detecting degradation in dna samples keith inman forensic analytical specialties, inc dayton, ohio...
TRANSCRIPT
![Page 1: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/1.jpg)
Detecting Degradation in DNA samples
Keith Inman
Forensic Analytical Specialties, Inc
Dayton, Ohio
August 11, 2006
![Page 2: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/2.jpg)
Intact and degraded DNA
![Page 3: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/3.jpg)
“Wedge” effect
![Page 4: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/4.jpg)
How To Identify Challenging Samples?
experience (analyst, intra-lab, inter-lab, literature)
unsuccessful analysis using routine methods i.e., partial or null typing results
• inefficient use of analyst time
![Page 5: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/5.jpg)
Degradation of DNA
Random breaking of DNA molecule into numerous fragments of varying sizes
Can speak of “average fragment size”
![Page 6: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/6.jpg)
Loss of signal at high MW loci
Potential causes Uneven
amplification
• Preferential (allele)
• Differential (locus)
![Page 7: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/7.jpg)
Loss of signal at high MW loci
Potential causes Uneven
amplification
• Preferential (allele)
![Page 8: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/8.jpg)
Loss of signal at high MW loci
Potential causes Uneven
amplification
• Differential (locus)
![Page 9: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/9.jpg)
Uneven signal response
Differential dye sensitivity
![Page 10: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/10.jpg)
Fewer intact molecules - degradation
• Exposure to environmental insult
• Time
• Heat
• Moisture
• Chemicals; microorganisms
• UV light
Loss of signal at high MW loci
![Page 11: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/11.jpg)
Effect of Heat on DNA
![Page 12: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/12.jpg)
Solutions Detection
• Prior to amplification
• Knowledge of sample
• Age
• Condition
• Substrate
![Page 13: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/13.jpg)
Solutions
Adjustment of primer concentrations and amp conditions Done by mfg during developmental validation
Solves problem of uneven amplification and dye sensitivity
![Page 14: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/14.jpg)
Solutions
Detection Prior to amplification
• Differential quantitation
• Use of two primers, one for long and one for short molecules
![Page 15: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/15.jpg)
Nuclear nuTH01 qPCR Target
target sequence spans TH01 CODIS STR locus
(2 copies/diploid genome) FAM-labeled TaqMan detection probe target sequence length: ~170 – 190 bp
STRs
probe
![Page 16: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/16.jpg)
Nuclear nuCSF qPCR Target
target sequence flanks the CODIS CSF STR region (2 copies/diploid genome)
VIC-labeled TaqManMGB detection probe target sequence length: 67 bp
probe
STR
![Page 17: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/17.jpg)
Using Short and Long Nuclear Targets to Assess DNA Fragmentation
nuCSF assay – detects and quantifies DNA fragments larger than ~67bp
nuTH01 assay – detects and quantifies DNA fragments larger than ~180bp
LH 0 1 2 3 4 5 15 30 45 60 LD LH
10 kbp
1.5 kb
1 kbp 800 bp
600 bp
400 bp
200 bp
Minutes of DNase Treatment
~67 bpnuCSFar
nuTH01
![Page 18: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/18.jpg)
qPCR Degradation Ratio = nuCSF Quantity (ng) nuTH01 Quantity (ng)
For high-molecular weight DNA, expect the Degradation Ratio to be ~ 1.
For highly-degraded DNA, expect the Degradation Ratio to be > 1.
The bigger the qPCR Degradation Ratio, the more fragmented the DNA.
