globalfilertm extra cycle evaluation report...this chapter provides detailed information about...

GlobalFiler extra PCR cycles evaluation report

GlobalFiler

TM Extra Cycle Evaluation Report

Table of Content

Chapter 1 Summary

1. Introduction .................................................................................................................................... 2

2. Systems, Kits and Conditions .................................................................................................... 3

3. Studies ............................................................................................................................................... 4

Chapter 2 Minimum Threshold and Background Study

1. Study Overview .............................................................................................................................. 7

2. Experimental Design and Data Analysis ............................................................................... 7

3. Results ............................................................................................................................................... 8

4. Conclusions .................................................................................................................................... 13

Chapter 3 Sensitivity and Stochastic Study

1. Study Overview ............................................................................................................................ 14

2. Experimental Design and Data Analysis ............................................................................. 14

3. Results ............................................................................................................................................. 18

4. Conclusions .................................................................................................................................... 42

5. Attachment .................................................................................................................................... 44

Chapter 4 Inhibited Sample Study

1. Study Overview ............................................................................................................................ 49


3. Results ............................................................................................................................................. 51

4. Conclusion ...................................................................................................................................... 59

5. Attachment .................................................................................................................................... 60

Chapter 5 Degraded Sample Study

1. Study Overview ............................................................................................................................ 64


3. Results ............................................................................................................................................. 67

4. Conclusions .................................................................................................................................... 77

5. Attachment .................................................................................................................................... 78


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 v1.5.

1. 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.

Chapter 1: Summary


2. Systems, Kits and Conditions The following components were used to perform the studies summarized in this report:

Kits

Quantification kit Quantifiler

TM Trio DNA Quantification Kit

Part Number 4482910

Amplification kit GlobalFiler PCR Amplification Kit Part Number 4476135

Instruments

Real-time PCR system Applied Biosystems 7500 Real-Time PCR System Serial no. 275007979

Thermal cycler GeneAmp™ PCR System 9700 Thermal Cycler Serial no. G96S2013139, G96S8100368, G96S5080107

Genetic analyzer Applied Biosystems 3500xl Genetic Analyzer Serial no. 21101-031

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

Parameters

Amplification 29, 30, 31 cycles

Injection 1.2 kV 24 seconds

The following conditions were used to perform the studies summarized in this report:

Process Conditions

Quantification

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

Amplification

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

Genetic analysis

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.

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.

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).

29 cycles 30 cycles 31 cycles 29 cycles 30 cycles 31 cycles

Blue (6-FAM™) 5 6 5 35 55 65

Green (VIC™) 10 11 8 60 80 75

Yellow (NED™) 4 5 4 40 50 45

Red (TAZ™) 8 8 7 40 45 40

Purple (SID™) 9 9 7 40 45 35

Calculated Minimum ThresholdDye

Average Peak Height of Background

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

Goal

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.

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.

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. 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 0.031 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.

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.


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 v1.5.

1. 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.


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.

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) 54 5.35 2.85 13.91 33.88 35

Green (VIC) 96 9.97 5.22 25.63 62.14 60

Yellow (NED) 96 4.44 3.69 15.52 41.36 40

Red (TAZ) 80 7.76 3.18 17.29 39.55 40

Purple (SID) 50 8.63 3.13 18.01 39.89 40




Standard Deviation




Blue (6-FAM) 54 6.24 4.69 20.31 53.13 55

Green (VIC) 99 10.96 6.71 31.09 78.06 80

Yellow (NED) 100 4.80 4.74 19.02 52.20 50

Red (TAZ) 78 8.27 3.71 19.40 45.38 45

Purple (SID) 38 9.16 3.52 19.72 44.38 45






Standard Deviation




Blue (6-FAM) 78 5.26 6.10 23.57 66.28 65

Green (VIC) 85 8.45 6.71 28.57 75.52 75

Yellow (NED) 81 4.10 4.18 16.63 45.86 45

Red (TAZ) 80 6.54 3.59 17.32 42.46 40

Purple (SID) 31 6.66 2.84 15.18 35.08 35

Figure 2-1. Comparison of maximum peak height of back ground noise of three cycle conditions.

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.

0

20

40

60

80

100

120

Blue(6-FAM)

Green(VIC)

Yellow(NED)

Red(TAZ)

Purple(SID)

Maximum Peak Height of Background (RFU)

29 cycles

30 cycles

31 cycles



Figure 2-2. Comparison of average peak height of back ground noise of three cycle conditions.

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.

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.

2

4

6

8

10

12

Blue(6-FAM)

Green(VIC)

Yellow(NED)

Red(TAZ)

Purple(SID)


29 cycles

30 cycles

31 cycles

5

10

15

20

25

30

35

Blue(6-FAM)

Green(VIC)

Yellow(NED)

Red(TAZ)

Purple(SID)

LOD (RFU)

29 cycles

30 cycles

31 cycles



Figure 2-4. Comparison of LOQ of the three cycle conditions.

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.

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.

10

20

30

40

50

60

70

80

90

100

Blue(6-FAM)

Green(VIC)

Yellow(NED)

Red(TAZ)

Purple(SID)

LOQ (RFU)

29 cycles

30 cycles

31 cycles

0

10

20

30

40

50

60

70

80

90

100

Blue(6-FAM)

Green(VIC)

Yellow(NED)

Red(TAZ)

Purple(SID)


29 cycles

30 cycles

31 cycles



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





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.


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 v1.5.

1. Study Overview


3. Results

4. Conclusions

1. Study Overview

Goal

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.

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.


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.

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 7.195 ng/µL 7057 stock.

Dilution tube Final Target

concentration (ng/µL)

DNA from tube DNA volume (µL)

from stock or previous tube

TE-4

buffer volume (µL)

M-A 2.0 stock 83.4 216.6

M-B 1.0 A 150 150

M-C 0.5 B 150 150

M-D 0.25 C 150 150

M-E 0.125 D 150 150

M-F 0.0625 E 150 150

M-G 0.0313 F 150 150

M-H 0.0156 G 150 150

3. 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 8.417 ng/µL IMR-90 stock.

Dilution tube Final Target

concentration (ng/µL)

DNA from tube DNA volume (µL)

from stock or previous tube

TE-4

buffer volume (µL)

F-A 2.0 stock 71.3 228.7

F-B 1.0 A 150 150

F-C 0.5 B 150 150

F-D 0.25 C 150 150

F-E 0.125 D 150 150

F-F 0.0625 E 150 150

F-G 0.0313 F 150 150

F-H 0.0156 G 150 150



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, 0.0625, 0.03125, and 0.0156 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 7057

DNA input

Number of replicates of Male DNA 7057

Female IMR-90

DNA input

Number of replicates of Female DNA IMR90

29 cycles 30 cycles 31 cycles 29 cycles 30 cycles 31 cycles

2ng 3 3 3 2ng 3 3 3

1ng 3 3 3 1ng 3 3 3

0.5ng 3 3 3 0.5ng 3 3 3

0.25ng 3 3 3 0.25ng 3 3 3

0.125ng 3 3 3 0.125ng 3 3 3

0.063ng 3 3 3 0.063ng 3 3 3

0.031ng 3 3 3 0.031ng 3 3 3

0.016ng 3 3 3 0.016ng 3 3 3

1. 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.

7057 DNA dilutions from step 2

Quant Trio Results (ng/µL)

Final Input

(ng)/rxn

Volume (µL)/rxn

Master tubes for 12 reactions

TE (µL) DNA (µL) Master

Mix (µL)

M-Neg1 180.0 0.0 120

M-Neg2 180.0 0.0 120

M-A_2.0ng 1.800 2 1.1 166.7 13.3 120

M-B_1.0ng 1.050 1 1.0 168.6 11.4 120

M-C_0.5ng 0.525 0.5 1.0 168.6 11.4 120

M-D_0.25ng 0.259 0.25 1.0 168.4 11.6 120

M-E_0.125ng 0.126 0.125 1.0 168.1 11.9 120

M-F_0.063ng 0.070 0.0625 0.9 169.3 10.7 120

M-G_0.031ng 0.036 0.03125 0.9 169.6 10.4 120

M-H_0.016ng 0.017 0.015625 0.9 169.0 11.0 120

M-POS 60.0 120.0 120



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 dilutions from step 3

Quant Trio Results (ng/µL)

Final Input

(ng)/rxn

Volume (µL)/rxn


TE (µL) DNA (µL) Master

Mix (µL)

F-Neg1 180.0 0.0 120

F-Neg2 180.0 0.0 120

F-A_2.0ng 1.740 2 1.15 166.2 13.8 120

F-B_1.0ng 0.960 1 1.04 167.5 12.5 120

F-C_0.5ng 0.478 0.5 1.05 167.4 12.6 120

F-D_0.25ng 0.230 0.25 1.09 167.0 13.0 120

F-E_0.125ng 0.115 0.125 1.09 167.0 13.0 120

F-F_0.063ng 0.054 0.0625 1.16 166.0 14.0 120

F-G_0.031ng 0.031 0.03125 1.01 167.9 12.1 120

F-H_0.016ng 0.016 0.015625 0.98 168.2 11.8 120

F-POS 60.0 120.0 120

3. 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 re-labeled 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.



3. Results

3.1 Peak heights

Figure 3-1. Average heterozygous peak heights (RFU) for the 7057 dilution series -29 cycles

Figure 3-2. Average heterozygous peak heights (RFU) for the IMR-90 dilution series -29 cycles

9 303 575 1,485

2,771

5,085

10,318

16,412

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0.016ng 0.031ng 0.063ng 0.125ng 0.25ng 0.5ng 1ng 2ngAv

era

ge H

ete

rozyg

ou

s P

eak H

eig

ht

(RF

U)

Input DNA

7057 GlobalFiler (29c - 24s) Sensitivity

28 207 685 1,411

3,479

6,101

12,276

18,564

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0.016ng 0.031ng 0.063ng 0.125ng 0.25ng 0.5ng 1ng 2ng

Av

era

ge H

ete

rozyg

ou

s P

eak H

eig

ht

(RF

U)

Input DNA

IMR-90 GlobalFiler (29c - 24s) Sensitivity





224 516 958

2,521

5,221

9,678

18,737

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0.016ng 0.031ng 0.063ng 0.125ng 0.25ng 0.5ng 1ng

Av

era

ge H

ete

rozyg

ou

s P

eak H

eig

ht

(RF

U)

Input DNA


164 448 1,172

2,734

5,624

10,504

21,252

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

0.016ng 0.031ng 0.063ng 0.125ng 0.25ng 0.5ng 1ng

Av

era

ge H

ete

rozyg

ou

s P

eak H

eig

ht

(RF

U)

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.

