height (RFU) Average Heterozygous Peak Height (RFU) Chapter 3: Sensitivity and Stochastic Study Figure 3-5. Average heterozygous peak heights (RFU) for the 7057 dilution series -31 cycles 7057 GlobalFiler (31c - 24s) Sensitivity , , ,470 1, ng 0.031ng 0.063ng 0.125ng 0.25ng 0.5ng Input DNA Figure 3-6. Average heterozygous peak heights (RFU) for the IMR-90 dilution series -31 cycles IMR-90 GlobalFiler (31c - 24s) Sensitivity , , ,796 2, ng 0.031ng 0.063ng 0.125ng 0.25ng 0.5ng Input DNA Peak heights for all samples are depicted in figure 3-7 through figure 3-12, as well as highlighted occurrences of drop-out and the peak heights of surviving sister alleles. Dropout of alleles will be discussed in detail in section 3.5. Page 20

21 Chapter 3: Sensitivity and Stochastic Study Figure 3-7. Peak heights (RFU) for each allele detected in the 7057 dilution series -29 cycles 29 cycles AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 DYS391 FGA SE33 TH01 TPOX vwa Yindel Male X Y ng ng ng ng ng ng Figure 3-8. Peak heights (RFU) for each allele detected in the 7057 dilution series -30 cycles 30 cycles AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 DYS391 FGA SE33 TH01 TPOX vwa Yindel Male X Y ng ng ng ng ng ng Figure 3-9. Peak heights (RFU) for each allele detected in the 7057 dilution series -31 cycles 31 cycles AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 DYS391 FGA SE33 TH01 TPOX vwa Yindel Male X Y ng ng ng ng ng ng Page 21

22 Chapter 3: Sensitivity and Stochastic Study Figure Peak heights (RFU) for each allele detected in the IMR-90 dilution series -29 cycles 29 cycles AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vwa Female X ng ng ng ng ng ng Figure Peak heights (RFU) for each allele detected in the IMR-90 dilution series -30 cycles 30 cycles AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vwa Female X ng ng ng ng ng ng Figure Peak heights (RFU) for each allele detected in the IMR-90 dilution series -31 cycles 31 cycles AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vwa Female X ng ng ng ng ng ng Page 22

23 Chapter 3: Sensitivity and Stochastic Study 3.2 Peak Height Ratio Figure Peak height ratios (PHR) for heterozygous loci for the 7057 dilution series -29 cycles Male 29 cycles AMEL CSF1PO D10S124 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 SE33 TH01 vwa 0.5ng % 92.76% 99.98% 79.47% 96.63% 93.78% 75.11% 97.60% 66.56% 96.37% 92.77% 79.89% 95.81% 91.10% 95.27% 69.91% 77.75% 69.33% 89.32% % 86.76% % 86.68% 93.01% 87.17% 85.96% 93.64% 73.71% 90.09% 63.29% 96.42% 80.66% 76.14% 84.23% 93.57% 76.18% 91.47% 93.55% % 81.34% 89.18% 83.31% 99.05% 98.06% 84.11% 90.56% 73.17% 72.61% 90.20% 84.67% 87.14% 82.72% 86.38% 99.92% 63.98% 79.21% 83.72% 0.25ng % 94.70% 89.02% 95.53% 74.82% 94.04% 84.44% 90.49% 58.58% 99.11% 89.81% 71.26% 63.95% 96.34% 78.23% 88.66% 71.06% 86.16% 93.67% % 80.64% 87.11% 81.97% 95.48% 81.54% 67.80% 74.02% 98.07% 73.82% 64.37% 88.63% 86.78% 86.00% 85.08% 90.98% 87.73% 99.32% 94.35% % 79.63% 74.13% 90.92% 78.63% 93.97% 99.17% 90.92% 79.64% 81.37% 82.71% 90.76% 81.36% 84.33% 88.54% 98.01% 95.75% 90.99% 95.55% 0.125ng % 92.75% 90.43% 55.03% 66.97% 60.44% 89.50% 55.95% 56.79% 90.48% 53.28% 90.05% 98.70% 73.25% 61.83% 76.25% 95.17% 70.87% 83.28% % 76.23% 63.82% 47.38% 56.79% 63.88% 73.09% 92.79% 52.46% 72.76% 79.75% 92.34% 69.41% 91.13% 40.51% 86.82% 71.16% 84.17% 86.99% % 96.94% 54.00% 66.43% 82.31% 83.81% 71.76% 49.43% 67.48% 78.82% 89.31% 35.07% 89.92% 52.53% 88.83% 67.01% 56.02% 77.63% 95.09% 0.063ng % 71.83% 69.46% 30.87% 91.60% 77.40% 44.90% 30.15% 71.08% 81.27% 96.44% 38.61% 76.90% 91.06% 57.14% 80.44% 81.03% 36.12% 58.76% % 90.03% 38.10% 55.85% 61.55% 38.33% 59.32% 82.45% 99.45% 74.90% 82.50% 59.53% 50.20% 51.08% 70.67% 53.43% 38.39% 98.21% % 68.79% 42.23% 72.46% 49.18% 57.78% 81.66% 50.69% 65.53% 45.22% 81.54% 66.56% 65.56% 48.96% 71.06% 80.79% 91.57% 77.01% 0.031ng % 85.65% 51.34% 64.86% 70.29% 72.06% % 98.98% 65.78% 73.70% 90.51% 32.68% 67.15% 55.90% 93.63% % 79.44% 52.61% 58.38% 76.04% 21.02% 65.71% 75.18% 78.19% 68.19% 43.21% 0.016ng Figure Peak height ratios (PHR) for heterozygous loci for the 7057 dilution series -30 cycles Male 30 cycles AMEL CSF1PO D10S124 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 SE33 TH01 vwa 0.5ng % 76.63% 57.70% 94.37% 83.63% 85.84% 82.60% 77.74% 73.40% 96.36% 99.47% 98.60% 91.80% 96.52% 91.19% 59.29% 91.44% 89.36% 71.41% % 88.11% 82.05% 98.90% 86.40% 66.25% 71.10% 87.82% 88.96% 83.16% 89.13% 65.70% 82.90% 89.32% 82.45% 94.71% 83.96% 87.61% 84.65% % 95.26% 88.62% 80.25% 94.67% 74.29% 87.38% 83.07% 99.93% 89.91% 74.74% 78.30% 86.09% 84.78% 78.78% 80.55% 94.92% 85.66% 84.22% 0.25ng % 74.24% 90.12% 78.54% 72.52% 68.01% 87.07% 79.67% 93.55% 96.01% 86.23% 77.83% 83.62% 84.73% 95.63% 87.27% 99.13% 87.20% 94.13% % 83.79% 71.27% 92.79% 84.21% 80.92% 83.77% 93.48% 54.32% 98.72% 99.54% 81.91% 82.76% 84.27% 79.84% 63.95% 97.72% 68.05% 81.35% % 91.12% 91.03% 53.42% 85.29% 75.16% 93.83% 80.29% 73.27% 88.70% 75.13% 94.22% 66.88% 66.82% 72.04% 72.96% 61.86% 70.96% 91.63% 0.125ng % 70.85% 95.04% 62.54% 84.81% 92.38% 83.01% 72.48% 61.41% 92.49% 80.54% 56.06% 45.45% 88.08% 65.65% 89.64% 63.68% 63.15% 49.80% % 52.44% 92.52% 73.99% 68.06% 75.95% 91.97% 89.21% 87.75% 48.98% 43.72% 71.39% 90.25% 70.47% 80.64% 70.57% 85.35% 85.73% 73.82% % 67.11% 72.69% 81.23% 47.27% 87.09% 93.28% 77.85% 69.47% 75.25% 86.54% 76.64% 27.73% 96.51% 52.86% 70.92% 94.73% 91.75% 74.89% 0.063ng % 86.72% 46.25% 52.74% 51.38% 55.56% 61.00% 44.21% 71.41% 54.89% 73.18% 80.24% 51.02% 17.22% 56.09% 71.09% 61.02% 86.38% 99.04% % 29.10% 44.12% 77.88% 81.21% 58.13% 29.82% 73.57% 81.25% 60.02% 32.21% 47.71% 64.52% 99.50% 46.08% 47.86% 69.63% 81.28% 80.74% % 34.46% 56.08% 79.46% 66.92% 96.74% 29.21% 58.59% 85.89% 78.47% 86.85% 67.00% 75.57% 69.67% 86.13% 89.14% 87.93% 57.12% 0.031ng % 27.18% 37.28% 61.15% 55.97% 89.27% 50.79% 40.72% 66.90% 74.27% 29.30% 59.33% 55.65% 51.80% % 79.51% 85.76% 93.04% 55.87% 85.07% 96.36% 55.89% 71.95% 41.61% 33.10% 68.16% 80.00% 51.75% 23.71% 48.93% % 80.80% 34.49% 43.70% 51.95% 85.78% 59.65% 92.32% 93.84% 90.74% 82.53% 55.13% 42.88% 64.43% 0.016ng % 62.66% 77.27% 73.94% 57.30% 78.07% % 48.76% 52.84% % 90.29% 67.01% 97.67% 88.80% 99.57% Figure Peak height ratios (PHR) for heterozygous loci for the 7057 dilution series -31 cycles Male 31 cycles AMEL CSF1PO D10S124 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 SE33 TH01 vwa 0.5ng % 93.99% 94.55% 90.69% 81.57% 81.47% 94.99% 99.10% 81.81% 95.28% 66.02% 86.09% 86.44% 86.73% 78.46% 96.85% 81.59% 89.94% 66.19% % 84.67% 68.56% 94.06% 93.31% 80.31% 98.50% 90.57% 50.27% 97.24% 96.40% 73.34% 91.21% 85.48% 71.82% 89.22% 83.76% 65.29% 78.23% % 99.61% 90.70% 93.24% 80.47% 83.51% 84.92% 97.68% 63.73% 95.00% 99.63% 75.21% 88.41% 94.62% 77.11% 86.32% 81.54% 69.71% 83.14% 0.25ng % 92.44% 74.85% 81.12% 99.99% 86.58% 95.08% 99.26% 92.95% 82.93% 97.79% 82.57% 91.59% 65.76% 71.88% 89.47% 94.76% 67.11% 90.48% % 95.58% 67.06% 65.38% 83.15% 91.67% 89.73% 80.48% 73.75% 91.41% 93.06% 70.52% 89.24% 90.67% 81.37% 93.56% 75.96% 76.88% 95.10% % 60.08% 81.70% 81.16% 77.85% 78.82% 89.06% 74.89% 90.75% 94.07% 55.06% 80.71% 94.99% 91.83% 98.47% 75.87% 73.81% 69.50% 96.34% 0.125ng % 86.35% 73.83% 87.98% 62.82% 78.67% 93.35% 65.05% 88.58% 72.72% 90.74% 92.37% 82.17% 76.89% 83.28% 70.20% 92.00% 73.96% 96.46% % 86.44% 61.84% 49.03% 66.35% 45.74% 92.21% 87.64% 91.46% 53.95% 79.83% 74.15% 55.55% 98.16% 86.47% 88.78% 95.12% 86.54% 93.66% % 66.13% 75.07% 98.91% 92.05% 77.95% 88.94% 89.70% 67.14% 88.58% 83.78% 80.06% 83.87% 58.59% 85.00% 71.84% 57.20% 92.24% 76.40% 0.063ng % 69.71% 58.13% 72.34% 30.59% 47.54% 27.71% 93.51% 48.24% 59.66% 67.58% 56.85% 79.91% 59.33% 83.91% 51.15% 42.17% 64.14% 94.45% % 63.56% 76.75% 87.00% 43.65% 66.20% 62.05% 48.42% 53.84% 97.92% 85.95% 76.88% 45.93% 81.28% 56.77% 97.50% 44.67% 36.61% 27.79% % 25.17% 43.82% 74.27% 49.16% 38.54% 93.34% 18.46% 96.06% 98.93% 58.93% 93.87% 57.20% 9.47% 94.14% 69.27% 34.30% 67.01% 0.031ng % 71.85% 38.55% 58.97% 41.91% 72.72% 34.63% 46.93% 82.57% 86.83% 55.37% 87.62% 44.95% 38.74% 56.59% 70.55% 37.35% % 63.36% 83.58% 68.17% 32.15% 28.78% 77.50% 81.73% 49.30% 83.92% 40.79% 17.02% 47.25% % 20.68% 45.58% 17.94% 81.88% 66.97% 76.65% 32.71% 27.31% 28.32% 22.34% 27.56% 86.57% 46.98% 52.91% 42.63% 82.21% 0.016ng % 87.54% 90.89% 33.45% 37.61% 75.07% 84.62% 65.30% % 57.31% 59.20% 64.33% 67.81% 83.52% 71.88% % 63.40% 67.24% 50.22% 34.57% 43.80% 78.31% 81.38% Page 23

