Comparative Tolerance of Short Tandem Repeat and Massively … · 2017. 5. 31. · • STR...
1
Comparative Tolerance of Short Tandem Repeat and Massively Parallel Sequencing Chemistries to Inhibited Samples Kyleen Elwick, BA*; Charity Beherec, MSFS; David Gangitano, PhD; Sheree Hughes-Stamm, PhD Department of Forensic Science, Sam Houston State University, Huntsville, TX 77340 RESULTS AND DISCUSSION CONCLUSIONS REFERENCES [1] Brough AL, Morgan B, Rutty GN. The basics of disaster victim identification. Journal of Forensic Radiology and Imaging. 2015;3(1):29- 37. [2] Goodwin WH, Simmons T, Houck JASJSM. Disaster Victim Identification. Encyclopedia of Forensic Sciences. Waltham: Academic Press; 2013;332-8. [3] Dhanardhono T, Wulandari N, Bhima SKL, Ahmad HJ, Widodo PT. DNA profiling of disaster victim identification in Trenggalek shipwreck case. Forensic Science International: Genetics Supplement Series. 2013;4(1):e5-e6. [4] Budowle B, Bieber FR, Eisenberg AJ. Forensic aspects of mass disasters: Strategic considerations for DNA-based human identification. Legal Medicine. 2005;7(4):230-43. [5] Thompson RE, Duncan G, McCord BR. An Investigation of PCR Inhibition Using Plexor®-Based Quantitative PCR and Short Tandem Repeat Amplification. [6] Kemp BM, Monroe C, Smith DG. Repeat silica extraction: a simple technique for the removal of PCR inhibitors from DNA extracts. Journal of Archaeological Science. 2006;33(12):1680-9. [7] Schmedes S, Marshall P, King J, Budowle B. Effective removal of co-purified inhibitors from extracted DNA samples using synchronous coefficient of drag alteration (SCODA) technology. [8] Hu Q, Liu Y, Yi S, Huang D. A comparison of four methods for PCR inhibitor removal. Forensic Science International: Genetics. 2015;16:94-7. [9] Alaeddini R. Forensic implications of PCR inhibition—A review. Forensic Science International: Genetics. 2012;6(3):297-305. [10] Børsting C, Fordyce SL, Olofsson J, Mogensen HS, Morling N. Evaluation of the Ion Torrent™ HID SNP 169-plex: A SNP typing assay developed for human identification by second generation sequencing. Forensic Science International: Genetics. 2014;12:144-54. [11] Churchill JD, Chang J, Ge J, Rajagopalan N, Wootton SC, Chang C-W, et al. [12] Zeng X, King JL, Stoljarova M, Warshauer DH, LaRue BL, Sajantila A, et al. High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing. Forensic Science International: Genetics. 2015;16:38-47. [13] Bottino CG, Chang CW, Wootton S, Rajagopalan N, Langit R, Lagacé RE, et al. STR genotyping using ion torrent PGM and STR 24-plex system: Performance and data interpretation. Forensic Science International: Genetics Supplement Series. 2015;5:e325-e6. [14] Eduardoff M, Santos C, de la Puente M, Gross TE, Fondevila M, Strobl C, et al. Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™. Forensic Science International: Genetics. 2015;17:110-21. [15] Daniel R, Santos C, Phillips C, Fondevila M, van Oorschot RAH, Carracedo Á, et al. A SNaPshot of next generation sequencing for forensic SNP analysis. Forensic Science International: Genetics. 2015;14:50-60. This work was supported by Award #2015-DN-BX-K066 (National Institute of Justice, Office of Justice Programs, U.S. Department of Justice). The opinions, findings, and conclusions or recommendations expressed in this presentation are those of the author(s) and do not necessarily reflect those of the Department of Justice. INTRODUCTION ACKNOWLEDGEMENTS MATERIALS AND METHODS MATERIALS AND METHODS Sample Preparation DNA samples (N=3) included control DNA 2372 Component A Male (NIST, Gaithersburg, MD) and DNA sourced from buccal swabs provided by two anonymous donors. Samples were collected in accordance with the Sam Houston State University Institutional Review Board (#2015-12-26123). Inhibitor Preparation A range of inhibitor concentrations was used to test the tolerance of STR typing and MPS-based methods for HID purposes. Final concentrations for each inhibitor were prepared based on previously published studies in literature (Table 1). All subsequent working solutions were made with deionized water. Inhibitors were added to the PCR amplification to achieve the desired final inhibitor concentration in each 25 µL PCR reaction. The concentrations chosen for PCR amplification are shown in Table 1. STR Amplification Amplification was performed using the GlobalFiler® PCR Amplification Kit (Thermo Fisher Scientific) in a 25 µL reaction volume as per manufacturer instructions. Capillary