LH 0 1 2 3 4 5 15 30 45 60 LD LH
10 kbp
1.5 kb
1 kbp 800 bp
600 bp
400 bp
200 bp
Minutes of DNase Treatment
nuCSFar
nuTH01
~67 bp
![Page 19: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/19.jpg)
qPCR Degradation Ratio ~ 25:“1 ng” (nuTH01) Identifiler STR Results
![Page 20: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/20.jpg)
Interpreting the qPCR Degradation Ratio
Degradation Ratio
STR Implications
1 – 3 none
3 – 5 “wedge” effect,
possible cross-dye pull-up
>5
(>10 artifacts
expected to be significant)
increasing “wedge” effect, pull-up,
dropped-out alleles at larger loci,
off-scale peaks, called stutter peaks,
-A shouldering
![Page 21: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/21.jpg)
Solutions
Post amplification• Yield gel
![Page 22: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/22.jpg)
Solutions
Post Typing
•Assessment of PHR’s between loci
•At this point, a visual assessment
![Page 23: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/23.jpg)
Solutions Increase injection time
• Increases likelihood of saturated data
• Artifacts created
• Doesn’t really work with degraded samples
![Page 24: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/24.jpg)
Saturated data and artifacts
![Page 25: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/25.jpg)
Solutions Amplify more DNA
Increases likelihood of saturated data
• Frequently must combine data from two amps to get full profile
![Page 26: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/26.jpg)
New (Non-Routine) Analysis Tools for Challenging and Compromised Samples
* miniSTRs* SNPs* mitochondrial sequencing/linear-array typing enhanced PCR conditions (e.g., extra Taq, BSA) Y-STR analysis for male/female mixtures low-volume PCR amplifications increased PCR cycle numbers
![Page 27: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/27.jpg)
Solutions
Consideration of PHR’s between loci Use of positive
controls
• Likely undegraded
• Establishes a baseline for good samples
![Page 28: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/28.jpg)
Strategy for post-typing diagnosis of degradation
Consider the slope between loci as indicator of drop-off of signal within colors
Calculate a single summary value from the three normalized slopes as another parameter of normal undegraded sample
![Page 29: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/29.jpg)
For each dye color, 6 data points were used to calculate the slope Y coordinate is RFU
X coordinate is peak data collection point (as determined by Genescan)
![Page 30: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/30.jpg)
Strategy
Calculation of slope by best fit linear regression
Intercompare slopes between dye colors using correlation coefficients (r2) and paired-T tests
![Page 31: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/31.jpg)
Results
Distribution of slopes is approximately normally distributed
A
0
5
10
15
20
25
30
35
40
-12 -11 -10-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5
Slope (RFU/data collection point)
Count
B
0
5
10
15
20
25
30
35
40
-12 -11 -10-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5
Slope (RFU/data collection point)
Count
C
0
5
10
15
20
25
30
35
40
-12 -11 -10-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5
Slope (RFU/data collection point)
Count
![Page 32: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/32.jpg)
All slopes are negative Due to differential dye sensitivity and
multiplex complexities summarized earlier
Slopes between the three colors are not correlated Each color shows a different pattern of drop-
off in intensity between the loci
![Page 33: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/33.jpg)
![Page 34: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/34.jpg)
One number for evaluation
Slopes for each samples were normalized against the max and min slopes for each dye, then added to give a single normalized sum of slopes value
mnorm = (m – mmin)/(mmax – mmin)
![Page 35: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/35.jpg)
0
5
10
15
20
25
0
0.2 0.4 0.6 0.8
1
1.2 1.4 1.6 1.8
2
2.2 2.4 2.6 2.8
3
Normalized Sum
Count
![Page 36: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/36.jpg)
Results The average and standard deviation of the samples
can be used to calculate thresholds of departure from normal at both the 5% and 1% levels for each color
The same statistic can be used with the normalized sum to determine departures from normal at the 5% and 1% level for a single sample
Can now determine if, post typing, a sample deviates from our expectation of a normal, undegraded sample.
![Page 37: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/37.jpg)
Threshold levels and significance levels
Avg PH Avg PH Std Dev Slope Avg
Slope Std Dev = 0.05 = 0.01
Blue 1320 515 -3.82 2.26 -7.54 -9.08 Green 1790 682 -1.00 1.67 -3.75 -4.88 Yellow 1570 592 -4.53 1.98 -7.79 -9.14 Normalized Sum 1.49 0.42 0.80 0.51
![Page 38: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/38.jpg)
Threshold levels and significance levels
Avg PH Avg PH Std Dev Slope Avg
Slope Std Dev = 0.05 = 0.01
Blue 1320 515 -3.82 2.26 -7.54 -9.08 Green 1790 682 -1.00 1.67 -3.75 -4.88 Yellow 1570 592 -4.53 1.98 -7.79 -9.14 Normalized Sum 1.49 0.42 0.80 0.51
![Page 39: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/39.jpg)
Avg PH Avg PH Std Dev Slope Avg
Slope Std Dev = 0.05 = 0.01
Blue 1320 515 -3.82 2.26 -7.54 -9.08 Green 1790 682 -1.00 1.67 -3.75 -4.88 Yellow 1570 592 -4.53 1.98 -7.79 -9.14 Normalized Sum 1.49 0.42 0.80 0.51
![Page 40: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/40.jpg)
Next step Prepare degraded samples and apply the same analysis
Artificially degrade samples with DNAse
Monitor level of degradation via a yield gel
• Gives information about average base pair size when compared to a standard ladder
![Page 41: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/41.jpg)
Next Step
Amplify and type the samples Amplify normal
amounts (1.5 – 2 ng)
Amplify larger amounts to bring up larger, more degraded loci
![Page 42: Detecting Degradation in DNA samples Keith Inman Forensic Analytical Specialties, Inc Dayton, Ohio August 11, 2006](https://reader035.vdocuments.net/reader035/viewer/2022070305/55146762550346b0158b4bc8/html5/thumbnails/42.jpg)
Acknowledgements
Dan Krane Jason Gilder Cristian Orrego Zach Gaskin