24 Chapter 3: Sensitivity and Stochastic Study Figure Peak height ratios (PHR) for heterozygous loci for the IMR-90 dilution series -29 cycles Female 29 cycles CSF1PO D10S1248 D12S391 D13S317 D16S539 D19S433 D1S1656 D21S11 D22S104 5 D2S1338 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vwa 0.5ng % 70.62% 77.44% 72.52% 87.14% 97.77% 99.84% 78.07% 81.14% 87.86% 97.44% 84.34% 88.26% 78.58% 97.72% 90.66% 94.80% 96.36% 86.58% % 82.83% 97.89% 94.10% 97.46% 97.31% 98.71% 94.68% 82.76% 99.43% 84.36% 97.27% 72.46% 91.80% 77.68% 91.66% 98.53% 79.17% 81.49% % 80.37% 83.86% 84.67% 71.99% 94.73% 80.36% 97.08% 81.63% 95.80% 90.96% 81.43% 90.05% 95.22% 83.92% 98.09% 77.33% 94.94% 92.23% 0.25ng % 76.62% 93.28% 95.87% 84.66% 94.42% 93.42% 76.20% 63.96% 92.72% 78.18% 73.43% 83.93% 88.13% 92.55% 94.48% 78.35% 95.28% 82.91% % 92.48% 84.49% 95.09% 69.56% 83.92% 89.12% 86.91% 88.34% 90.05% 90.16% 85.87% 95.96% 81.09% 98.21% 79.42% 92.52% 64.81% 75.66% % 99.72% 91.63% 91.23% 92.26% 89.95% 97.97% 93.93% 95.47% 99.33% 93.39% 93.71% 96.84% 95.31% 73.75% 87.87% 90.79% 99.92% 90.84% 0.125ng % 92.61% 54.14% 95.16% 52.40% 99.52% 82.22% % 53.54% 87.75% 92.52% 92.70% 86.31% 59.11% 60.83% 89.99% 70.60% 70.84% 81.10% % 73.80% 98.05% 78.71% 85.81% 63.37% 93.04% 87.35% 93.80% 95.85% 40.91% 83.89% 63.29% 63.73% 98.03% 97.62% 89.69% 91.71% 83.95% % 93.23% 71.76% 70.78% 86.54% 73.78% 98.08% 70.59% 73.09% 67.50% 69.60% 82.38% 66.84% 89.43% 86.30% 72.08% 89.30% 49.09% 87.50% 0.063ng % 82.00% 66.03% 90.80% 56.43% 62.25% 50.60% 72.53% 49.87% 64.85% 91.94% 84.47% 85.04% 87.76% 50.18% 99.84% 63.49% 95.32% 81.90% % 66.92% 51.33% 87.81% 81.65% 67.10% 53.77% 71.29% 83.58% 47.47% 97.58% 89.78% 65.40% 82.55% 89.38% 83.35% 59.96% 71.63% 46.62% % 69.56% 95.97% % 98.13% 88.74% 63.89% 60.00% 72.71% 44.79% 65.78% 63.93% 58.73% 60.61% 78.74% 98.70% 77.26% 63.68% 84.46% 0.031ng % 96.20% 77.08% % 94.69% 99.65% 68.32% % 98.72% % 59.63% 59.26% 85.54% 86.72% 98.31% 63.69% 52.42% 75.30% 0.016ng % 2 3 Figure Peak height ratios (PHR) for heterozygous loci for the IMR-90 dilution series -30 cycles Female 30 cycles CSF1PO D10S1248 D12S391 D13S317 D16S539 D19S433 D1S1656 D21S11 D22S104 5 D2S1338 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vwa 0.5ng % 93.22% 84.41% 77.64% 90.16% 93.32% 97.62% 94.47% 88.25% 94.56% 99.71% 83.54% 85.35% 90.37% 94.05% 88.07% 92.08% 93.81% 78.53% % 89.08% 94.82% 99.72% 81.61% 86.06% 76.60% 80.67% 80.00% 80.79% 85.10% 97.81% 70.64% 85.63% 92.70% 90.67% 83.27% 91.29% 99.36% % 76.89% 99.39% 99.92% 79.37% 95.62% 95.76% 72.60% 85.87% 70.35% 97.49% 92.01% 95.48% 92.80% 83.99% 74.32% 99.77% 89.83% 90.55% 0.25ng % 96.21% 84.35% 96.63% 85.34% 89.43% 92.88% 97.15% 70.52% 94.13% 82.77% 94.00% 90.19% 84.74% 91.32% 68.20% 82.54% 79.66% 90.67% % 84.52% 81.41% 93.03% 90.46% 85.40% 87.65% 92.99% 97.51% 87.95% 80.59% 98.82% 73.77% 81.00% 74.06% 92.45% 81.33% 94.57% 86.68% % 90.40% 65.08% 86.84% 70.65% 96.45% 68.90% 85.57% 95.08% 77.14% 91.46% 97.76% 84.04% 78.94% 86.14% 95.92% 75.42% 90.11% 92.77% 0.125ng % 87.93% 84.08% 80.73% 81.19% 60.57% 39.93% 39.03% 77.34% 73.38% 87.17% 94.90% 59.95% 96.70% 86.58% 96.29% 81.77% 81.82% 98.41% % 92.33% 91.62% 69.83% 95.23% 91.44% 95.37% 80.40% 60.42% 97.73% 96.27% 75.94% 74.45% 92.59% 99.86% 91.35% 81.72% 96.41% 85.67% % 92.61% 86.79% 93.61% 79.41% 69.61% 40.62% 77.86% 74.21% 54.69% 91.00% 81.12% 98.68% 84.47% 97.64% 88.10% 83.46% 79.02% 98.00% 0.063ng % 93.41% 50.69% 71.80% 52.04% 92.01% 52.30% 50.08% 84.94% 80.98% 70.80% 73.55% 81.07% 85.93% 68.08% 98.18% 76.56% 98.19% 86.69% % 76.42% 76.44% 76.44% 86.04% 76.09% 76.25% 98.73% 56.64% 50.89% 94.39% 96.12% 86.38% 98.97% 34.91% 46.36% 99.91% 65.26% 77.43% % 47.38% 80.43% 70.22% 45.05% 62.17% 71.29% 56.06% 43.37% 91.54% 85.88% 74.28% 65.77% 83.46% 73.56% 90.94% 71.59% 43.50% 82.06% 0.031ng % 64.60% 86.97% 43.78% 59.53% 92.37% 66.19% 33.99% 89.16% 67.71% 62.56% 94.23% 68.17% 71.32% 93.91% 93.38% % 78.13% 63.26% 71.51% 37.57% 83.60% 96.50% 50.32% 54.37% 88.08% 65.33% 87.32% % 97.75% 87.04% 80.78% 53.74% 85.22% 87.33% 65.79% 51.87% 52.44% 66.61% 47.83% 69.22% 73.60% 84.96% 0.016ng % 49.91% 47.21% 66.78% 97.39% 77.04% 93.53% % 94.62% % 55.56% 71.21% 84.25% 89.07% Figure Peak height ratios (PHR) for heterozygous loci for the IMR-90 dilution series -31 cycles Female 31 cycles CSF1PO D10S1248 D12S391 D13S317 D16S539 D19S433 D1S1656 D21S11 D22S104 5 D2S1338 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vwa 0.5ng % 95.83% 82.90% 83.57% 92.77% 86.08% 82.91% 81.06% 76.58% 93.84% 87.50% 96.73% 99.78% 90.86% 94.18% 96.35% 90.90% 90.38% 90.04% % 92.11% 99.87% 93.59% 80.14% 69.93% 80.16% 83.37% 81.14% 87.59% 97.19% 92.20% 87.24% 88.77% 92.23% 79.09% 88.19% 90.73% 99.61% % 75.05% 81.74% 79.88% 84.93% 99.63% 91.78% 84.03% 95.30% 74.37% 87.60% 98.83% 81.13% 92.37% 79.79% 90.79% 91.98% 96.05% 88.19% 0.25ng % 97.73% 98.24% 81.81% 96.47% 96.41% 85.86% 95.84% 95.36% 89.72% 92.92% 86.66% 93.75% 85.80% 59.67% 88.13% 80.34% 91.99% 84.23% % 87.58% 87.39% 90.83% 93.10% 92.21% 79.06% 96.52% 80.77% 93.14% 97.49% 85.51% 95.49% 83.38% 90.19% 84.21% 90.76% 98.29% 98.25% % 68.96% 81.04% 78.77% 89.13% 80.76% 99.54% 92.12% 96.95% 86.14% 94.95% 74.73% 67.73% 66.95% 82.41% 64.15% 85.86% 94.95% 72.76% 0.125ng % 68.71% 69.24% 79.17% 78.35% 91.22% 67.49% 93.30% 93.13% 87.82% 93.75% 86.56% 83.46% 56.15% 98.71% 69.38% 75.41% 92.51% 63.80% % 76.73% 64.74% 87.23% 77.96% 59.19% 67.47% 81.25% 87.14% 42.65% 83.48% 80.45% 83.93% 59.89% 92.01% 96.49% 83.02% 70.14% 84.03% % 77.45% 65.24% 94.34% 81.84% 99.46% 91.73% 65.14% 85.92% 77.56% 76.37% 76.50% 82.66% 93.11% 98.54% 90.46% 80.55% 77.93% 41.66% 0.063ng % 96.39% 43.09% 35.47% 77.43% 69.77% 51.82% 79.00% 90.34% 90.57% 88.10% 87.85% 81.26% 73.69% 94.87% 74.06% 94.37% 93.95% 84.80% % 90.56% 57.32% 94.27% 74.22% 64.35% 70.26% 54.44% 79.44% 74.86% 88.38% 69.35% 69.59% 67.04% 98.99% 75.05% 74.59% 50.03% 88.86% % 52.06% 91.58% 81.77% 89.48% 85.25% 84.54% 62.87% 80.59% 64.50% 98.93% 42.49% 44.10% 87.28% 90.67% 71.83% 82.58% 56.84% 86.04% 0.031ng % 41.87% 42.71% 49.42% 56.31% 59.41% 53.93% 84.70% 99.85% 87.29% 73.24% 82.38% 42.62% 66.58% 47.39% 44.63% 47.83% 76.45% 70.38% % 52.08% 44.98% 77.80% 40.04% 88.11% 47.51% 57.22% 99.88% 34.54% 59.86% 51.13% 56.95% 92.96% 92.89% 60.65% 67.99% 52.23% % 29.62% 56.64% 23.96% 97.68% 74.25% 35.67% 93.23% 66.38% 70.23% 73.94% 66.61% 80.96% 51.95% 41.14% 91.92% 44.89% 70.95% 0.016ng % 90.00% 67.97% 97.45% 71.78% 96.82% 88.57% 57.23% 39.36% 71.48% 71.14% 95.07% % 80.56% 62.23% 68.13% 89.49% 76.71% 52.37% % 96.49% 80.22% 79.69% 83.77% The PHR heat maps depict sister allele balance across the dilution series, and were used to assess the point at which intra-locus imbalance occurs. The heat maps are color coded from green (>70% PHR) to red (drop-out of a sister allele has occurred). The data showed that extra cycles did not improve the PHRs for the input at 0.063ng and above. The PHRs from samples with less than 0.063ng input were significantly below 70% or missing PHRs at 29 cycles, demonstrating the variability associated with low template stochastic amplification effects. With extra cycles, the PHRs of the low input samples improved because more alleles were detected. Page 24