Electrophoresis PCR products were separated and detected via capillary electrophoresis using the 3500™ Genetic Analyzer (Thermo Fisher Scientific). STRs were analyzed using GeneMapper ID-X v. 1.4 (Thermo Fisher Scientific). • STR chemistry (GlobalFiler®) was more tolerant to inhibitors with 1 ng DNA than 0.1 ng. • GlobalFiler® was more tolerant to the PCR inhibitors tested in this study compared to the Ion PGM sequencing chemistry for the HID-Ion AmpliSeq TM Library Kit and Identity Panel. • With MPS the inhibitors most vulnerable were hematin, humic acid, and melanin, but was more tolerant to samples spiked with collagen and calcium than STR analysis. • Samples inhibited with collagen and calcium behaved similarly, while humic acid, melanin, and hematin showed similar patterns of inhibition and amplification success. Massively Parallel Sequencing For this preliminary study, MPS was performed with a DNA template amount of 1 ng. Samples were prepared for amplification using the HID-Ion AmpliSeq TM Library Kit and Identity Panel (Thermo Fisher Scientific). After amplification, primer sequences were partially digested. Following the partial digestion of the primer sequences barcode adapters were ligated to the amplicons (a different barcode for every sample). After the ligation of the barcode adapters, Agencourt® AMPure® XP Reagent was added to each library and placed on a magnetic rack. Ethanol (70% solution) was added to the libraries to purify the DNA adhered to the beads. After the ethanol was removed from the libraries, Low TE was added to elute the DNA from the beads. Following library purification, the samples were quantified using the Ion Library TaqMan® Quantitation Assay (Thermo Fisher Scientific) using the 7500 Real-Time PCR System (Thermo Fisher Scientific). After quantification, libraries were diluted to 20 pM and pooled together. The pooled library was added to the Ion Chef™ System (Thermo Fisher Scientific) and loaded onto 316 barcoded semiconductor chips. Once the Ion Chef™ run was complete, the chips were placed on the Ion PGM™ to be sequenced. Data analysis including ISP loading, percent enrichment, percent library, and total usable reads was completed using Torrent Suite Software v4.6. SNP calls, coverage, and number of reads were analyzed using HID_SNP_Genotyper v4.3 plugin. • STR success decreased as the concentration of each of the 5 inhibitors increased at both 1 and 0.1 ng of DNA input (Fig. 1). • Peak heights decreased as the concentration of inhibitor increased for both 1 and 0.1 ng of DNA input. Heterozygote peak height ratios did not appear to notably decrease as locus size increased (data not shown). • STR analysis (GlobalFiler® kit) was most vulnerable to samples inhibited with high levels of humic acid and hematin. MPS Inhibitor Tolerance Mass disasters can be categorized as environmental, natural disasters, medical, vehicle, industrial, and terrorist attacks (1-3). Missing persons cases can also be referred to as mass disasters taking place over a longer period of time (4). Mass disasters, missing persons, and forensic cases may present with fragmented and highly decomposed or skeletonized human remains, comingled, contaminated with environmental elements, and/or intense heat damage (1). DNA in these samples may be highly degraded, damaged, and/or inhibited because remains may be exposed to adverse conditions, which can also increase the rate of decomposition (3). Highly degraded, damaged or environmentally effected samples can be problematic and may reduce the success of downstream DNA typing for identification purposes. Inhibitors are chemical or biological matrix interferences that can affect DNA extraction and/or PCR amplification processes during DNA analysis (5). Common co-extracted inhibitors include humic acid, hematin, collagen, calcium, melanin, indigo, bile salt, and urea and have different mechanisms by which they inhibit DNA amplification (5-9). This study will focus on PCR inhibitors most commonly encountered in skeletal and decomposed remains. Human Identification (HID) is traditionally performed using capillary electrophoresis-based STR typing and mitochondrial DNA analyses. Massively parallel sequencing (MPS) has offered an alternative method to Sanger sequencing for mitochondrial genome sequencing, STR, and SNP typing for HID purposes (10). With MPS, DNA molecules are sequenced in parallel, which increases throughput and can also provide more genetic information for each