268 879

1,824

4,470

11,521

15,426

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0.016ng 0.031ng 0.063ng 0.125ng 0.25ng 0.5ng

Av

era

ge H

ete

rozyg

ou

s P

eak H

eig

ht

(RF

U)

Input DNA


272 731

2,279

4,796

11,626

15,591

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0.016ng 0.031ng 0.063ng 0.125ng 0.25ng 0.5ng

Av

era

ge H

ete

rozyg

ou

s P

eak H

eig

ht

(RF

U)

Input DNA




Figure 3-7. Peak heights (RFU) for each allele detected in the 7057 dilution series -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

X Y 11 14 13 16 21 13 14 10 12 10 13 13 14.2 15.3 18.3 28 30 15 16 19 24 10 14 15 17 10 12 10 12 12 13 11 19 22 31.2 6 9.3 8 16 17 2

0.5ng 1 5946 5939 3787 3513 5589 5590 8464 7014 5574 4295 4445 5757 6139 2704 2031 4410 4304 3413 5128 4512 4348 4211 4539 5265 4206 4749 4550 4911 4474 5460 5202 4929 7050 4962 8910 4406 5667 4799 3327 6360 4063 3629 6898

2 6050 6220 3886 4479 5541 5541 9356 7491 6493 5305 4934 6253 5451 3666 4265 6702 7157 3888 5275 5312 5896 7733 4894 5387 5587 5635 4545 5740 7539 6924 5832 6808 7276 7606 10256 7375 5618 5038 5508 6667 4976 5319 6730

3 6066 6125 3697 4545 5507 4911 9515 7943 6617 3955 3993 5351 5247 3058 2572 5369 4862 4805 3516 5768 4188 5274 4757 3503 4137 5149 5909 4294 5191 5110 5916 5953 5948 5609 8331 6833 4372 4511 3573 5212 4674 3913 5539

0.25ng 1 3774 3256 2999 2840 3380 3009 4555 4118 3934 3495 2615 3122 2936 1851 1563 3219 2913 1777 1041 2815 2790 3700 3323 1396 1959 2588 4047 2785 2683 4331 3388 2712 3059 3847 5105 3725 2647 2558 2204 3371 2638 2471 3258

2 4917 3852 1978 1595 2561 2231 4460 3445 2824 1967 1878 2244 2752 1615 1095 1795 2425 1322 1348 2991 2208 3270 2105 1816 2049 2625 3025 2943 2531 2395 2815 3637 3309 2542 4209 2645 3015 1777 1765 2918 2016 1902 3003

3 4064 3064 2956 2354 3923 2908 6272 4195 3814 3037 2388 3334 3133 1918 1934 3533 3886 2308 2898 2323 2855 2162 2614 2877 3170 3568 2903 3696 4383 2712 3063 4268 4183 2111 4878 3175 3040 2587 2354 4113 3098 2960 4797

0.125ng 1 2724 2074 1394 1293 2070 1872 3033 3044 1675 876 1308 1934 1169 1152 1031 1682 941 1768 1004 1776 1607 2380 1268 1167 1296 1615 1594 1536 2097 1247 771 1573 2063 1758 2749 1617 1699 1476 1046 1522 1148 956 1528

2 1338 1394 1132 1485 1272 1993 3274 2877 1363 895 1576 1056 1653 554 758 1429 1540 661 1260 1083 788 2375 1894 1032 953 984 683 1477 1346 813 2007 1324 1525 1351 2156 1342 1886 1175 1396 1350 1437 1652 1887

3 2666 2416 1646 1698 1322 2448 2963 2446 1625 1255 1033 2026 1698 1009 1406 1216 2460 1168 1731 1728 1362 1747 1956 633 1805 2122 2360 1721 904 1026 1155 2346 1572 1344 2966 2815 1577 892 1149 1527 1221 1161 1825

0.063ng 1 765 427 640 891 429 298 994 1095 338 536 491 822 1062 176 392 713 215 322 453 525 646 872 841 200 518 596 775 397 436 742 424 854 687 779 841 485 393 634 229 602 354 208 1097

2 425 345 732 659 1147 437 1679 234 419 814 501 947 363 472 280 405 530 437 361 363 749 1000 623 514 178 299 248 494 926 473 424 600 730 1162 788 421 276 719 771 602 613 716

3 788 188 708 487 201 476 1243 571 788 850 418 561 971 374 458 799 405 386 589 227 502 1208 985 619 412 630 413 954 709 1448 577 410 400 1101 757 937 439 402 1146 593 770 780

0.031ng 1 565 251 780 470 220 309 340 332 265 370 209 179 214 228 191 372 336 518 849 491 276 194 476 343 232

2 571 310 483 246 309 586 580 488 321 227 308 267 295 409 323 667 218 297 274 184 234 178 270 483 261 548 204 191 272 229 292

3 819 399 295 319 1037 271 360 286 272 517 178 634 370 216 273 359 923 194 462 276 420 427 321 577 441 564 206 387 1006 268 393 344 210 486 651

0.016ng 1 190 176 236 188

2 347 228 176 287 182

3 177 294 188 175 178

29 cycles

Male


X Y 11 14 13 16 21 13 14 10 12 10 13 13 14.2 15.3 18.3 28 30 15 16 19 24 10 14 15 17 10 12 10 12 12 13 11 19 22 31.2 6 9.3 8 16 17 2

0.5ng 1 19471 10411 9542 7312 13237 7638 16194 13573 14382 9644 8065 11910 10224 7845 6480 8486 10916 6396 8714 10185 10570 8332 8288 8957 8832 10348 9499 11931 11516 11803 12943 15395 9127 9933 19371 10603 11595 7427 6637 10566 11624 8301 13541

2 16570 12442 6772 7686 11895 9760 16419 14378 14538 7746 8965 12257 8120 7797 5544 9025 7926 8330 7410 8917 7415 7882 8843 6123 9320 8741 10544 11845 10580 10537 8688 10136 9600 9883 14116 11317 13479 6575 7505 11675 7999 9449 14969

3 12548 13165 7806 7436 10161 9005 18456 14063 11285 9389 8889 11912 8850 6059 6934 9351 7768 8016 8010 10174 9147 12692 9486 11077 8673 13670 11769 9248 10908 10068 7932 11250 13966 8626 19446 7971 8398 6935 8096 12054 7509 8916 11046

0.25ng 1 9463 5533 5804 4309 6531 5886 12316 6167 7852 5418 3929 4704 6917 3388 2950 5188 6512 3377 3610 3852 4012 3653 3150 5643 4392 6726 5624 5743 4866 6227 5955 8685 7579 4317 9159 5032 5076 4638 5319 6697 4614 4343 8673

2 5879 5269 4811 4031 3812 5349 8728 5705 6148 5637 4747 5416 6693 2952 3524 6612 7073 4433 2408 5918 5995 5422 5397 4615 3780 4780 5776 4890 4121 5480 4375 6219 3977 6180 10556 5913 5778 4488 3054 5697 3995 4911 6732

3 6806 5982 4148 4552 4047 3684 9267 10056 5372 4668 5473 3758 5000 3858 3620 6600 5299 2957 4036 4970 5603 6433 4833 5803 6159 4659 6966 5088 7615 6702 4828 8875 6475 5588 12615 4265 6895 5731 4067 4986 3883 3558 6747

0.125ng 1 4650 4329 2350 1665 3143 2987 7612 3967 2481 1474 1738 2113 1952 1854 1539 3463 2510 3081 1892 2512 2716 1718 2133 2647 1484 2702 1228 2682 3045 2074 3159 2614 2916 1520 3813 4920 3133 1840 1162 2857 2968 1478 2509

2 2904 2538 2849 1494 2932 3169 4732 4360 3226 3275 2229 1763 1339 2189 2380 1753 1965 2662 2336 2395 1173 2022 884 1405 1968 1843 2042 4091 2883 2632 3264 3829 2702 2214 4136 1480 1734 2602 3035 2629 2540 1875 2317

3 4161 3073 2873 1928 4075 2962 5362 3627 4465 2378 1124 2757 2401 1928 2067 3143 4037 2571 1786 2375 3156 2170 1878 2813 2156 1004 3621 2811 2713 4419 2336 4711 3341 2387 4573 3093 3265 2776 2547 3768 3075 2303 2847

0.063ng 1 2077 859 806 699 1161 537 2183 1432 2715 428 833 1501 834 513 841 864 382 497 696 1359 746 1182 865 1129 1407 1078 550 1051 181 576 1027 782 1100 1425 1883 885 540 896 774 472 720 727 1319

2 1710 791 298 1024 1097 484 1722 2193 1708 804 990 1655 962 959 286 787 579 720 585 1183 710 604 1875 698 333 522 809 1795 1786 1875 864 1960 938 591 2409 1089 1564 1342 1651 1351 1724 1392 633

3 1774 387 471 1367 636 1134 2875 1076 855 910 609 982 950 886 474 1623 949 556 822 706 1542 1210 515 593 1194 800 1138 860 943 657 1062 1233 583 1770 1125 1262 685 779 726 373 653 797

0.031ng 1 1587 901 541 287 913 1527 415 507 189 520 318 267 477 872 233 208 288 567 889 362 578 864 534 719 257 877 459 604 1018 706 1050 271 487 651 672 639 331 233

2 288 522 516 649 512 597 303 194 503 468 409 732 572 221 188 344 357 299 535 490 681 846 352 1124 372 715 1049 355 284 207 400 348 508 1067 253 206 421 188 775 492

3 1045 501 400 672 543 662 407 1180 691 302 591 307 219 626 537 865 844 1415 664 613 259 276 713 647 332 274 553 1003 1075 461 1296 982 322 582 375 1437 628 495

0.016ng 1 579 346 230 707 443 396 261 266 310 381 242 187 376 278 212 370 790 833 452 579 258 423 549

2 234 191 301 880 553 268 324 361 323 205 482 235 398 347 357 381 721 368 216 199 407 305

3 243 271 502 556 266 237 260 388 494 385 210 215 434 277 414 220 444 428 482 277 464 466 176 219

30 cycles

Male


X Y 11 14 13 16 21 13 14 10 12 10 13 13 14.2 15.3 18.3 28 30 15 16 19 24 10 14 15 17 10 12 10 12 12 13 11 19 22 31.2 6 9.3 8 16 17 2