25 Heterozygous Peak Heights (RFU) AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 DYS391 FGA SE33 TH01 TPOX vwa Yindel AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 DYS391 FGA SE33 TH01 TPOX vwa Yindel Heterozygous Peak Heights (RFU) Chapter 3: Sensitivity and Stochastic Study 3.3 Inter-locus Balance To assess inter-locus balance among DNA input levels, an average peak height for each sample was calculated by averaging the two peak heights of heterozygous alleles and by halving the peak heights of homozygous alleles. Inter-locus balance among DNA input levels is shown in Figure 3-19 through Figure 3-24 for 7057 samples and Figure 3-25 through Figure 3- for IMR-90 samples. Figure Inter-locus peak height balance for the 7057 dilution series -29 cycles Globalfiler Inter-locus Balance -29 cycles 2ng 1ng 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng Locus Figure Inter-locus peak height balance for low-level 7057 samples -29 cycles Globalfiler Inter-locus Balance -29 cycles ng 0.063ng 0.031ng 0.016ng Locus Page 25

26 Heterozygous Peak Heights (RFU) AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 DYS391 FGA SE33 TH01 TPOX vwa Yindel AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 DYS391 FGA SE33 TH01 TPOX vwa Yindel Heterozygous Peak Heights (RFU) Chapter 3: Sensitivity and Stochastic Study Figure Inter-locus peak height balance for the 7057 dilution series -30 cycles Globalfiler Inter-locus Balance 30 cycles ng 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng Locus Figure Inter-locus peak height balance for low-level 7057 samples -30 cycles Globalfiler Inter-locus Balance -30 cycles ng 0.063ng 0.031ng 0.016ng 0 Locus Page 26

27 AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 DYS391 FGA SE33 TH01 TPOX vwa Yindel Heterozygous Peak Heights (RFU) AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 DYS391 FGA SE33 TH01 TPOX vwa Yindel Heterozygous Peak Heights (RFU) Chapter 3: Sensitivity and Stochastic Study Figure Inter-locus peak height balance for the 7057 dilution series -31 cycles Globalfiler Inter-locus Balance -31 cycles ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng Locus Figure Inter-locus peak height balance for low-level 7057 samples -31 cycles Globalfiler Inter-locus Balance -31 cycles 0.125ng 0.063ng 0.031ng 0.016ng Locus Page 27

28 Heterozygous Peak Heights (RFU) AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vwa AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vwa Heterozygous Peak Heights (RFU) Chapter 3: Sensitivity and Stochastic Study Figure Inter-locus peak height balance for the IMR-90 dilution series -29 cycles IMR-90 Globalfiler Inter-locus Balance -29cycles ng 1ng 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng Locus Figure Inter-locus peak height balance for low-level IMR-90 samples -29 cycles IMR-90 Globalfiler Inter-locus Balance -29 cycles ng 0.063ng 0.031ng 0.016ng 0 Locus Page 28

29 Heterozygous Peak Heights (RFU) AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vwa AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vwa Heterozygous Peak Heights (RFU) Chapter 3: Sensitivity and Stochastic Study Figure Inter-locus peak height balance for the IMR-90 dilution series -30 cycles IMR-90 Globalfiler Inter-locus Balance -30 cycles ng 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng Locus Figure Inter-locus peak height balance for low-level IMR-90 samples -30 cycles 4000 IMR-90 Globalfiler Inter-locus Balance -30 cycles ng 0.063ng 0.031ng 0.016ng Locus Page 29

30 Heterozygous Peak Heights (RFU) AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vwa AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vwa Heterozygous Peak Heights (RFU) Chapter 3: Sensitivity and Stochastic Study Figure Inter-locus peak height balance for the IMR-90 dilution series -31 cycles IMR-90 Globalfiler Inter-locus Balance -31 cycles ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng Locus Figure Inter-locus peak height balance for low-level IMR-90 samples -31 cycles IMR-90 Globalfiler Inter-locus Balance -31 cycles 0.125ng 0.063ng 0.031ng 0.016ng Locus Page 30

31 Chapter 3: Sensitivity and Stochastic Study 3.4 Allele calls Full profile was generated and 43 out of 43 (expected) alleles were called from male DNA input at 125pg and above. Full profile was observed and 41 out of 41 expected alleles were called from female DNA input at 63pg and above. Figure Number of alleles called in Male DNA 7057 dilution series Number of alleles called - Male ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng 29cyc 30cyc 31cyc Figure Number of alleles called in Female DNA IMR-90 dilution series Number of alleles called - Female IMR90 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng 29cyc 30cyc 31cyc Page 31

32 Chapter 3: Sensitivity and Stochastic Study Table 3-6. The number of alleles detected in each replicate of Male 7057 and Female IMR-90 DNA dilution series: Number of alleles called Male7057 Female IMR90 Input Replicate 29cyc 30cyc 31cyc Input Replicate 29cyc 30cyc 31cyc ng ng ng ng ng ng ng ng ng ng ng ng Allele calls were 100% (number of actual allele calls/expected allele calls) for male DNA input at 125pg and above. With lowest input at 16pg, the allele call rates were 11% at 29 cycles, and increased to 54% at 30 cycles and 62% at 31 cycles. Allele calls were 100% for female DNA input at 63pg and above. With lowest input at 16pg, the allele call rates were 6.5% at 29 cycles, and increased to 51% at 30 cycles and 65% at 31 cycles. The data demonstrated that one more cycle (30 cycles) did improve the allele call rate at low input of DNA significantly. Over 40% of more alleles were called by one extra cycle. However, two more cycles (31 cycles) did not improve the allele call rate in a linear manner; only about 10% more alleles were called at 31 cycles compared to 30 cycles. Page 32

33 Figure Percentage of alleles called in Male DNA 7057 dilution series Chapter 3: Sensitivity and Stochastic Study 100% Percentage of alleles called - Male % 60% 40% 20% 0% 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng 29 cyc 30 cyc 31 cyc Figure Percentage of alleles called in Female DNA IMR-90 dilution series 100% Percentage of alleles called - Female IMR90 80% 60% 40% 20% 0% 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng 29 cyc 30 cyc 31 cyc Table 3-7. Percentage of alleles detected in Male 7057 and Female IMR-90 DNA dilution series: Average allele call percentage at low level input (total called alleles/total expected alleles) Male cyc 30 cyc 31 cyc Female IMR90 29 cyc 30 cyc 31 cyc 0.5ng 100% 100% 100% 0.5ng 100% 100% 100% 0.25ng 100% 100% 100% 0.25ng 100% 100% 100% 0.125ng 100% 100% 100% 0.125ng 100% 100% 100% 0.063ng 98.4% 99.2% 99.2% 0.063ng 100% 100% 100% 0.031ng 70.5% 89.9% 92.2% 0.031ng 56.1% 88.6% 98.4% 0.016ng 10.9% 53.5% 62.0% 0.016ng 6.5% 51.2% 65.0% Page 33

34 Chapter 3: Sensitivity and Stochastic Study 3.5 Allele dropout No allele dropout was observed at male DNA input 125pg and above, nor at female DNA input 63pg and above. Allele dropout presented at male DNA input 63pg and below, and at female DNA input 31pg and below, in all three cycling conditions. Figure Number of allele dropout in Male DNA 7057 dilution series Number of allele dropout - Male ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng 29cyc 30cyc 31cyc Figure Number of allele dropout in Female DNA IMR-90 dilution series Number of allele dropout - Female IMR ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng 29cyc 30cyc 31cyc Page 34

35 Chapter 3: Sensitivity and Stochastic Study Table 3-8. The number of allele dropout in each replicate of Male 7057 and Female IMR-90 DNA dilution series: Number of allele dropout Male7057 Female IMR90 Input Replicate 29cyc 30cyc 31cyc Input Replicate 29cyc 30cyc 31cyc ng ng ng ng ng ng ng ng ng ng ng ng > No allele dropout was observed at male DNA input 125pg and above, nor at female DNA input 63pg and above. With lowest input at 16pg of male DNA, allele dropout rate (number of dropped alleles/expected allele calls) was 89% at 29 cycles, and improved to 46.5% at 30 cycles and 38% at 31 cycles. With lowest input at 16pg of female DNA, the allele dropout rate was 93.5% at 29 cycles, and improved to 49% at 30 cycles and 35% at 31 cycles. The data showed that one more cycle (30 cycles) decrease significantly the allele dropout rate at low input of DNA. Over 40% of dropped alleles at 29 cycles were rescued by one extra cycle. However, two more cycles (31 cycles) did not rescue dropped alleles twice as many. Page 35

36 Figure Percentage of allele dropout in Male DNA 7057 dilution series Chapter 3: Sensitivity and Stochastic Study 100.0% Percentage of allele dropout - Male % 60.0% 40.0% 20.0% 0.0% 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng 29 cyc 30 cyc 31 cyc Figure Percentage of allele dropout in Female DNA IMR-90 dilution series 100.0% Percentage of allele dropout - Female IMR % 60.0% 40.0% 20.0% 0.0% 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng 29 cyc 30 cyc 31 cyc Table 3-9. Percentage of allele dropout in Male 7057 and Female IMR-90 DNA dilution series: Average allele dropout percentage at low level input (total dropped alleles/total expected alleles) Male cyc 30 cyc 31 cyc Female IMR90 29 cyc 30 cyc 31 cyc 0.5ng 0.0% 0.0% 0.0% 0.5ng 0.0% 0.0% 0.0% 0.25ng 0.0% 0.0% 0.0% 0.25ng 0.0% 0.0% 0.0% 0.125ng 0.0% 0.0% 0.0% 0.125ng 0.0% 0.0% 0.0% 0.063ng 1.6% 0.8% 0.8% 0.063ng 0.0% 0.0% 0.0% 0.031ng 29.5% 10.1% 7.8% 0.031ng 43.1% 11.4% 1.6% 0.016ng 89.1% 46.5% 38.0% 0.016ng 93.5% 48.8% 35.0% Page 36

37 Chapter 3: Sensitivity and Stochastic Study 3.6 Off-scale peaks Significant off-scale peaks were observed with extra cycles. Off-scale peaks presented in all the male and female DNA samples as well as in the positive control samples at 1ng input and some samples at 0.5ng input with 30 cycles. With 31 cycles, all the samples at 0.5ng input and some samples even at 0.25ng input had off-scale peaks. The problems with off-scale peaks are that they generated a lot of pull-up peaks in other color channel and increased peak height of stutter peaks, which are further discussed in section 3.7 and 3.8. Figure Number of off-scale peaks in each sample of Male DNA 7057 dilution series Number of Off-scale peaks in each sample - Male ng 1ng 0.5ng 0.25ng 0.125ng 0.063ng POS-1ng 29cyc 30cyc 31cyc Figure Number of off-scale peaks in each sample of Female DNA IMR-90 dilution series Number of Off-scale peaks in each sample - Female IMR90 2ng 1ng 0.5ng 0.25ng 0.125ng 0.063ng POS-1ng 29cyc 30cyc 31cyc Page 37

38 Chapter 3: Sensitivity and Stochastic Study Table Number of off-scale peaks in each sample of Male 7057 and Female IMR-90 DNA dilution series: Number of off-scale peaks in each sample Male7057 Female IMR90 Input Replicate 29cyc 30cyc 31cyc Input Replicate 29cyc 30cyc 31cyc ng ng ng ng ng ng ng ng ng ng ng ng POS-1ng POS-1ng > Table Percentage of off-scale peaks in each sample of Male 7057 and Female IMR-90 DNA dilution series: Average percentage of off-scale peaks (total number of OS peaks/total number of expected peaks) female DNA input 29cyc 30cyc 31cyc male DNA input 29cyc 30cyc 31cyc 2ng 8.1% 61.0% 22.8% 2ng 4.7% 48.1% 17.1% 1ng 3.3% 10.6% 41.5% 1ng 0.0% 6.2% 34.9% 0.5ng 0.0% 1.6% 4.9% 0.5ng 0.0% 0.0% 4.7% 0.25ng 0.0% 0.0% 1.6% 0.25ng 0.0% 0.0% 0.0% Page 38

39 Chapter 3: Sensitivity and Stochastic Study 3.7 Pull-ups Pull-up peaks were observed in the male DNA samples at 0.5ng input and above, and the female DNA samples at 1ng input and above with 29 cycles. Pull-up peaks presented in the male DNA samples at 0.25ng input and above, and the female DNA samples at 0.5ng input and above with 30 cycles. When cycle number was increased to 31 cycles, the pull-up peaks increased dramatically, and shown up in both male and female DNA at as low as 0.125pg input. Pull-up peaks created flags in the genotyping quality by the software and required manually reviewing and editing by the analyst. Figure Number of pull-up peaks in each sample of Male DNA 7057 dilution series Number of pull-up peaks - Male cyc 30cyc 31cyc ng 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng Figure Number of pull-up peaks in each sample of Female DNA IMR-90 dilution series Number of pull-up peaks - Female IMR cyc 30cyc 31cyc ng 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng Page 39