0.5ng 1 26223 16631 11498 10807 12764 12069 24950 17498 19294 14332 11691 15627 12732 7967 8387 13812 13938 12229 10004 11932 12523 15194 10031 16506 14210 15653 13531 15597 13527 13620 17360 19427 20059 11191 30016 14109 11512 13417 12067 15830 15238 10086 24343

2 29724 16757 13277 15680 18425 12633 30706 20615 21916 14745 15802 20812 16714 11842 12022 18345 20256 18577 9339 17460 16978 16548 15953 17797 13053 19217 17528 15771 18449 21753 15623 24025 26928 13270 31236 17481 20870 18784 12264 21069 16175 12654 22587

3 23886 19561 10601 10643 14975 13583 25620 18220 19542 16035 12903 17840 14899 12357 10494 15563 15932 8710 13668 14542 15307 17520 17456 15763 11855 14346 12684 15275 16143 15392 11869 23744 20497 17774 30144 13208 16198 17219 12003 17156 13010 10816 23102

0.25ng 1 13933 10076 7529 6960 9502 7112 15033 12184 15019 7876 7875 10615 9190 6555 6894 9890 9817 6991 6498 10139 8408 10305 10538 9193 11134 8946 9767 11430 7516 8007 11139 10102 9038 6672 18378 10467 9919 6675 9947 10557 7762 8579 13206

2 15121 19254 10773 11271 10226 6858 23583 24176 15807 13385 11130 12978 14157 6687 7452 13367 10758 10680 7877 11966 13091 13888 12924 13202 9310 13152 14738 14288 12955 13012 10588 18359 17176 13643 21027 15802 12003 14034 10790 12392 11274 10722 15534

3 18535 19828 13185 7922 15837 12939 28246 13710 16892 9180 11792 14687 11576 11213 9986 11070 14782 10730 9738 8151 8665 16016 8818 13452 10857 13673 14394 13334 14520 10331 10173 16045 12173 16268 24487 17884 24229 12992 9029 19862 11441 11876 12197

0.125ng 1 7756 4066 2953 2550 3390 2503 8321 5218 5931 5452 3425 3866 4914 2443 2617 3648 5608 3226 3642 4395 3196 3978 4384 4404 4068 4471 5441 3321 4319 4486 3736 3401 4845 1538 8025 4387 4036 3329 2462 4209 4148 4001 3036

2 4845 4101 3349 2895 3298 5333 5072 3847 7847 3406 2260 4250 1944 2491 2297 4133 3622 3128 2861 3422 1846 3569 2849 3923 2909 3323 1846 3962 3889 4855 4198 3797 4277 1374 8535 3720 3911 4012 3472 3938 3395 3625 6008

3 6781 8837 4558 3014 6566 4929 10373 6213 6145 5278 5734 6833 8766 4793 4263 4825 5379 4017 5983 6763 7635 5284 4427 4693 5862 5297 6316 7355 4309 7308 6212 9502 6826 8329 8090 7902 4520 6973 7560 8595 5937 7771 7949

0.063ng 1 2990 3946 1781 2555 1784 1037 3573 1858 1344 1064 3478 528 251 384 1386 1369 1464 1820 878 1065 1785 1259 1863 1073 610 2444 1953 1313 2213 1765 1481 957 1871 3447 5148 1031 2445 1252 1952 1753 1753 1856 2152

2 2562 1671 1707 1085 2155 1654 2313 3469 3018 564 1292 1500 993 2440 1514 3007 1456 1242 2307 1504 1536 840 722 1001 1302 1274 2774 1949 2398 5276 2995 2000 1950 2046 2858 1056 2364 1811 663 2933 913 3285 1172

3 2589 3585 1871 471 538 3419 1084 2474 1411 1048 1837 3737 2226 858 1576 1471 1934 357 1584 1649 1501 1485 1468 2491 2021 2153 2479 4334 463 4889 2811 2986 2411 3087 1693 2444 2921 1002 1967 1119 1670 2523

0.031ng 1 803 883 513 714 416 1079 805 365 619 902 378 557 766 441 1363 472 290 618 701 849 873 758 232 419 848 743 298 663 313 808 1237 700 629 1254 726 1029 485 535 822 307 1175

2 701 1501 1583 1003 1297 1084 1853 2268 1546 815 262 1139 218 474 1647 1140 1471 535 881 1078 564 2209 1089 487 342 287 350 858 789 963 2068 352 1200 567 1184 1070 865

3 734 1532 249 1204 1503 777 2431 1108 301 1678 1062 1297 294 439 1910 1464 772 2360 941 257 1829 518 262 1173 1408 388 1258 1089 1641 771 2421 1281 1133 2709 720 1689 450 406 1085 892 1475

0.016ng 1 180 225 281 366 343 288 329 483 439 189 565 411 420 670 252 280 373 273 231 501 246 175 268 221

2 292 272 213 669 408 192 499 286 193 326 232 220 342 183 279 269 238 351 294 352 371 182 384 276 325 203 660

3 351 574 655 296 334 500 317 312 464 305 226 450 307 888 431 984 309 191 295 231 782 986 252 647 795 723 239 322 1142

31 cycles

Male



Figure 3-10. Peak heights (RFU) for each allele detected in the IMR-90 dilution series -29 cycles



AMEL CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vWA

X 11 13 13 14 18 21 11 13 10 13 17 10 13 11 15 30.2 31 12 16 19 25 11 14 15 12 13 12 9 13 14 25 26 18 26.2 8 9.3 8 9 16 19

0.5ng 1 13818 4711 3964 7290 5148 3619 4673 5908 8147 5017 4372 10567 3984 4075 6321 6331 3432 4396 5609 4551 5811 6614 9672 5663 5518 6506 5487 5155 5841 7746 6087 4281 4381 5784 6380 5004 4744 3820 3681 4665 4039

2 16430 4929 5202 8577 7104 5760 5884 7842 8334 4725 4605 11774 3839 3945 7099 7192 5694 5391 6253 5175 7530 7573 10795 7099 5989 6952 6762 6429 8873 8293 7613 5833 4531 6910 7539 5173 5250 3618 4570 4093 5023

3 18629 5765 4599 8180 6574 5369 6402 10295 8717 5822 4191 14094 5299 5020 7947 6386 6256 6444 6969 5689 8599 8976 12428 6600 7256 7205 8848 6967 7737 9130 8694 6529 5479 8097 8255 4990 6453 4541 4783 5226 4820

0.25ng 1 10548 4244 2011 5475 4195 3708 3459 4502 4696 2894 2450 7644 2474 2336 5170 4830 3366 2565 2756 4309 5269 5683 6435 3505 4483 2830 3854 4325 5153 3808 4321 3879 3590 5272 4981 3649 2859 2604 2733 3750 3109

2 7689 2230 2183 3655 3380 2490 2947 3930 4133 2901 2018 5492 1996 1675 3897 3473 2225 2560 2872 2537 3187 3539 4496 3181 2868 3970 3409 3274 3412 4034 3271 1870 1904 3102 3906 2193 2029 1586 2447 2576 1949

3 9693 2890 4018 4656 4669 3655 3349 5129 5622 2682 2907 6604 2369 2131 5020 5124 3188 3394 3001 2865 5310 5346 6439 3728 3992 4338 4629 4078 3949 3640 3819 3101 2287 4788 4207 2857 2594 2660 2662 2959 2688

0.125ng 1 3710 841 1454 1354 1254 1620 877 1509 1436 1500 786 2431 830 826 1420 1727 1150 1150 1483 794 1714 1504 1523 1525 1411 1118 1206 1274 1476 1861 1100 1583 963 1196 1329 790 1119 668 943 725 894

2 4280 1410 875 1912 1411 1659 1692 1697 2156 782 671 2620 1002 635 1236 1150 1399 1222 1408 1501 1783 1709 1911 2222 909 1477 1239 1179 1863 1326 845 1318 1292 1684 1725 992 1106 763 832 1240 1041

3 3820 2004 1469 2025 1888 1377 1919 1800 2543 1672 1447 2955 1598 1179 1723 1690 1401 989 1005 1375 2975 2008 2596 1911 1330 1875 2276 1939 2901 2057 2300 1285 1489 2120 1528 1476 1318 510 1039 1136 994

0.063ng 1 2290 515 632 820 1000 379 574 1046 1152 583 329 1248 282 453 505 998 466 338 762 380 891 1374 1149 730 794 882 745 881 1036 784 688 550 276 609 610 808 513 385 367 629 768

2 1204 678 405 789 528 423 824 927 814 632 516 1068 257 383 729 392 707 504 728 871 413 870 1110 702 685 747 832 501 766 854 705 446 499 751 901 340 567 631 452 255 547

3 2096 799 643 1337 930 690 719 1135 1135 526 536 1922 675 599 453 709 695 417 678 493 922 413 956 640 973 824 1289 673 1146 614 1013 474 602 909 921 676 875 603 384 549 650

0.031ng 1 323 180 219 179 283 663 209 184 177 483 283 269 349 240 238 266 217 279

2 483 333 339 243 272 278 400 247 276 179 287 396 414 392 282 283 234 262 179 337 337 231 234 216

3 661 247 456 192 322 192 324 230 471 320 325 278 186 265 418 482 587 214 291 296 300 471 195 372 226 189 251

0.016ng 1 211 180 212 197

2 404 210 209 218

3

29 cycles

Female


X 11 13 13 14 18 21 11 13 10 13 17 10 13 11 15 30.2 31 12 16 19 25 11 14 15 12 13 12 9 13 14 25 26 18 26.2 8 9.3 8 9 16 19

0.5ng 1 27379 8004 8575 11314 10547 6954 8238 10891 14027 8492 7656 20337 7228 6745 10223 9980 8194 7741 9524 8405 11693 11057 17124 8616 8591 9629 11526 9552 11192 10388 9388 8829 8304 10699 12148 9008 9783 6039 5665 8676 6813

2 30292 9796 9346 13800 12293 9292 8811 14076 14037 8902 10908 22736 9452 8134 12245 9380 9721 7842 10142 8114 14750 11916 20670 11910 10135 11272 11524 10867 15383 15681 13427 8557 9231 11795 13009 9598 11527 6788 6197 8014 7963

3 30114 10372 8129 12640 9719 10484 10548 15678 15665 10807 8578 23439 8348 7982 12266 11746 9543 6928 9246 7940 16312 11476 21002 11245 10963 10865 11809 13471 14108 14854 13785 11223 9426 14897 11071 10973 10948 7591 6819 9884 8950