40 Chapter 3: Sensitivity and Stochastic Study Table Number of pull-up peaks in each sample of Male 7057 and Female IMR-90 DNA dilution series: Number of pull-up peaks in each sample Male7057 Female IMR90 input Replicate 29cyc 30cyc 31cyc input Replicate 29cyc 30cyc 31cyc ng ng ng ng ng ng ng ng ng ng ng ng ng ng Page 40

41 3.8 Increased Stutter Peaks and Additional Stutter peaks Chapter 3: Sensitivity and Stochastic Study GlobalFiler marker-specific stutter filter set in GeneMapper ID-X Software was applied when analyzing the data. Peaks in the stutter position that were above the stutter filter percentage specified in the software are reported and evaluated in the section. Additional stutter peaks (e.g. 2 nd and 3 rd stutter peaks) are also included in this section. Increased stutter peaks and additional stutter peaks caused failure in the genotyping quality and lead to inconclusive genotypes. Figure Number of extra stutter peaks in each sample of Male DNA 7057 dilution series Number of extra stutter peaks - Male ng 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng 29cyc 30cyc 31cyc Figure Number of extra stutter peaks in each sample of Female DNA IMR-90 dilution series Number of extra stutter peaks - Female IMR90 1ng 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng 29cyc 30cyc 31cyc Page 41

42 Chapter 3: Sensitivity and Stochastic Study Table Number of extra stutter peaks in each sample of Male 7057 and Female IMR-90 DNA dilution series: Number of stutter peaks in each sample Male7057 Female IMR90 input Replicate 29cyc 30cyc 31cyc input Replicate 29cyc 30cyc 31cyc ng ng ng ng ng ng ng ng ng ng ng ng ng ng Conclusions One extra cycle (30 cycles) could improve the genotyping results in single source low-input DNA samples at 0.031ng and lower, in which one extra cycle could recover 20% to 44% of allele calls compared to standard 29 cycles while generating no or minimal artifacts. The extra cycle did not benefit the samples with input at 0.125ng or higher, because full profiles can be generated from these samples at 29 cycles; and extra cycle can only generate extra pull-up peaks, extra stutter peaks and extra artifacts. Two extra cycles (31 cycles) added less benefit to low input samples. Although two extra cycles could recover 2% to 14% more allele calls in samples with input at and lower compared to one extra cycle, they also added extra stutter peaks and other artifacts, which could cause problem in interpreting the genotype of samples. Two extra cycles did not add any benefit to samples with input at 0.063ng and above, because they did not add recovered alleles, but added off-scale peaks, pull-up peaks and extra stutter peaks. Page 42

43 Chapter 3: Sensitivity and Stochastic Study Figure Summary of limitation of extra cycles Summary map for limitation of extra cycles Male7057 Female IMR90 Input Replicate 29cyc 30cyc 31cyc Input Replicate 29cyc 30cyc 31cyc 1 Off-scale Off-scale Off-scale 1 Off-scale Off-scale Off-scale 2ng 2 Off-scale Off-scale Off-scale 2ng 2 Off-scale Off-scale Off-scale 3 Off-scale Off-scale Off-scale 3 Off-scale Off-scale Off-scale 1 pull-up Off-scale Off-scale 1 Off-scale Off-scale Off-scale 1ng 2 pull-up Off-scale Off-scale 1ng 2 Off-scale Off-scale Off-scale 3 pull-up Off-scale Off-scale 3 Off-scale Off-scale Off-scale 1 pull-up pull-up Off-scale 1 full profile pull-up Off-scale 0.5ng 2 pull-up pull-up Off-scale 0.5ng 2 full profile Off-scale Off-scale 3 pull-up pull-up Off-scale 3 full profile Off-scale Off-scale 1 full profile pull-up pull-up 1 full profile full profile Off-scale 0.25ng 2 full profile pull-up pull-up 0.25ng 2 full profile Stutter Off-scale 3 full profile pull-up pull-up 3 Stutter full profile pull-up 1 full profile Stutter Stutter 1 full profile full profile Stutter 0.125ng 2 full profile full profile Stutter 0.125ng 2 Stutter full profile Stutter 3 full profile Stutter pull-up 3 full profile full profile pull-up 1 full profile full profile Stutter 1 full profile full profile Stutter 0.063ng 2 dropout full profile Stutter 0.063ng 2 full profile full profile Stutter 3 dropout dropout dropout 3 full profile Stutter full profile 1 dropout dropout dropout 1 dropout dropout Stutter 0.031ng 2 dropout dropout dropout 0.031ng 2 dropout dropout dropout 3 dropout dropout dropout 3 dropout dropout dropout 1 dropout dropout dropout 1 dropout dropout dropout 0.016ng 2 dropout dropout dropout 0.016ng 2 dropout dropout dropout 3 dropout dropout dropout 3 dropout dropout dropout Table Summary of limitation of extra cycles Effects and characterizations Instrument saturation Increased artifacts Full profiles Decreased artifacts Stochastic amplification Allelic drop-out and dropin Input template DNA 29 cycles 30 cycles 31 cycles 1ng 0.5ng 0.125ng ng ng ng 0.063ng 0.063ng Page 43

44 Chapter 3: Sensitivity and Stochastic Study 5. Attachment Figure Electropherogram shows five (5) alleles were detected in male DNA sample with 0.016ng input at 29 cycles. Page 44

45 Chapter 3: Sensitivity and Stochastic Study Figure Electropherogram shows 24 alleles were detected in male DNA sample with 0.016ng input at 30 cycles in comparison to 5 alleles at 29 cycles for the same DNA input. Page 45

46 Chapter 3: Sensitivity and Stochastic Study Figure Electropherogram shows 29 alleles were detected in male DNA sample with 0.016ng input at 31 cycles in comparison to 5 alleles at 29 cycles and 24 alleles at 30 cycles for the same DNA input. Page 46

47 Chapter 3: Sensitivity and Stochastic Study Figure Electropherogram shows increased stutter peaks presenting in male DNA sample with 0.063ng input amplified at 31 cycles stutter stutter stutter Page 47

48 Chapter 3: Sensitivity and Stochastic Study Figure Electropherogram shows increased stutter peaks presenting in male DNA sample with 0.031ng input amplified at 31 cycles. Page 48

49 Chapter 4 Inhibited Sample Study Contents This chapter provides detailed information about Inhibited Sample Study performed in the evaluation of the GlobalFiler extra PCR cycle protocol, using GlobalFiler Amplification Kit, Applied Biosystems 3500xl Genetic Analyzer, and GeneMapper ID-X Software v Study Overview 2. Experimental Design and Data Analysis 3. Results 4. Conclusion 5. Attachment 1. Study Overview Goal Evaluate the advantages and limitations of the extra PCR cycles with GlobalFiler kit for single source inhibited DNA Samples analyzed on the Applied Biosystems 3500xl Genetic Analyzer with GeneMapper ID-X Software v1.5. Provide a foundation for understanding the limitations of the extra cycles used with GlobalFiler Kit and the artifacts that are observed when extra cycles are used for PCR amplification. Two inhibitors (Hematin and Humic Acid) and three concentrations of each inhibitor were included in this study. Three replicates of each inhibitor and each concentration were amplified with GlobalFiler kit under three cycle conditions: 29, 30 and 31 cycles (see table 4-3). 2. Experimental Design and Data Analysis 2.1 PCR Reaction mix with Inhibitors Hematin and humic acid were mixed with male DNA sample and GlobalFiler reaction mix and primer set to generate three different concentrations of each inhibitor in the final GlobalFiler PCR reaction mix. Table 4-1 and 4-2 list the volumes of each reagent required to generate the final PCR reaction mixes containing three different concentrations of hematin and humic acid for one (1) PCR reaction. Page 49

50 Chapter 4: Inhibited Sample Study Table 4-1. GlobalFiler PCR Reaction Mix with Hematin. GlobalFiler PCR Reaction Mix with Hematin Target concentration 150 um 300 um 450 um GF Reaction Mix GF Primer Mix DNA (1ng/uL) Hematin (5 mm) TE Table 4-2. GlobalFiler PCR Reaction Mix with Humic Acid. GlobalFiler PCR Reaction Mix with Humic Acid Target concentration 90 ng/ul 150 ng/ul 210 ng/ul GF Reaction Mix GF Primer Mix DNA (1ng/uL) Humic Acid (1000 ng/ul) TE Amplification Table 4-3. Samples, replicates and PCR conditions included in the inhibited Sample Study. Samples, Replicates and Cycle Conditions for inhibited sample study Inhibitor Sample 29 cycles 30 cycles 31 cycles 150 um Hematin 300 um um ng/ul Humic Acid 150 ng/ul ng/ul Capillary Electrophoresis The Amplified PCR products were run on 3500xl Genetic Analyzer instrument with standard injection parameters specified in the GlobalFiler PCR Amplification kit User Guide. Data Collection Software v3.1 was used for data collection. 2.4 Data analysis 1. GeneMapper ID-X v1.5 was used for data analysis. Number of allele calls, dropout of alleles, and extra non-allele peaks in electropherograms were analyzed by using the threshold at 175 RFU. Page 50