0.25ng 1 15920 3787 4370 7571 7284 4917 5829 8478 8192 5894 5030 11706 4264 4768 8079 7504 5825 5996 4304 3035 7597 7151 10023 6749 5586 6817 7252 6347 7037 6948 8199 5379 4912 8060 5497 5000 4127 4360 3473 4954 5464

2 13257 4399 3755 5479 4631 4647 3783 7299 7846 4478 4051 10030 3727 3183 5035 4413 3834 4123 4335 4227 6051 5322 8055 4065 5044 5518 5453 4210 5707 4763 5880 3901 2889 4585 4239 3917 4816 2663 2816 3642 3157

3 19304 5028 5802 7580 8385 6000 3905 8257 7170 6865 4850 12447 4449 4291 8457 5827 3936 4600 5502 5787 8726 6731 10326 7575 6928 7631 7806 7360 8758 7392 5835 5210 4488 6824 7114 4685 6212 3831 3452 5244 4865

0.125ng 1 8240 2848 1916 3802 3343 2538 2134 2560 3171 2089 2573 5429 1544 2549 1267 3173 3182 1242 2321 3001 3082 4200 4884 2946 2568 2622 2763 3643 2184 2663 2754 2489 2155 3036 3153 2467 3017 1595 1305 2905 2952

2 5728 2674 1965 3717 3432 3211 2942 2641 3782 2577 2706 6204 1893 1731 2534 2657 1780 2214 2870 1734 3523 3443 5377 2455 2550 3146 2389 2200 2955 3130 2898 2762 2758 3617 3304 2319 1895 1557 1615 2044 2386

3 7480 2528 1596 4437 4109 2301 1997 5024 5367 2542 3201 5566 1681 2415 3592 1459 2100 1635 1879 2532 2601 4756 4320 2355 2588 3592 4428 3869 3818 2355 2788 2528 2589 3579 3153 1786 2140 2326 1838 2208 2253

0.063ng 1 4503 1116 1478 1275 1191 1235 626 1347 1876 1130 588 1844 726 668 842 1610 627 314 1448 1230 1162 1435 1981 1531 1084 1176 865 972 1199 1038 892 1037 706 878 862 771 1007 884 868 827 954

2 3275 1261 1266 2031 1552 821 1074 1269 970 746 867 2163 786 1033 1499 1143 1242 1258 1356 768 1739 885 3114 1978 1867 824 792 1307 1129 1653 1636 2025 707 777 1676 1136 1135 901 588 1143 885

3 3515 1155 1322 497 1049 1119 900 1790 2549 396 879 2011 825 1327 879 1233 1106 620 1508 654 1005 920 1865 1558 1338 1567 1164 1560 1026 1917 1600 1102 1498 1254 1379 1239 887 465 1069 1605 1317

0.031ng 1 1152 738 674 517 519 839 261 404 989 327 376 989 433 403 677 459 424 368 556 1287 207 609 288 323 239 353 406 649 312 294 911 621 460 645 586 624 282 302

2 1203 573 420 512 400 210 580 351 183 272 430 551 394 527 198 316 378 303 314 354 239 475 366 199 266 303 344 176 439 179 274 287 276 241

3 719 316 627 489 599 579 566 463 403 1109 307 248 668 359 588 323 379 1279 1117 679 436 702 1067 1043 541 333 635 389 584 922 441 479 692 262 356 452 384

0.016ng 1 790 456 200 377 579 289 281 432 203 430 201 301 603 270 264 256 447 459 433 198 257 232 217 314

2 535 272 264 199 258 204 227 300 444 209 191 260 246 192 264 199

3 526 266 179 252 211 280 504 264 188 380 209 259 414 278 192 246 292 277 319 201 311 277 339

30 cycles

Female


X 11 13 13 14 18 21 11 13 10 13 17 10 13 11 15 30.2 31 12 16 19 25 11 14 15 12 13 12 9 13 14 25 26 18 26.2 8 9.3 8 9 16 19

0.5ng 1 30327 9680 14346 16368 17081 19083 15820 19385 23195 16657 15452 29459 15055 12959 17644 14629 16145 13087 16326 12503 21152 22540 31357 18097 15834 16609 17170 17691 17730 22106 24331 14873 14008 17984 17328 16240 17865 13387 12099 15018 13522

2 29524 12872 12916 19027 17526 16640 16619 22810 21348 12988 16207 30891 16559 11580 14916 18608 17656 14720 16100 13064 24506 21465 31451 15925 15477 19187 17691 18723 21461 22518 19989 19139 17652 25789 20397 17007 19285 13196 11973 13680 13626

3 30856 10433 10287 15184 11395 8230 10069 15543 19457 12718 10802 27387 11217 11259 14600 15908 13075 10987 11835 11279 17684 13152 27146 15697 13750 13795 13958 13005 16029 18389 19909 13259 10579 15243 13839 13137 14283 8825 8476 9570 10851

0.25ng 1 30534 10952 11121 16529 16153 12976 12747 22125 18101 11536 11958 29217 7831 8123 18544 15922 14287 13692 10471 9985 18380 16490 23152 12324 13263 16668 14444 16615 17722 19641 16852 16684 9955 16401 18609 9773 12164 9223 8484 11812 14024

2 30781 9112 8236 12261 10738 9680 8459 16200 14714 10006 10748 22113 8313 9015 12290 9716 9692 10041 10013 8088 14464 15529 21363 11419 11132 10795 12624 10730 10246 11995 14386 9866 8898 13230 11141 9002 9918 7357 7231 9270 9108

3 25789 9008 7718 12421 8565 11209 9084 10616 13478 8955 7982 22305 5899 7304 11575 11522 8239 8944 7779 8024 16316 14055 16294 10142 9630 12670 9468 14653 9924 15369 10289 10457 8618 12568 8063 9503 8159 6629 6294 7901 5749

0.125ng 1 12862 4495 5133 5721 3931 3045 4398 9233 7310 2978 3801 7873 3190 3497 5370 3624 4254 3969 4395 4093 6649 5839 6672 4062 3808 4717 4083 3945 4727 3765 6705 4683 4744 6116 4243 3732 4949 2904 3139 4448 2838

2 13666 6184 3711 4485 5845 5666 3668 5835 6689 5136 4004 11090 2572 4345 6068 4094 5153 6342 5286 4606 6089 2597 10011 5525 6618 7922 6373 4330 5159 8055 4824 4896 4505 5832 6044 5266 4372 2866 4086 3352 3989

3 13104 3387 4135 3791 4895 5719 3731 5510 5198 2672 3265 7841 3872 3851 5989 5494 3597 5522 3807 4431 5564 7174 11923 4907 6425 3473 4540 3685 4458 4867 5227 4661 4730 6307 5705 4045 5022 2691 3453 2165 5197

0.063ng 1 7901 1807 1299 2269 2354 2866 1235 1358 3829 2645 2048 5576 2180 1521 2779 1440 2209 1745 2371 2142 2853 2584 3664 3782 3332 2544 2235 2193 1782 2081 2824 2320 2201 2406 1782 1475 1563 1520 1428 1645 1395

2 5632 2349 1956 2427 2198 1853 3233 3621 3841 4387 3256 7273 1688 2623 3235 2273 1803 3312 2208 1754 3510 4689 4107 3295 2912 1821 2626 2723 1895 3231 2166 2742 2770 3207 4273 2292 3073 827 1653 1770 1992

3 4523 2210 1697 2432 1266 1284 1402 3517 2876 1701 1901 3534 2285 1948 2232 1887 2257 1419 1495 1855 2276 1468 2774 2321 2346 1824 775 1304 2957 2296 2004 1331 1468 2116 1520 2847 2351 922 1622 1331 1547

0.031ng 1 1922 486 490 1323 554 249 583 428 866 1831 1031 1024 363 611 625 1159 371 438 681 680 735 842 1219 908 665 1084 893 355 833 769 512 631 299 519 1163 646 309 539 705 952 670

2 1264 450 437 828 818 426 779 1732 554 431 1304 197 492 326 370 200 421 452 790 814 815 1057 1022 353 349 583 429 839 1238 705 1008 937 575 619 447 737 395 581 919 480

3 2437 448 755 1558 942 279 1137 644 276 1152 1397 690 674 831 617 290 813 620 578 1378 2076 1539 802 1142 921 681 1165 776 974 1203 492 947 529 1286 1052 967 382 851 718 1012

0.016ng 1 737 176 265 322 481 700 630 191 281 828 235 229 411 295 283 274 423 403 455 282 346 232 297 519 220 559 176 512 366 451 634 253 345 328

2 446 233 315 406 296 245 255 318 257 319 287 318 413 257 320 218 295 264 224 292 217 422 221 343

3 443 196 215 339 431 354 220 183 220 228 794 262 215 240 278 223 264 255 320 265 222 568

31 cycles

Female



3.2 Peak Height Ratio

Figure 3-13. Peak height ratios (PHR) for heterozygous loci for the 7057 dilution series -29 cycles



AMEL CSF1POD10S124

8D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11

D22S104

5D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 SE33 TH01 vWA

0.5ng 1 99.88% 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%

2 97.27% 86.76% 100.00% 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%

3 99.04% 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 1 86.27% 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%

2 78.34% 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%

3 75.39% 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 1 76.14% 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%

2 95.98% 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%

3 90.62% 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 1 55.82% 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%

2 81.18% 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%

3 23.86% 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 1 90.88% 85.65% 51.34% 64.86% 70.29% 72.06%

2 54.29% 98.98% 65.78% 73.70% 90.51% 32.68% 67.15% 55.90% 93.63%

3 92.48% 79.44% 52.61% 58.38% 76.04% 21.02% 65.71% 75.18% 78.19% 68.19% 43.21%

0.016ng 1

2

3

Male 29 cycles

AMEL CSF1POD10S124

8D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11

D22S104


0.5ng 1 53.47% 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%

2 75.09% 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%

3 95.31% 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 1 58.47% 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%

2 89.62% 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%

3 87.89% 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 1 93.10% 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%

2 87.40% 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%

3 73.85% 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 1 41.36% 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%

2 46.26% 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%

3 21.82% 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 1 60.04% 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%

2 55.17% 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%

3 79.84% 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 1 59.76% 62.66% 77.27% 73.94% 57.30% 78.07%

2 81.62% 48.76% 52.84%

3 89.67% 90.29% 67.01% 97.67% 88.80% 99.57%

Male 30 cycles

AMEL CSF1POD10S124

8D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11

D22S104


0.5ng 1 63.42% 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%

2 56.38% 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%

3 81.89% 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 1 72.32% 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%

2 78.53% 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%

3 93.48% 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 1 52.42% 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%

2 84.64% 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%

3 76.73% 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 1 75.77% 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%

2 65.22% 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%

3 72.22% 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 1 90.94% 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%

2 46.70% 63.36% 83.58% 68.17% 32.15% 28.78% 77.50% 81.73% 49.30% 83.92% 40.79% 17.02% 47.25%

3 47.91% 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 1 93.72% 87.54% 90.89% 33.45% 37.61% 75.07% 84.62% 65.30%

2 93.15% 57.31% 59.20% 64.33% 67.81% 83.52% 71.88%

3 88.62% 63.40% 67.24% 50.22% 34.57% 43.80% 78.31% 81.38%

Male 31 cycles



Figure 3-16. Peak height ratios (PHR) for heterozygous loci for the IMR-90 dilution series -29 cycles



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.

CSF1PO D10S1248 D12S391 D13S317 D16S539 D19S433 D1S1656 D21S11D22S104

5D2S1338 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vWA

0.5ng 1 84.14% 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%

2 94.75% 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%

3 79.77% 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 1 47.38% 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%

2 97.89% 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%

3 71.93% 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 1 57.84% 92.61% 54.14% 95.16% 52.40% 99.52% 82.22% 100.00% 53.54% 87.75% 92.52% 92.70% 86.31% 59.11% 60.83% 89.99% 70.60% 70.84% 81.10%

2 62.06% 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%

3 73.30% 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 1 81.49% 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%

2 59.73% 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%

3 80.48% 69.56% 95.97% 100.00% 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 1 63.25% 96.20% 77.08%

2 97.84% 94.69% 99.65% 68.32% 100.00% 98.72%

3 54.17% 59.63% 59.26% 85.54% 86.72% 98.31% 63.69% 52.42% 75.30%

0.016ng 1 92.92%

2

3

Female 29 cycles



0.5ng 1 93.34% 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%

2 95.41% 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%

3 78.37% 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 1 86.66% 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%

2 85.36% 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%

3 86.66% 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 1 67.28% 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%

2 73.49% 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%

3 63.13% 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 1 75.51% 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%

2 99.61% 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%

3 87.37% 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 1 76.71% 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%

2 73.30% 78.13% 63.26% 71.51% 37.57% 83.60% 96.50% 50.32% 54.37% 88.08% 65.33% 87.32%

3 77.99% 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 1 43.86% 49.91% 47.21% 66.78% 97.39% 77.04% 93.53%

2 75.67% 94.62%

3 71.03% 55.56% 71.21% 84.25% 89.07%

Female 30 cycles



0.5ng 1 67.48% 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%

2 99.66% 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%

3 98.60% 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 1 98.48% 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%

2 90.39% 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%

3 85.68% 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 1 87.57% 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%

2 60.01% 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%

3 81.91% 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 1 71.89% 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%

2 83.27% 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%

3 76.79% 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 1 99.18% 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%

2 97.11% 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%

3 59.34% 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 1 82.30% 90.00% 67.97% 97.45% 71.78% 96.82% 88.57% 57.23% 39.36% 71.48% 71.14% 95.07%

2 96.08% 80.56% 62.23% 68.13% 89.49% 76.71% 52.37%

3 63.42% 96.49% 80.22% 79.69% 83.77%

Female 31 cycles



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 3-19. Inter-locus peak height balance for the 7057 dilution series -29 cycles

Figure 3-20. Inter-locus peak height balance for low-level 7057 samples -29 cycles

0

5000

10000

15000

20000

25000

AM

EL

CS

F1P

O

D1

0S

1248

D1

2S

391

D1

3S

317

D1

6S

539

D1

8S

51

D1

9S

433

D1

S16

56

D2

1S

11

D2

2S

1045

D2

S13

38

D2

S44

1

D3

S13

58

D5

S81

8

D7

S82

0

D8

S11

79

DY

S3

91

FG

A

SE

33

TH

01

TP

OX

vW

A

Yin

del

Hete

rozyg

ou

s P

eak H

eig

hts

(R

FU

)

Locus

7057 Globalfiler Inter-locus Balance -29 cycles

2ng

1ng

0.5ng

0.25ng

0.125ng

0.063ng

0.031ng

0.016ng

0

500

1000

1500

2000

2500

AM

EL

CS

F1P

O

D1

0S

1248

D1

2S

391

D1

3S

317

D1

6S

539

D1

8S

51

D1

9S

433

D1

S16

56

D2

1S

11

D2

2S

1045

D2

S13

38

D2

S44

1

D3

S13

58

D5

S81

8

D7

S82

0

D8

S11

79

DY

S3

91

FG

A

SE

33

TH

01

TP

OX

vW

A

Yin

del

Hete

rozyg

ou

s P

eak H

eig

hts

(R

FU

)

Locus


0.125ng

0.063ng

0.031ng

0.016ng





0

5000

10000

15000

20000

25000

30000

AM

EL

CS

F1P

O

D1

0S

1248

D1

2S

391

D1

3S

317

D1

6S

539

D1

8S

51

D1

9S

433

D1

S16

56

D2

1S

11

D2

2S

1045

D2

S13

38

D2

S44

1

D3

S13

58

D5

S81

8

D7

S82

0

D8

S11

79

DY

S3

91

FG

A

SE

33

TH

01

TP

OX

vW

A

Yin

delH

ete

rozyg

ou

s P

eak H

eig

hts

(R

FU

)

Locus

7057 Globalfiler Inter-locus Balance 30 cycles

1ng

0.5ng

0.25ng

0.125ng

0.063ng

0.031ng

0.016ng

0

1000

2000

3000

4000

AM

EL

CS

F1P

O

D1

0S

1248

D1

2S

391

D1

3S

317

D1

6S

539

D1

8S

51

D1

9S

433

D1

S16

56

D2

1S

11

D2

2S

1045

D2

S13

38

D2

S44

1

D3

S13

58

D5

S81

8

D7

S82

0

D8

S11

79

DY

S3

91

FG

A

SE

33

TH

01

TP

OX

vW

A

Yin

del

Hete

rozyg

ou

s P

eak H

eig

hts

(R

FU

)

Locus


0.125ng

0.063ng

0.031ng

0.016ng





0

5000

10000

15000

20000

25000

AM

EL

CS

F1P

O

D1

0S

1248

D1

2S

391

D1

3S

317

D1

6S

539

D1

8S

51

D1

9S

433

D1

S16

56

D2

1S

11

D2

2S

1045

D2

S13

38

D2

S44

1

D3

S13

58

D5

S81

8

D7

S82

0

D8

S11

79

DY

S3

91

FG

A

SE

33

TH

01

TP

OX

vW

A

Yin

del

Hete

rozyg

ou

s P

eak H

eig

hts

(R

FU

)

Locus


0.5ng

0.25ng

0.125ng

0.063ng

0.031ng

0.016ng

0

1000

2000

3000

4000

5000

6000

7000

AM

EL

CS

F1P

O

D1

0S

1248

D1

2S

391

D1

3S

317

D1

6S

539

D1

8S

51

D1

9S

433

D1

S16

56

D2

1S

11

D2

2S

1045

D2

S13

38

D2

S44

1

D3

S13

58

D5

S81

8

D7

S82

0

D8

S11

79

DY

S3

91

FG

A

SE

33

TH

01

TP

OX

vW

A

Yin

delHete

rozyg

ou

s P

eak H

eig

hts

(R

FU

)

Locus


0.125ng

0.063ng

0.031ng

0.016ng



Figure 3-25. Inter-locus peak height balance for the IMR-90 dilution series -29 cycles

Figure 3-26. Inter-locus peak height balance for low-level IMR-90 samples -29 cycles

0

5000

10000

15000

20000

25000

AM

EL

CS

F1P

O

D1

0S

1248

D1

2S

391

D1

3S

317

D1

6S

539

D1

8S

51

D1

9S

433

D1

S16

56

D2

1S

11

D2

2S

1045

D2

S13

38

D2

S44

1

D3

S13

58

D5

S81

8

D7

S82

0

D8

S11

79

FG

A

SE

33

TH

01

TP

OX

vW

A

Hete

rozyg

ou

s P

eak H

eig

hts

(R

FU

)

Locus

IMR-90 Globalfiler Inter-locus Balance -29cycles

2ng

1ng

0.5ng

0.25ng

0.125ng

0.063ng

0.031ng

0.016ng

0

500

1000

1500

2000

2500

AM

EL

CS

F1P

O

D1

0S

1248

D1

2S

391

D1

3S

317

D1

6S

539

D1

8S

51

D1

9S

433

D1

S16

56

D2

1S

11

D2

2S

1045

D2

S13

38

D2

S44

1

D3

S13

58

D5

S81

8

D7

S82

0

D8

S11

79

FG

A

SE

33

TH

01

TP

OX

vW

A

Hete

rozyg

ou

s P

eak H

eig

hts

(R

FU

)

Locus

IMR-90 Globalfiler Inter-locus Balance -29 cycles

0.125ng

0.063ng

0.031ng

0.016ng





0

5000

10000

15000

20000

25000

30000

AM

EL

CS

F1P

O

D1

0S

1248

D1

2S

391

D1

3S

317

D1

6S

539

D1

8S

51

D1

9S

433

D1

S16

56

D2

1S

11

D2

2S

1045

D2

S13

38

D2

S44

1

D3

S13

58

D5

S81

8

D7

S82

0

D8

S11

79

FG

A

SE

33

TH

01

TP

OX

vW

AHete

rozyg

ou

s P

eak H

eig

hts

(R

FU

)

Locus


1ng

0.5ng

0.25ng

0.125ng

0.063ng

0.031ng

0.016ng

0

1000

2000

3000

4000

AM

EL

CS

F1

PO

D1

0S

1248

D1

2S

391

D1

3S

317

D1

6S

539

D1

8S

51

D1

9S

433

D1

S16

56

D2

1S

11

D2

2S

1045

D2

S13

38

D2

S44

1

D3

S13

58

D5

S81

8

D7

S82

0

D8

S11

79

FG

A

SE

33

TH

01

TP

OX

vW

A

Hete

rozyg

ou

s P

eak H

eig

hts

(R

FU

)