51 Chapter 4: Inhibited Sample Study 2. The profiles were visually assessed, and extraneous peaks and extra peaks were reviewed and re-labeled manually with allele edit comments. The known genotype was used to assess the electropherograms. 3. The GeneMapper ID-X data was exported to excel for further analysis and graphs. 3. Results 3.1 Allele calls Full profile was generated and 43 out of 43 (expected) alleles were detected from all the samples with hematin regardless the levels of hematin concentrations. With humic acid inhibited samples, full profile was generated in samples containing low (90 ng/ul) and medium (150 ng/ul) levels of humic acid, while only partial profile was generated, and alleles out of 43 expected alleles were detected in the sample containing high concentration (210 ng/ul) of humic acid. The data demonstrated that the numbers of alleles detected were equal or similar among the three cycle conditions. Extra cycles did not improve the allele call rate in humic acid inhibited samples. Figure 4-1. Number of alleles called in Inhibited Samples Number of alleles detected in inhibited samples cycles 30 cycles 31 cycles uM 300uM 450uM 90ng/ul 150ng/ul 210ng/ul Hematin Humic Acid Page 51

52 Chapter 4: Inhibited Sample Study Table 4-4. Number of alleles called in Inhibited Samples Number of alleles detected in inhibited samples Inhibitor conc. replicates 29 cycles 30 cycles 31 cycles uM Hematin 300uM uM ng/ul Humic Acid 150ng/ul ng/ul Allele dropout No allele dropout was observed in any of the samples containing hematin and the samples containing low (90 ng/ul) and medium (150 ng/ul) levels of humic acid. Allele dropout presented in all the replicates containing 210 ng/ul of humic acid in all three cycling conditions. With the allele dropout in the samples caused by humic acid, two phenomena was observed: 1. The numbers of allele dropout are equal or similar among the three cycle conditions; in other words, the number of allele dropout did not decrease as the cycle number increased. This implied that the allele dropout caused by inhibition could not be rescued by extra cycles. 2. The allele dropout always happened with specific markers (CSF1PO, D18S51, D7S820, and SE33 are affected in the case of humic acid), which are not necessarily the longest fragments. This is different from the dropout observed in the degraded samples. Page 52

53 Chapter 4: Inhibited Sample Study Figure 4-2. Number of allele dropout in each replicate of inhibited samples Number of allele dropout in inhibited samples cycles 30 cycles 31 cycles uM 300uM 450uM 90ng/ul 150ng/ul 210ng/ul Hematin Humic Acid Table 4-5. The number of allele dropout in each replicate of inhibited samples Number of allele dropout in inhibited samples Inhibitor conc. replicates 29 cycles 30 cycles 31 cycles uM Hematin 300uM uM ng/ul Humic Acid 150ng/ul ng/ul Page 53

54 Chapter 4: Inhibited Sample Study 3.3 Off-scale peaks No off-scale peaks presented in any samples with either hematin or humic acid at 29 cycles. At 30 cycles, only samples containing low concentration of hematin had off-scale peaks. However, significant off-scale peaks were observed at 31 cycles in almost all the samples except for one replicate with 210 ng/ul of humic acid. The problems with off-scale peaks are that they generated a lot of pull-up peaks in other color channel and increased peak height of stutter peaks, which are further discussed in sections 4-4 and 4-5. Figure 4-3. Number of off-scale peaks in each replicate of inhibited samples Number of off-scale peaks in inhibited samples uM 300uM 450uM 90ng/ul 150ng/ul 210ng/ul 29 cycles 30 cycles 31 cycles Hematin Humic Acid Table 4-6. Number of off-scale peaks in each replicate of inhibited samples Number of off-scale peaks in inhibited samples Inhibitor conc. replicates 29 cycles 30 cycles 31 cycles uM Hematin 300uM uM ng/ul Humic Acid 150ng/ul ng/ul Page 54

55 Chapter 4: Inhibited Sample Study 3.4 Pull-ups Pull-up peaks were observed in a few replicates with low level of hematin at 29 cycles. Pull-up peaks were presented across all the samples and replicates at 30 cycles. The number of pull-up peaks increased dramatically at 31 cycles, especially in samples with hematin and low-medium level of humic acid. These pull-up peaks made all the genotyping results fail and had to be manually reviewed and edited. Figure 4-4. Number of pull-up peaks in each replicates of inhibited samples Number of pull-up peaks in inhibited samples 0 150uM 300uM 450uM 90ng/ul 150ng/ul 210ng/ul 29 cycles 30 cycles 31 cycles Hematin Humic Acid Table 4-7. Number of pull-up peaks in each replicates of inhibited samples Number of pull-up peaks in inhibited samples Inhibitor conc. replicates 29 cycles 30 cycles 31 cycles uM Hematin 300uM uM ng/ul Humic Acid 150ng/ul ng/ul Page 55

56 Chapter 4: Inhibited Sample Study 3.5 Increased Stutter Peaks and Additional Stutter peaks GlobalFiler marker-specific stutter filter set in GeneMapper ID-X Software was applied when analyzing the data. Peaks in the stutter position that were above the stutter filter percentage specified in the software are reported and evaluated in this section. Additional stutter peaks (e.g. 2 nd and 3 rd stutter peaks) are also included in this section. Extra stutter peaks presented in all the samples with hematin and humic acid at 31 cycles, and in samples with low-medium levels of hematin and humic acid at 30 cycles, and was occasionally observed in samples with low level of hematin and humic acid at 29 cycles. Increased stutter peaks and additional stutter peaks caused failure in the genotyping quality and lead to inconclusive genotypes. Figure 4-5. Number of extra stutter peaks in each replicates of inhibited samples Number of stutter peaks in inhibited samples cycles 30 cycles 31 cycles uM 300uM 450uM 90ng/ul 150ng/ul 210ng/ul Hematin Humic Acid Page 56

57 Chapter 4: Inhibited Sample Study Table 4-8. Number of extra stutter peaks in each replicates of inhibited samples Number of increased and additional stutter peaks in inhibited samples Inhibitor conc. replicates 29 cycles 30 cycles 31 cycles uM Hematin 300uM uM ng/ul Humic Acid 150ng/ul ng/ul Other extra peaks In addition to pull-up peaks, increased stutter peaks and additional 2 nd stutter peaks, other artifacts and minus-a peaks were observed in all the samples at 31 cycles, with humic acid inhibited samples being more significant, but were not observed at 29 cycles or 30 cycles. Example electropherograms of samples with the artifacts and minus-a peaks are attached to the report in the attachment section. Page 57

58 Chapter 4: Inhibited Sample Study Figure 4-6. Number of other extra peaks/artifacts in each replicates of inhibited samples Number of other extra peaks/artifacts in inhibited samples cycles 30 cycles 31 cycles uM 300uM 450uM 90ng/ul 150ng/ul 210ng/ul Hematin Humic Acid Table 4-9. Number of other extra peaks/artifacts in each replicates of inhibited samples Number of other extra peaks/artifacts in inhibited samples Inhibitor conc. replicates 29 cycles 30 cycles 31 cycles uM Hematin 300uM uM ng/ul Humic Acid 150ng/ul ng/ul Page 58

59 Chapter 4: Inhibited Sample Study 4. Conclusion Allele dropout was observed in samples with high concentration (210 ng/ul) of humic acid, but not in samples with low (90 ng/ul) or medium (150 ng/ul) concentration of humic acid, or in any samples with hematin regardless the concentration of hematin. One extra cycle (30 cycles) or two extra cycles (31 cycles) did not rescue the allele dropout presented at 29 cycles in humic acid inhibited samples, thus had no effect in improving the genotyping results. Moreover, two extra cycles (31 cycles) increased the numbers of off-scale peaks, pull-up peaks, extra stutter peaks, minus-a peaks and other artifacts, making the genotyping fail and data not usable. Page 59

60 Chapter 4: Inhibited Sample Study 5. Attachment Figure 4-7. Full profile was generated in samples with 450 um of hematin. 43 alleles out of 43 expected alleles were detected at 29 cycles. No off-scale peak, pull-up peak, extra stutter peak or artifact peak presented. Page 60

61 Chapter 4: Inhibited Sample Study Figure 4-8. Electropherogram of a sample with 210 ng/ul of humic acid shows allele dropout presented at 29 cycles. 35 alleles out of 43 expected alleles were detected and 8 alleles were dropped out at 29 cycles. dropout dropout dropout dropout Page 61

62 Chapter 4: Inhibited Sample Study Figure 4-9. Extra cycle did not rescue allele dropout in humic acid inhibited sample. Electropherogram of a sample with 210 ng/ul of humic acid shows allele dropout presented at 30 cycles. The same number of alleles (8 alleles) was dropped out at 30 cycles as at 29 cycles. Therefore, extra cycle did not rescue the dropped alleles; moreover, pull-up peaks and additional stutter peaks were observed at 30 cycles. dropout pull-up pull-up pull-up pull-up dropout stutter dropout dropout Page 62