Locus


0.125ng

0.063ng

0.031ng

0.016ng





0

5000

10000

15000

20000

25000

AM

EL

CS

F1P

O

D1

0S

1248

D1

2S

391

D1

3S

317

D1

6S

539

D1

8S

51

D1

9S

433

D1

S16

56

D2

1S

11

D2

2S

1045

D2

S13

38

D2

S44

1

D3

S13

58

D5

S81

8

D7

S82

0

D8

S11

79

FG

A

SE

33

TH

01

TP

OX

vW

AHete

rozyg

ou

s P

eak H

eig

hts

(R

FU

)

Locus


0.5ng

0.25ng

0.125ng

0.063ng

0.031ng

0.016ng

0

1000

2000

3000

4000

5000

6000

7000

AM

EL

CS

F1P

O

D1

0S

1248

D1

2S

391

D1

3S

317

D1

6S

539

D1

8S

51

D1

9S

433

D1

S16

56

D2

1S

11

D2

2S

1045

D2

S13

38

D2

S44

1

D3

S13

58

D5

S81

8

D7

S82

0

D8

S11

79

FG

A

SE

33

TH

01

TP

OX

vW

A

Hete

rozyg

ou

s P

eak H

eig

hts

(R

FU

)

Locus


0.125ng

0.063ng

0.031ng

0.016ng



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 3-31. Number of alleles called in Male DNA 7057 dilution series

Figure 3-32. Number of alleles called in Female DNA IMR-90 dilution series

0

5

10

15

20

25

30

35

40

45

0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng

Numberofallelescalled-Male7057

29cyc 30cyc 31cyc

0

5

10

15

20

25

30

35

40

45

0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng

Numberofallelescalled-FemaleIMR90

29cyc 30cyc 31cyc



Table 3-6. The number of alleles detected in each replicate of Male 7057 and Female IMR-90 DNA dilution series:

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.

Input Replicate 29cyc 30cyc 31cyc Input Replicate 29cyc 30cyc 31cyc

1 43 43 43 1 41 41 41

2 43 43 43 2 41 41 41

3 43 43 43 3 41 41 41

1 43 43 43 1 41 41 41

2 43 43 43 2 41 41 41

3 43 43 43 3 41 41 41

1 43 43 43 1 41 41 41

2 43 43 43 2 41 41 41

3 43 43 43 3 41 41 41

1 43 43 43 1 41 41 41

2 42 43 43 2 41 41 41

3 42 42 42 3 41 41 41

1 25 38 41 1 18 38 41

2 31 40 37 2 24 34 40

3 35 38 41 3 27 37 40

1 4 23 24 1 4 24 34

2 5 22 27 2 4 16 24

3 5 24 29 3 0 23 22

Male7057 Female IMR90

Number of alleles called

0.5ng

0.25ng

0.016ng 0.016ng

0.031ng 0.031ng

0.125ng

0.063ng

0.5ng

0.25ng

0.125ng

0.063ng



Figure 3-33. Percentage of alleles called in Male DNA 7057 dilution series

Figure 3-34. Percentage of alleles called in Female DNA IMR-90 dilution series

Table 3-7. Percentage of alleles detected in Male 7057 and Female IMR-90 DNA dilution series:

0%

20%

40%

60%

80%

100%

0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng

Percentageofallelescalled-Male7057

29cyc 30cyc 31cyc

0%

20%

40%

60%

80%

100%

0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng

Percentageofallelescalled-FemaleIMR90

29cyc 30cyc 31cyc

Male7057 29cyc 30cyc 31cyc FemaleIMR90 29cyc 30cyc 31cyc

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%

Averageallelecallpercentageatlowlevelinput(totalcalledalleles/totalexpectedalleles)



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 3-35. Number of allele dropout in Male DNA 7057 dilution series

Figure 3-36. Number of allele dropout in Female DNA IMR-90 dilution series

0

10

20

30

40

50

0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng

Numberofalleledropout-Male7057

29cyc 30cyc 31cyc

0

10

20

30

40

50

0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng

Numberofalleledropout-FemaleIMR90

29cyc 30cyc 31cyc



Table 3-8. The number of allele dropout in each replicate of Male 7057 and Female IMR-90 DNA dilution series:

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.


1 0 0 0 1 0 0 0

2 0 0 0 2 0 0 0

3 0 0 0 3 0 0 0

1 0 0 0 1 0 0 0

2 0 0 0 2 0 0 0

3 0 0 0 3 0 0 0

1 0 0 0 1 0 0 0

2 0 0 0 2 0 0 0

3 0 0 0 3 0 0 0

1 0 0 0 1 0 0 0

2 1 0 0 2 0 0 0

3 1 1 1 3 0 0 0

1 18 5 2 1 22 3 0

2 12 3 6 2 17 7 1

3 8 5 2 3 14 4 1

1 39 20 19 1 37 17 7

2 38 21 16 2 37 25 17

3 38 19 14 3 41 18 19

>20 1 - 20 0

0.031ng 0.031ng

0.016ng 0.016ng


0.25ng 0.25ng

0.125ng 0.125ng

0.063ng 0.063ng

Number of allele dropout

0.5ng 0.5ng



Figure 3-37. Percentage of allele dropout in Male DNA 7057 dilution series

Figure 3-38. Percentage of allele dropout in Female DNA IMR-90 dilution series

Table 3-9. Percentage of allele dropout in Male 7057 and Female IMR-90 DNA dilution series:

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng

Percentageofalleledropout-Male7057

29cyc 30cyc 31cyc

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng

Percentageofalleledropout-FemaleIMR90

29cyc 30cyc 31cyc

Male7057 29cyc 30cyc 31cyc FemaleIMR90 29cyc 30cyc 31cyc

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%

Averagealleledropoutpercentageatlowlevelinput(totaldroppedalleles/totalexpectedalleles)



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 3-39. Number of off-scale peaks in each sample of Male DNA 7057 dilution series

Figure 3-40. Number of off-scale peaks in each sample of Female DNA IMR-90 dilution series

0

5

10

15

20

25

30

35

2ng 1ng 0.5ng 0.25ng 0.125ng 0.063ng POS-1ng

Number of Off-scale peaks in each sample - Male 7057

29cyc

30cyc

31cyc

0

5

10

15

20

25

30

35

2ng 1ng 0.5ng 0.25ng 0.125ng 0.063ng POS-1ng

Number of Off-scale peaks in each sample - Female IMR90

29cyc

30cyc

31cyc



Table 3-10. Number of off-scale peaks in each sample of Male 7057 and Female IMR-90 DNA dilution series:

Table 3-11. Percentage of off-scale peaks in each sample of Male 7057 and Female IMR-90 DNA dilution series:


1 2 25 11 1 3 24 9

2 3 24 4 2 1 19 13

3 1 13 7 3 6 32 6

1 0 4 8 1 1 5 16

2 0 2 22 2 1 4 11

3 0 2 15 3 2 4 24

1 0 0 1 1 0 0 2

2 0 0 4 2 0 1 3

3 0 0 1 3 0 1 1

1 0 0 0 1 0 0 1

2 0 0 0 2 0 0 1

3 0 0 0 3 0 0 0

1 0 0 0 1 0 0 0

2 0 0 0 2 0 0 0

3 0 0 0 3 0 0 0

1 0 0 0 1 0 0 0

2 0 0 0 2 0 0 0

3 0 0 0 3 0 0 0

1 0 1 1 1 0 1 1

2 0 2 7 2 0 2 7

3 0 2 8 3 0 2 15

>5 1 - 5 0

POS-1ng POS-1ng


0.25ng 0.25ng

0.125ng 0.125ng

0.063ng 0.063ng

2ng 2ng

1ng 1ng

0.5ng 0.5ng

Number of off-scale peaks in each sample

Averagepercentageofoff-scalepeaks(totalnumberofOSpeaks/totalnumberofexpectedpeaks)

femaleDNAinput 29cyc 30cyc 31cyc maleDNAinput 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%



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 3-41. Number of pull-up peaks in each sample of Male DNA 7057 dilution series

Figure 3-42. Number of pull-up peaks in each sample of Female DNA IMR-90 dilution series

0

10

20

30

40

50

60

1ng 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng

Number of pull-up peaks - Male 7057

29cyc

30cyc

31cyc

0

10

20

30

40

50

60

1ng 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng

Number of pull-up peaks - Female IMR90

29cyc

30cyc

31cyc



Table 3-12. Number of pull-up peaks in each sample of Male 7057 and Female IMR-90 DNA dilution series:

input Replicate 29cyc 30cyc 31cyc input Replicate 29cyc 30cyc 31cyc

1 2 23 27 1 5 31 51

2 3 8 50 2 5 26 38

3 2 15 45 3 11 30 59

1 1 2 5 1 0 1 19

2 1 1 12 2 0 5 22

3 1 3 7 3 0 3 7

1 0 1 2 1 0 0 11

2 0 1 3 2 0 0 6

3 0 1 6 3 0 0 4

1 0 0 0 1 0 0 0

2 0 0 0 2 0 0 0

3 0 0 1 3 0 0 1

1 0 0 0 1 0 0 0

2 0 0 0 2 0 0 03 0 0 0 3 0 0 0

1 0 0 0 1 0 0 0

2 0 0 0 2 0 0 0

3 0 0 0 3 0 0 0

1 0 0 0 1 0 0 0

2 0 0 0 2 0 0 0

3 0 0 0 3 0 0 0

0.016ng 0.016ng

0.125ng 0.125ng

0.063ng 0.063ng

0.031ng 0.031ng

0.25ng

1ng 1ng

Numberofpull-uppeaksineachsample

0.5ng 0.5ng

0.25ng

Male7057 FemaleIMR90



3.8 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. 2nd and 3rd 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 3-43. Number of extra stutter peaks in each sample of Male DNA 7057 dilution series

Figure 3-44. Number of extra stutter peaks in each sample of Female DNA IMR-90 dilution series

0

2

4

6

8

10

12

14

16

18

20

1ng 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng

Number of extra stutter peaks - Male 7057

29cyc

30cyc

31cyc

0

2

4

6

8

10

12

14

16

18

20

1ng 0.5ng 0.25ng 0.125ng 0.063ng 0.031ng 0.016ng

Number of extra stutter peaks - Female IMR90

29cyc

30cyc

31cyc



Table 3-13. Number of extra stutter peaks in each sample of Male 7057 and Female IMR-90 DNA dilution series:

4. 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 0.031 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.