63 Chapter 4: Inhibited Sample Study Figure Two extra cycles did not rescue allele dropout in humic acid inhibited sample. Electropherogram of a sample with 210 ng/ul of humic acid shows allele dropout presented at 31 cycles. The same number of alleles (8 alleles) was dropped out at 31 cycles as at 29 cycles and 30 cycles. Therefore, two extra cycles did not rescue the dropped alleles; moreover, pull-up peaks, additional stutter peaks and minus-a peaks were observed at 31 cycles. -A peaks dropout artifact pull-up pull-up pull-up pull-up dropout -A peaks artifact -A peaks artifact dropout dropout -A peaks -A peaks Page 63

64 Chapter 5 Degraded Sample Study Contents This chapter provides detailed information about Degraded Sample Study performed in the evaluation of the GlobalFiler extra PCR cycle protocol, using GlobalFiler Amplification Kit, Applied Biosystems 3500xl Genetic Analyzer, and GeneMapper ID-X Software v Study Overview 2. Experimental Design and Data Analysis 3. Results 4. Conclusions 5. Attachment 1. Study Overview Goal Evaluate the advantages, and limitations of the extra PCR cycles with GlobalFiler kit for single source degraded DNA Samples analyzed on the Applied Biosystems 3500xl Genetic Analyzer with GeneMapper ID-X Software v1.5. Provide a foundation for understanding the limitations of the extra cycles using with GlobalFiler Kit and the artifacts that are observed when different inputs of degraded DNA samples are amplified with standard and extra cycles. Artificially degraded male DNA samples (prepared by HID R&D scientists) were used in this study. Two levels of degraded (low-degraded and medium-degraded) DNA samples at two levels of input amount (1ng and 0.5ng) were amplified with GlobalFiler kit under three different PCR conditions: standard 29 cycles, 29 cycles with additional Taq Polymerase and BSA, and 30 cycles. Three replicates were amplified for each sample/condition as shown in table Experimental Design and Data Analysis 2.1 Quantification In this Degraded Sample Study, artificially degraded male DNA samples prepared by HID R&D scientist were quantified with the Quantifiler Trio DNA Quantification Kit on a 7500 Real-Time PCR System and analyzed with HID Real-Time PCR Analysis Software v1.2. The quantities of the small autosomal target were used in the subsequent calculation for the input of the GlobalFiler amplification. Page 64

65 Chapter 5: Degraded Sample Study 2.2 Amplification 1. Samples, inputs and replicates for the three GlobalFiler amplification conditions are listed in table 5-1. Table 5-1. Samples, replicates and PCR conditions included in the Degraded Sample Study. 2. The standard reaction mix was prepared with GlobalFiler Master Mix and Primer Set for standard protocol at 29 cycles and 30 cycles (see table 5-2). The additional reaction mix was prepared with GlobalFiler Master Mix and Primer Set plus Taq Polymerase and BSA (see table 5-3). Table 5-2. Standard Reaction Mix. Reagents Number of Reactions (N) N + 10% Volume per Reaction (μl) Calculated Volume (μl) GlobalFiler Master Mix GlobalFiler Primer Set Table 5-3. Additional Reaction Mix. Reagents GlobalFiler Master Mix Number of Reactions (N) N + 10% Volume per Reaction (μl) Calculated Volume (μl) GlobalFiler Primer Set Taq BSA Input DNA and TE buffer were added to the 1.5-mL tubes. Based on the Quantifiler Trio results (listed in table 5-4 and 5-5), GlobalFiler amplification reactions were prepared using the volumes listed in Table 3-4 for standard reaction protocol to cover enough reaction mix for eight (8) replicates of the samples running 29 and 30 PCR cycles. 4. Additional GlobalFiler amplification reactions were prepared using the volumes listed in Table 3-5 for additional reaction protocol to cover enough reaction mix for four (4) replicates of each sample running 29 PCR cycles. Page 65

66 Chapter 5: Degraded Sample Study Table 5-4. Quantities of DNA and TE buffer added for standard reaction protocol. Master tubes for 8 reactions Dilution Average Volume Final Input DNA Conc. per Amp TE -4 Master (µl) DNA (µl) (ng) Mix (µl) (ng/µl) (µl) Total NTC MD LD CTR MD LD CTR POS POS Table 5-1. Quantities of DNA and TE buffer added for IMR-90 amplification. Concentration measured by the Quantifiler Trio DNA Quantification Kit. Master tubes for 4 reactions Dilution Average DNA Conc. (ng/µl) Final Input (ng) Volume per Amp (µl) TE -4 (µl) DNA (µl) Master Mix (µl) Total NTC MD LD CTR MD LD CTR POS POS Amplification controls were prepared: Control DNA 007 was added to the positive amplification control (POS) TE buffer was added to the negative control (NTC) 6. Each 1.5-mL tube of reaction mix was distributed into three replicate wells on a 96-well plate and two plates (for 29 and 30 cycles) were prepared. 2.3 Capillary Electrophoresis The Amplified PCR products were run on 3500xl Genetic Analyzer instrument with standard injection parameters specified in the GlobalFiler PCR Amplification kit User Guide. Data Collection Software v3.1 was used for data collection. 2.4 Data analysis 1. GeneMapper ID-X v1.5 was used for data analysis. Allele calls, dropout of alleles, and extra non-allele peaks in electropherograms were analyzed by using the threshold at 175 RFU. Page 66

67 Chapter 5: Degraded Sample Study 2. The profiles were visually assessed, and extraneous peaks were reviewed and relabeled manually with allele edit comments. The known genotype was used to assess the electropherograms. 3. The GeneMapper ID-X data was exported to excel for further analysis. 3. Results 3.1 Quantities and Degradation Index of Artificially Degraded Samples Table 5-6. Quantities and Degradation Index of Artificially Degraded Samples measured by the Quantifiler Trio DNA Quantification Kit. Sample Name T.Small Autosomal T.Large Autosomal Degradation Degradation Cт Quantity Qty Mean Cт Quantity Qty Mean Index Index Mean Control DNA Control DNA CTRL CTRL CTRL low-degraded low-degraded low-degraded medium-degraded medium-degraded medium-degraded Allele calls Full profile was generated and 43 out of 43 (expected) alleles were detected from the non-degraded control male DNA sample at both inputs of 1ng and 0.5ng. Neither lowdegraded nor medium degraded DNA samples generated full profile at input of 1ng or 0.5ng in any PCR conditions. In general, more alleles were detected in low-degraded DNA than medium degraded DNA in all the three PCR conditions and at both inputs of 1ng and 0.5ng. In comparison of 29 cycles with additional Taq polymerase and BSA to standard 29- cycle protocol, the additional Taq and BSA was not proved to improve the allele call rate; it actually decreased the number of allele calls in most samples, whereas 30 cycles did increase the number of allele calls in both low-degraded and medium degraded DNA samples and in both inputs of 1ng and 0.5 ng. Page 67

68 Chapter 5: Degraded Sample Study Figure 5-1. Number of alleles called in Degraded Samples Number of alleles detected in degraded samples cyc-std 29cyc- TaqBSA 30cyc-std CTRL LD MD CTRL LD MD 1ng 0.5ng Table 5-7. Number of alleles called in Degraded Samples Number of alleles detected in degraded samples Input sample 29cyc-std 29cyc-TaqBSA 30cyc-std difference (30cyc-29cyc) CTRL ng LD MD CTRL ng LD MD CTRL=non-degraded control LD=low degraded DNA MD=medium degraded DNA Page 68

69 Chapter 5: Degraded Sample Study Figure 5-2. Percentage of alleles called in Degraded Samples 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Percentage of alleles detected in degraded samples CTRL LD MD CTRL LD MD 29cyc-std 29cyc- TaqBSA 30cyc-std 1ng 0.5ng Table 5-8. Percentage of alleles called in Degraded Samples Percentage of alleles detected in degraded samples Input sample 29cyc-std 29cyc-TaqBSA 30cyc-std difference (30cyc-29cyc) 100% 100% 100% 0% CTRL 100% 100% 100% 0% 100% 100% 100% 0% 62.8% 65.1% 72.1% 9.3% 1ng LD 65.1% 65.1% 67.4% 2.3% 65.1% 65.1% 79.1% 14.0% 41.9% 41.9% 55.8% 14.0% MD 44.2% 37.2% 53.5% 9.3% 34.9% 41.9% 53.5% 18.6% 100% 100% 100% 0% CTRL 100% 100% 100% 0% 100% 100% 100% 0% 53.5% 51.2% 74.4% 20.9% 0.5ng LD 53.5% 39.5% 60.5% 7.0% 53.5% 44.2% 69.8% 16.3% 39.5% 30.2% 41.9% 2.3% MD 34.9% 30.2% 51.2% 16.3% 32.6% 34.9% 41.9% 9.3% Allele calls were 100% (number of actual allele calls/expected allele calls) from the nondegraded control male DNA sample at both inputs of 1ng and 0.5ng. Page 69

70 Chapter 5: Degraded Sample Study The data showed that additional Taq Polymerase and BSA did not improve the allele call rate in the degraded DNA samples at any input; it actually did not change or even decreased the allele call rate in most samples. However, 30 cycles increased allele call rates in both low-degraded and mediumdegraded samples at both 1ng and 0.5ng input compared to 29 cycles. In the low-degraded sample, the allele call rates were increased from 64% to 73% for 1ng input and from 54% to 68% for 0.5ng input by one more cycle (30 cycles). In the medium-degraded sample, the allele call rates were increased from 40% to 54% for 1ng input and from 36% to 45% for 0.5ng input by one more cycle. 3.3 Allele dropout No allele dropout was observed in the non-degraded control male DNA sample at 1ng input or 0.5ng input. Allele dropout presented in both low-degraded and medium-degraded DNA samples in all the three amplification conditions and in both 1ng and 0.5ng input, with more allele dropouts in medium degraded sample than in low-degraded samples in all the conditions. The longer fragments were more affected than the shorter fragments (ski slope). In comparison of 29 cycles with additional Taq polymerase and BSA to standard 29- cycle protocol, the additional Taq and BSA was not proven to rescue any dropped alleles. Compared to 29 cycles, 30 cycles decreased the number of allele dropout in both lowdegraded and medium degraded DNA samples and at both 1ng and 0.5 ng input. Figure 5-3. Number of allele dropout in each replicate of degraded samples Number of allele dropout in degraded samples CTRL LD MD CTRL LD MD 29cyc-std 29cyc-TaqBSA 30cyc-std 1ng 0.5ng Page 70

71 Chapter 5: Degraded Sample Study Table 5-9. The number of allele dropout in each replicate of degraded samples Number of allele dropout in degraded samples Input sample 29cyc-std 29cyc-TaqBSA 30cyc-std difference (30cyc-29cyc) CTRL ng LD MD CTRL ng LD MD The data showed that one extra cycle (30 cycles) decreased the allele dropout rate in both low-degraded and medium-degraded samples and at both 1ng and 0.5ng DNA input, compared to standard 29 cycles, though not as significantly as observed in Sensitivity Study where more dropped alleles were recovered in low input of non-degraded DNA by one extra cycle. In the low-degraded DNA sample, the allele dropout rates were decreased from 36% to 27% for 1ng input and from 46% to 32% for 0.5ng input by one extra cycle (30 cycles). In the medium-degraded sample, the allele dropout rates were decreased from 60% to 46% for 1ng input and from 64% to 55% for 0.5ng input by one extra cycle. The data showed that additional Taq Polymerase and BSA did not rescue the dropped alleles in the degraded DNA samples at any input; it actually did not change or even increased the allele dropout rate in most degraded samples. Page 71

72 Chapter 5: Degraded Sample Study Figure 5-4. Percentage of allele dropout in each replicates of degraded samples Percentage of allele dropout in degraded samples 100% 90% 80% 70% 60% 50% 40% 30% 20% 29cyc-std 10% 29cyc-TaqBSA 0% 30cyc-std CTRL LD MD CTRL LD MD 1ng 0.5ng Table Percentage of allele dropout in each replicates of degraded samples Percentage of allele dropout in degraded samples Input sample 29cyc-std 29cyc-TaqBSA 30cyc-std difference (30cyc-29cyc) 0% 0% 0% 0% CTRL 0% 0% 0% 0% 0% 0% 0% 0% 37.2% 34.9% 27.9% -9.3% 1ng LD 34.9% 34.9% 32.6% -2.3% 34.9% 34.9% 20.9% -14.0% 58.1% 58.1% 44.2% -14.0% MD 55.8% 62.8% 46.5% -9.3% 65.1% 58.1% 46.5% -18.6% 0% 0% 0% 0% CTRL 0% 0% 0% 0% 0% 0% 0% 0% 46.5% 48.8% 25.6% -20.9% 0.5ng LD 46.5% 60.5% 39.5% -7.0% 46.5% 55.8% 30.2% -16.3% 60.5% 69.8% 58.1% -2.3% MD 65.1% 69.8% 48.8% -16.3% 67.4% 65.1% 58.1% -9.3% Page 72

73 Chapter 5: Degraded Sample Study 3.4 Off-scale peaks Off-scale peaks were only observed in the medium-degraded sample at 1ng input amplified with extra cycle, and only in shorter amplicons. This was because when the DNA input amount was calculated based on the small autosomal target for degraded DNA samples, the reaction was actually overloaded with shorter template and underloaded with longer template, which explains why off-scale peaks and allele dropout presented at the same time in one sample. Table Number of off-scale peaks in each sample of Male 7057 and Female IMR-90 DNA dilution series: Number of off-scale peaks in degraded samples Input Sample Replicates 29cyc-std 29cyc-TaqBSA 30cyc-std LD ng MD LD ng MD Page 73

74 Chapter 5: Degraded Sample Study 3.5 Pull-ups Pull-up peaks were observed only in the medium-degraded DNA samples at 1ng input amplified with extra cycle, and only in the short fragment range. This was different from the pull-up peaks observed in the Sensitivity Study and in Inhibited Sample Study, in which the pull-up peaks spread out all over the range. Pull-up peaks created flags in the genotyping quality by the software and required manually reviewing and editing by the analyst. Figure 5-5. Number of pull-up peaks in each replicates of degraded samples 3 Number of pull-up peaks in degraded samples 2 29cyc-std 1 29cyc-TaqBSA 30cyc-std 0 LD MD LD MD 1ng 0.5ng Table Number of pull-up peaks in each replicates of degraded samples Number of pull-up peaks in degraded samples Input Sample Replicates 29cyc-std 29cyc-TaqBSA 30cyc-std LD ng MD LD ng MD Page 74

75 Chapter 5: Degraded Sample Study 3.6 Increased Stutter Peaks and Additional Stutter peaks GlobalFiler marker-specific stutter filter set in GeneMapper ID-X Software was applied when analyzing the data. Peaks in the stutter position that were above the stutter filter percentage specified in the software are reported and evaluated in the section. Additional stutter peaks (e.g. 2 nd and 3 rd stutter peaks) are also included in this section. Stutter peaks presented in both low-degraded and medium-degraded DNA samples, and at both 1ng and 0.5ng input, but only with extra cycle (except for one incident at 29 cycle). Electropherograms of the increased stutter peaks and additional stutter peaks caused by extra cycle are attached to the report in the attachment section. Figure 5-6. Number of extra stutter peaks in each replicates of degraded samples 3 Number of stutter peaks in degraded samples cyc-std 29cyc-TaqBSA 30cyc-std 0 LD MD LD MD 1ng 0.5ng Table Number of extra stutter peaks in each replicates of degraded samples Number of increased and additional stutter peaks in degraded samples Input Sample Replicates 29cyc-std 29cyc-TaqBSA 30cyc-std LD ng MD LD ng MD Page 75

76 Chapter 5: Degraded Sample Study 3.7 Other Artifact Peaks Figure 5-7. Number of other extra peaks/artifacts in each replicates of degraded samples 3 Number of other extra peaks/artifacts in degraded samples cyc-std 29cyc-TaqBSA 30cyc-std 0 LD MD LD MD 1ng 0.5ng Table Number of other extra peaks/artifacts in each replicates of degraded samples Number of other extra peaks/artifacts in degraded samples Input Sample Replicates 29cyc-std 29cyc-TaqBSA 30cyc-std 1ng 0.5ng LD MD LD MD Page 76

77 Chapter 5: Degraded Sample Study 4. Conclusions One extra cycle (30 cycles) decreased the allele dropout rate in both low-degraded and medium-degraded samples and at both 1ng and 0.5ng DNA input, compared to standard 29 cycles. The effectiveness of one extra cycle on degraded sample is less significant compared to that observed in Sensitivity Study where non-degraded DNA sample was used. In the degraded samples, 9% to 14% of alleles were recovered by one extra cycle compared to 20% to 44% in non-degraded DNA sample. One extra cycle could improve the genotyping result of low-degraded sample at both 1ng and 0.5ng input by recovering dropped alleles while generating minimal artifacts. It also added benefit medium-degraded sample at 0.5ng input in the same way. However, extra cycle did not work well on 1ng of medium-degraded sample, because of the off-scale peaks, pull-up peaks, extra stutter peaks and other artifacts it generated. Additional Taq Polymerase and BSA did not rescue the dropped alleles in the degraded DNA samples at any input; it either did not change or even increased the allele dropout rate in most degraded samples. Table Summary of limitation of extra cycle for degraded samples Summary of limitation of extra cycle for degraded samples Input Sample Replicates 29cyc-std 29cyc-TaqBSA 30cyc-std 1ng 0.5ng LD MD LD MD 1 2 ART ART 3 ST/ART 1 ST/PU/OS 2 PU/OS/ART 3 PU/OS/ART 1 ST 2 ST 3 ST 1 2 ST/ART 3 ST Page 77

78 Chapter 5: Degraded Sample Study 5. Attachment Figure 5-8. Full profile was generated in non-degraded control DNA sample at 29 cycles. Total detected allele number was 43. No dropout. Page 78

79 Chapter 5: Degraded Sample Study Figure 5-9. Low-degraded DNA sample at 1ng input was amplified at 29 cycles. Number of alleles detected was 28, and number of allele dropout was 15. No pull-up peak, extra stutter peak or artifacts presented. dropout dropout dropout dropout dropout dropout dropout Page 79

80 Chapter 5: Degraded Sample Study Figure Low-degraded DNA sample at 1ng input was amplified at 30 cycles. Number of allele call increased to 34 from 28 (at 29 cycles), and number of allele dropout decreased to 9 from 15 (at 29 cycles). A pull-up peak and an increased stutter peak presented. pull-up stutter Page 80