input Replicate 29cyc 30cyc 31cyc input Replicate 29cyc 30cyc 31cyc

1 2 9 9 1 1 11 18

2 1 2 13 2 1 5 14

3 2 6 12 3 1 7 18

1 0 2 1 1 0 1 3

2 1 1 3 2 0 1 5

3 0 2 4 3 0 1 4

1 0 0 3 1 0 0 4

2 0 2 5 2 0 2 3

3 0 1 2 3 1 0 4

1 0 1 1 1 0 0 1

2 0 0 1 2 1 0 1

3 0 1 4 3 0 0 5

1 0 0 3 1 0 0 1

2 0 0 1 2 0 0 13 0 0 2 3 0 1 0

1 0 0 0 1 0 0 1

2 0 0 1 2 0 0 0

3 0 0 3 3 0 0 0

1 0 0 0 1 0 0 0

2 0 0 0 2 0 0 0

3 0 0 0 3 0 0 0

Numberofstutterpeaksineachsample

0.125ng 0.125ng

0.063ng 0.063ng

1ng 1ng

0.5ng 0.5ng

0.25ng 0.25ng

Male7057 FemaleIMR90

0.016ng 0.016ng

0.031ng 0.031ng



Figure 3-45. Summary of limitation of extra cycles

Table 3-14. Summary of limitation of extra cycles

Effects and characterizations

Input template DNA

29 cycles 30 cycles 31 cycles

Instrument saturation

Increased artifacts ≥1ng ≥0.5ng ≥0.125ng

Full profiles

Decreased artifacts 0.063-0.5ng 0.063-0.25ng 0.063-0.125

Stochastic amplification

Allelic drop-out and drop-in

≤0.063ng ≤0.063ng ≤0.063ng


1 Off-scale Off-scale Off-scale 1 Off-scale Off-scale Off-scale



1 pull-up Off-scale Off-scale 1 Off-scale Off-scale Off-scale



1 pull-up pull-up Off-scale 1 fullprofile pull-up Off-scale

2 pull-up pull-up Off-scale 2 fullprofile Off-scale Off-scale

3 pull-up pull-up Off-scale 3 fullprofile Off-scale Off-scale

1 fullprofile pull-up pull-up 1 fullprofile fullprofile Off-scale

2 fullprofile pull-up pull-up 2 fullprofile Stutter Off-scale

3 fullprofile pull-up pull-up 3 Stutter fullprofile pull-up

1 fullprofile Stutter Stutter 1 fullprofile fullprofile Stutter

2 fullprofile fullprofile Stutter 2 Stutter fullprofile Stutter

3 fullprofile Stutter pull-up 3 fullprofile fullprofile pull-up

1 fullprofile fullprofile Stutter 1 fullprofile fullprofile Stutter

2 dropout fullprofile Stutter 2 fullprofile fullprofile Stutter

3 dropout dropout dropout 3 fullprofile Stutter fullprofile

1 dropout dropout dropout 1 dropout dropout Stutter

2 dropout dropout dropout 2 dropout dropout dropout



2 dropout dropout dropout 2 dropout dropout dropout3 dropout dropout dropout 3 dropout dropout dropout

0.031ng 0.031ng

0.016ng 0.016ng

0.125ng 0.125ng

0.063ng 0.063ng

1ng 1ng

0.5ng 0.5ng

0.25ng 0.25ng

SummarymapforlimitationofextracyclesMale7057 FemaleIMR90

2ng 2ng



5. Attachment

Figure 3-46. Electropherogram shows five (5) alleles were detected in male DNA sample with 0.016ng input at 29 cycles.



Figure 3-47. 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.



Figure 3-48. 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.



Figure 3-49. Electropherogram shows increased stutter peaks presenting in male DNA sample with 0.063ng input amplified at 31 cycles

stutter stutter stutter



Figure 3-50. Electropherogram shows increased stutter peaks presenting in male DNA sample with 0.031ng input amplified at 31 cycles.


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 v1.5.

1. Study Overview


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).


2.1 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.

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 7.5 7.5 7.5

GF Primer Mix 2.5 2.5 2.5

DNA (1ng/uL) 10 10 10

Hematin (5 mM) 0.75 1.5 2.25

TE 4.25 3.5 2.75

25 25 25

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 7.5 7.5 7.5

GF Primer Mix 2.5 2.5 2.5

DNA (1ng/uL) 10 10 10

Humic Acid (1000 ng/uL) 2.25 3.75 5.25

TE 2.75 1.25 0

25 25 25.25

2.2 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

Hematin

150 uM 3 3 3

300 uM 3 3 3

450 uM 3 3 3

Humic Acid

90 ng/uL 3 3 3

150 ng/uL 3 3 3

210 ng/uL 3 3 3



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.



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 34-35 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

0

5

10

15

20

25

30

35

40

45

150uM 300uM 450uM 90ng/ul 150ng/ul 210ng/ul

Hematin Humic Acid

Number of alleles detected in inhibited samples

29 cycles

30 cycles

31 cycles



Table 4-4. Number of alleles called in Inhibited Samples

3.2 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.

Inhibitor conc. replicates 29cycles 30cycles 31cycles1 43 43 432 43 43 433 43 43 431 43 43 432 43 43 433 43 43 431 43 43 432 43 43 433 43 43 431 43 43 432 43 43 433 43 43 431 43 43 432 43 43 433 43 43 431 35 34 352 35 35 353 35 34 35

Hematin

150uM

300uM

450uM

150ng/ul

210ng/ul

Numberofallelesdetectedininhibitedsamples

HumicAcid

90ng/ul



Figure 4-2. Number of allele dropout in each replicate of inhibited samples

Table 4-5. The number of allele dropout in each replicate of inhibited samples

0

5

10

15

20

25

30

35

40

45


Hematin Humic Acid

Number of allele dropout in inhibited samples

29 cycles

30 cycles

31 cycles


150ng/ul

210ng/ul

Hematin

150uM

300uM

450uM

Numberofalleledropoutininhibitedsamples

HumicAcid

90ng/ul



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

Table 4-6. Number of off-scale peaks in each replicate of inhibited samples

0

1

2

3

4

5

6

7

8

9

10


Hematin Humic Acid

Number of off-scale peaks in inhibited samples

29 cycles

30 cycles

31 cycles


150ng/ul

210ng/ul

450uM

90ng/ul

HumicAcid

Numberofoff-scalepeaksininhibitedsamples

150uM

300uMHematin



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

Table 4-7. Number of pull-up peaks in each replicates of inhibited samples

0

5

10

15

20

25

30

35

40


Hematin Humic Acid

Number of pull-up peaks in inhibited samples

29 cycles

30 cycles

31 cycles

Inhibitor conc. replicates 29cycles 30cycles 31cycles

1 0 9 352 1 12 353 3 18 391 0 6 31

2 0 8 313 0 7 271 0 4 222 0 9 23

3 0 6 221 0 9 212 0 10 243 0 12 271 0 4 172 0 4 143 1 9 171 0 5 82 0 7 83 0 5 8

Hematin

HumicAcid

210ng/ul

150ng/ul

150uM

300uM

450uM

Numberofpull-uppeaksininhibitedsamples

90ng/ul




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. 2nd and 3rd 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

0

5

10

15

20

25

30

35

40


Hematin Humic Acid

Number of stutter peaks in inhibited samples

29 cycles

30 cycles

31 cycles



Table 4-8. Number of extra stutter peaks in each replicates of inhibited samples

3.6 Other extra peaks In addition to pull-up peaks, increased stutter peaks and additional 2nd 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.


1 1 2 82 1 5 113 0 4 81 0 0 7

2 0 1 73 0 0 61 0 0 32 0 0 4

3 0 2 41 0 1 42 0 2 43 1 2 51 0 1 42 0 1 23 0 0 21 0 1 12 0 1 23 0 0 1

Numberofincreasedandadditionalstutterpeaksininhibitedsamples

450uM

90ng/ul

Hematin

HumicAcid 150ng/ul

210ng/ul

150uM

300uM



Figure 4-6. Number of other extra peaks/artifacts in each replicates of inhibited samples

Table 4-9. Number of other extra peaks/artifacts in each replicates of inhibited samples

0

5

10

15

20

25

30

35

40


Hematin Humic Acid

Number of other extra peaks/artifacts in inhibited samples

29 cycles

30 cycles

31 cycles


1 0 0 42 0 0 73 0 0 61 0 0 7

2 0 0 83 0 0 41 0 0 52 0 0 6

3 0 0 41 0 1 142 0 0 123 0 0 111 0 0 142 0 0 103 0 0 81 0 0 122 0 0 103 0 0 7

300uM

HumicAcid 150ng/ul

210ng/ul

90ng/ul

Numberofotherextrapeaks/artifactsininhibitedsamples

Hematin

450uM

150uM



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.



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.



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



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

dropout

dropout

dropout

stutter

pull-up pull-up pull-up pull-up



Figure 4-10. 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.

dropout

dropout

dropout

dropout

-A peaks

artifact

pull-up

pull-up

-A peaks

artifact

-A peaks

-A peaks

pull-up

pull-up

-A peaks

artifact


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 v1.5.

1. Study Overview


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 4-1.


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.

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 60 66

7.5 495.0

GlobalFiler Primer Set 2.5 165.0

Table 5-3. Additional Reaction Mix.

Reagents Number of

Reactions (N) N + 10%

Volume per Reaction (μL)

Calculated Volume (μL)

GlobalFiler Master Mix

30 33

7.5 247.5

GlobalFiler Primer Set 2.5 82.5

Taq 0.833 27.5

BSA 0.667 22

3. 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.



Table 5-4. Quantities of DNA and TE buffer added for standard reaction protocol.


Dilution Average

DNA Conc. (ng/µL)

Final Input (ng)

Volume per Amp

(µL) TE

-4 (µL) DNA (µL)

Master Mix (µL)

Total

NTC 90 0.0 60 150

MD 1.99 1 0.50 116 4.0 80 200

LD 1.00 1 1.00 112 8.0 80 200

CTR 1.00 1 1.00 112 8.0 80 200

MD 1.99 0.5 0.25 118 2.0 80 200

LD 1.00 0.5 0.50 116 4.0 80 200

CTR 1.00 0.5 0.50 116 4.0 80 200

POS-1 0.10 1 10.0 15 30.0 30 75

POS-0.5 0.10 0.5 5.0 30 15.0 30 75

Table 5-1. Quantities of DNA and TE buffer added for IMR-90 amplification. Concentration measured by the Quantifiler Trio DNA Quantification Kit.


Dilution Average

DNA Conc. (ng/µL)

Final Input (ng)

Volume per Amp

(µL) TE

-4 (µL) DNA (µL)

Master Mix (µL)

Total

NTC 45 0.0 34.5 79.5

MD 1.99 1 0.50 58 2.0 46 106.0

LD 1.00 1 1.00 56 4.0 46 106.0

CTR 1.00 1 1.00 56 4.0 46 106.0

MD 1.99 0.5 0.25 59 1.0 46 106.0

LD 1.00 0.5 0.50 58 2.0 46 106.0

CTR 1.00 0.5 0.50 58 2.0 46 106.0

POS-1 0.10 1 10.0 5 10.0 11.5 26.5

POS-0.5 0.10 0.5 5.0 10 5.0 11.5 26.5

5. 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.



2.4 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.



2. The profiles were visually assessed, and extraneous 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.

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.

3.2 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 low-degraded 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.

Cт Quantity Qty Mean Cт Quantity Qty Mean

Control DNA 007 30.32 0.10 0.11 28.07 0.15 0.15 0.71 0.71

Control DNA 007 30.25 0.11 0.11 28.02 0.15 0.15 0.72 0.71

CTRL 27.15 1.02 1.00 24.56 1.60 1.63 0.64 0.61

CTRL 27.17 1.01 1.00 24.53 1.64 1.63 0.62 0.61

CTRL 27.23 0.97 1.00 24.51 1.66 1.63 0.58 0.61

low-degraded 23.86 11.06 10.65 24.65 1.50 1.58 7.36 6.74

low-degraded 23.91 10.65 10.65 24.54 1.63 1.58 6.55 6.74

low-degraded 23.96 10.25 10.65 24.54 1.62 1.58 6.32 6.74

medium-degraded 26.25 1.97 1.99 31.90 0.01 0.01 182.47 154.19

medium-degraded 26.26 1.95 1.99 31.48 0.01 0.01 136.24 154.19

medium-degraded 26.19 2.05 1.99 31.49 0.01 0.01 143.87 154.19

T.Small Autosomal T.Large Autosomal Degradation

Index

Degradation

Index Mean Sample Name



Figure 5-1. Number of alleles called in Degraded Samples

Table 5-7. Number of alleles called in Degraded Samples

CTRL=non-degraded control LD=low degraded DNA MD=medium degraded DNA

0

5

10

15

20

25

30

35

40

45

CTRL LD MD CTRL LD MD

1ng 0.5ng

Number of alleles detected in degraded samples

29cyc-std

29cyc-TaqBSA

30cyc-std

Numberofallelesdetectedindegradedsamples

Input sample 29cyc-std 29cyc-TaqBSA 30cyc-std difference(30cyc-29cyc)

1ng

CTRL

43 43 43 0

43 43 43 0

43 43 43 0

LD

27 28 31 4

28 28 29 1

28 28 34 6

MD

18 18 24 6

19 16 23 4

15 18 23 8

0.5ng

CTRL

43 43 43 0

43 43 43 0

43 43 43 0

LD

23 22 32 9

23 17 26 3

23 19 30 7

MD

17 13 18 1

15 13 22 7

14 15 18 4



Figure 5-2. Percentage of alleles called in Degraded Samples

Table 5-8. Percentage of alleles called in Degraded Samples

Allele calls were 100% (number of actual allele calls/expected allele calls) from the non-degraded control male DNA sample at both inputs of 1ng and 0.5ng.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


1ng 0.5ng

Percentage of alleles detected in degraded samples

29cyc-std

29cyc-TaqBSA

30cyc-std

Percentageofallelesdetectedindegradedsamples


1ng

CTRL

100% 100% 100% 0%

100% 100% 100% 0%

100% 100% 100% 0%

LD

62.8% 65.1% 72.1% 9.3%

65.1% 65.1% 67.4% 2.3%

65.1% 65.1% 79.1% 14.0%

MD

41.9% 41.9% 55.8% 14.0%

44.2% 37.2% 53.5% 9.3%

34.9% 41.9% 53.5% 18.6%

0.5ng

CTRL

100% 100% 100% 0%

100% 100% 100% 0%

100% 100% 100% 0%

LD

53.5% 51.2% 74.4% 20.9%

53.5% 39.5% 60.5% 7.0%

53.5% 44.2% 69.8% 16.3%

MD

39.5% 30.2% 41.9% 2.3%

34.9% 30.2% 51.2% 16.3%

32.6% 34.9% 41.9% 9.3%



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 medium-

degraded 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 low-

degraded 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

0

5

10

15

20

25

30

35

40

45


1ng 0.5ng

Number of allele dropout in degraded samples

29cyc-std

29cyc-TaqBSA

30cyc-std



Table 5-9. The number of allele dropout in each replicate of degraded samples

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.

Numberofalleledropoutindegradedsamples


1ng

CTRL

0 0 0 0

0 0 0 0

0 0 0 0

LD

16 15 12 -4

15 15 14 -1

15 15 9 -6

MD

25 25 19 -6

24 27 20 -4

28 25 20 -8

0.5ng

CTRL

0 0 0 0

0 0 0 0

0 0 0 0

LD

20 21 11 -9

20 26 17 -3

20 24 13 -7

MD

26 30 25 -1

28 30 21 -7

29 28 25 -4



Figure 5-4. Percentage of allele dropout in each replicates of degraded samples

Table 5-10. Percentage of allele dropout in each replicates of degraded samples

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


1ng 0.5ng

Percentage of allele dropout in degraded samples

29cyc-std

29cyc-TaqBSA

30cyc-std

Percentageofalleledropoutindegradedsamples


1ng

CTRL

0% 0% 0% 0%

0% 0% 0% 0%

0% 0% 0% 0%

LD

37.2% 34.9% 27.9% -9.3%

34.9% 34.9% 32.6% -2.3%

34.9% 34.9% 20.9% -14.0%

MD

58.1% 58.1% 44.2% -14.0%

55.8% 62.8% 46.5% -9.3%

65.1% 58.1% 46.5% -18.6%

0.5ng

CTRL

0% 0% 0% 0%

0% 0% 0% 0%

0% 0% 0% 0%

LD

46.5% 48.8% 25.6% -20.9%

46.5% 60.5% 39.5% -7.0%

46.5% 55.8% 30.2% -16.3%

MD

60.5% 69.8% 58.1% -2.3%

65.1% 69.8% 48.8% -16.3%

67.4% 65.1% 58.1% -9.3%



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 3-11. 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

1ng

LD

1 0 0 0

2 0 0 0

3 0 0 0

MD

1 0 0 1

2 0 0 1

3 0 0 1

0.5ng

LD

1 0 0 0

2 0 0 0

3 0 0 0

MD

1 0 0 0

2 0 0 0

3 0 0 0



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

Table 5-12. Number of pull-up peaks in each replicates of degraded samples

Number of pull-up peaks in degraded samples


1ng

LD

1 0 0 0

2 0 0 0

3 0 0 0

MD

1 0 0 2

2 0 0 3

3 0 0 1

0.5ng

LD

1 0 0 0

2 0 0 0

3 0 0 0

MD

1 0 0 0

2 0 0 0

3 0 0 0

0

1

2

3

LD MD LD MD

1ng 0.5ng

Number of pull-up peaks in degraded samples

29cyc-std

29cyc-TaqBSA

30cyc-std




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. 2nd and 3rd 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

Table 5-13. Number of extra stutter peaks in each replicates of degraded samples

Number of increased and additional stutter peaks in degraded samples


1ng

LD

1 0 0 0

2 0 0 0

3 0 0 1

MD

1 0 0 1

2 0 0 0

3 0 0 0

0.5ng

LD

1 0 0 2

2 0 0 1

3 1 0 0

MD

1 0 0 0

2 0 0 1

3 0 0 2

0

1

2

3

LD MD LD MD

1ng 0.5ng

Number of stutter peaks in degraded samples

29cyc-std

29cyc-TaqBSA

30cyc-std



3.7 Other Artifact Peaks

Figure 5-7. Number of other extra peaks/artifacts in each replicates of degraded samples

Table 5-14. Number of other extra peaks/artifacts in each replicates of degraded samples

Number of other extra peaks/artifacts in degraded samples


1ng

LD

1 0 0 0

2 1 0 1

3 0 0 1

MD

1 0 0 0

2 0 0 2

3 0 0 2

0.5ng

LD

1 0 0 0

2 0 0 0

3 0 0 0

MD

1 0 0 0

2 0 0 1

3 0 0 0

0

1

2

3

LD MD LD MD

1ng 0.5ng

Number of other extra peaks/artifacts in degraded samples

29cyc-std

29cyc-TaqBSA

30cyc-std



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 5-15. Summary of limitation of extra cycle for degraded samples

Summary of limitation of extra cycle for degraded samples


1ng

LD

1

2 ART ART

3 ST/ART

MD

1 ST/PU/OS

2 PU/OS/ART

3 PU/OS/ART

0.5ng

LD

1 ST

2 ST

3 ST

MD

1

2 ST/ART

3 ST



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.



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



Figure 5-10. 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



Figure 5-11. Medium-degraded DNA sample at 1ng input was amplified at 29 cycles. Number of alleles detected was 18, and

number of allele dropout was 25. No pull-up peak, extra stutter peaks or artifacts presented.



Figure 5-12. Medium-degraded DNA sample at 1ng input was amplified at 30 cycles. Number of allele call increased to 24 from

18 (at 29 cycles), and number of allele dropout decreased to 19 from 25 (at 29 cycles). Two pull-up peaks and a secondary stutter peak presented.

pull-up

pull-up

stutter



Figure 5-13. Medium-degraded DNA sample at 1ng input was amplified at 30 cycles. Number of allele call increased to 23 from

19 (at 29 cycles), and number of allele dropout decreased to 20 from 24 (at 29 cycles). An off-scale peak was generated on Green channel. Three pull-up peaks and two artifact peaks presented in the short fragment range.

pull-up

pull-up

artifact

artifact

pull-up



Figure 5-14. Medium-degraded DNA sample at 0.5ng input was amplified at 29 cycles. Number of alleles detected was 15, and

number of allele dropout was 28. No pull-up peak, extra stutter peaks or artifacts presented.



Figure 5-15. Medium-degraded DNA sample at 0.5ng input was amplified at 30 cycles. Number of allele calls increased to22

from 15 (at 29 cycles), and number of allele dropout decreased to 21 from 28 (at 29 cycles). A pull-up peaks and an artifact peak presented.

stutter

Artifact


© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo

Fisher Scientific and its subsidiaries unless otherwise specified.

For Research, Forensic or Paternity use only. Not for use in diagnostic procedures.

globalfilertm extra cycle evaluation report...this chapter provides detailed information about...

Documents