sample than conventional STR typing, including but not limited to, sequence variations (or SNPs) within STRs, ancestral, and phenotypic information (11-15). The Ion Personal Genome Machine (PGM) is a MPS platform that can be used in combination with commercial library preparation kits for SNP-based HID panels, ancestry panels, and whole genome mitochondrial analysis for forensic and investigative purposes. The aim of this study was to determine the comparative tolerance to inhibitors for the chemistries used for the SNP-based HID panel via MPS and conventional STR analysis. Figure 1: Percentage of STR alleles reported with 1 ng (left) and 0.1 ng (right) of DNA input, at 5 different concentrations with 5 inhibitors (Table 1). Concentration 0 = no inhibitor. Data presented as average ± SD (N = 3). Table 2. Percentage of STRs vs SNP alleles reported for five PCR inhibitors at five concentrations. • In general, the HID-Ion AmpliSeq TM Library Kit and Identity Panel chemistry was less tolerant to the PCR inhibitors most relevant to skeletal samples than STR analysis using the GlobalFiler® PCR Amplification kit. • HID-Ion AmpliSeq TM Library and Identity Panel chemistries were the most susceptible to humic acid, melanin, and hematin (Table 2 and Fig. 2 B). • SNP typing with the HID-Ion AmpliSeq TM Library Kit and Identity Panel chemistry via MPS was more tolerant than STR analysis with collagen and calcium (Table 2). • Overall STR analysis produced more balanced amplification than the SNP markers as measured by heterozygote peak height ratios (Fig. 2 A & B). • Samples inhibited with hematin, humic acid, and melanin resulted in the least balanced SNP typing results. However, the HID-Ion AmpliSeq TM Identity Panel continued to produce balanced amplification for collagen and calcium samples as the level of inhibition increased (Fig. 2 B). STR Inhibitor Tolerance Humic Acid ng/µL 5 7 10 17 25 STRs 100.0% 100.0% 100.0% 100.0% 100.0% SNPs 94.0% 59.3% 33.0% 10.7% 14.3% 100% 70 - 99 % Melanin ng/µL 4 5 7 10 12 40 - 69 % STRs 100.0% 100.0% 100.0% 100.0% 100.0% < 39 % SNPs 96.3% 81.7% 91.0% 31.3% 65.0% Hematin µM 1 3 5 7 10 STRs 100.0% 100.0% 100.0% 100.0% 100.0% SNPs 99.0% 39.7% 7.7% 7.7% 20.3% Collagen ng/µL 50 100 113 130 160 STRs 100.0% 100.0% 100.0% 97.8% 48.8% SNPs 100.0% 100.0% 100.0% 100.0% 100.0% Calcium µM 250 350 500 650 850 STRs 100.0% 100.0% 87.2% 47.4% 36.6% SNPs 100.0% 100.0% 100.0% 83.0% 63.7% Inhibitor Units Inhibitor Concentrations 0 1 2 3 4 5 Humic Acid ng/μL 0 50 100 200 225 250 Melanin ng/μL 0 25 35 40 45 50 Hematin μM 0 300 500 1000 1050 1100 Collagen ng/μL 0 50 100 112.5 130 160 Calcium μM 0 250 350 500 650 850 Table 1. The various concentrations of the five PCR inhibitors tested in this study. 0 10 20 30 40 50 60 70 80 90 100 Humic Acid Melanin Hematin Collagen Calcium No. Alleles (%) 0 1 2 3 4 5 0 10 20 30 40 50 60 70 80 90 100 Humic Acid Melanin Hematin Collagen Calcium No. Alelles (%) Heterozygote Allele Balance Figure 2: Heterozygote allele balance, with 1 ng DNA input for five inhibitors at five different concentrations using the A) STRs (GlobalFiler®), and B) SNPs (AmpliSeq TM Identity Panel) . Concentration 0 = no inhibitor. 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 PHR (%) A B 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 PHR (%) Humic Acid Melanin Hematin Collagen Calcium
Transcript of Comparative Tolerance of Short Tandem Repeat and Massively … · 2017. 5. 31. · • STR...
-
Comparative Tolerance of Short Tandem Repeat and Massively Parallel Sequencing Chemistries to Inhibited Samples
Kyleen Elwick, BA*; Charity Beherec, MSFS; David Gangitano, PhD; Sheree Hughes-Stamm, PhD
Department of Forensic Science, Sam Houston State University, Huntsville, TX 77340
R E S U LT S A N D D I S C U S S I O N
C O N C L U S I O N S
R E F E R E N C E S[1] Brough AL, Morgan B, Rutty GN. The basics of disaster victim identification. Journal of Forensic Radiology and Imaging. 2015;3(1):29-37.[2] Goodwin WH, Simmons T, Houck JASJSM. Disaster Victim Identification. Encyclopedia of Forensic Sciences. Waltham: AcademicPress; 2013;332-8.[3] Dhanardhono T, Wulandari N, Bhima SKL, Ahmad HJ, Widodo PT. DNA profiling of disaster victim identification in Trenggalekshipwreck case. Forensic Science International: Genetics Supplement Series. 2013;4(1):e5-e6.[4] Budowle B, Bieber FR, Eisenberg AJ. Forensic aspects of mass disasters: Strategic considerations for DNA-based human identification.Legal Medicine. 2005;7(4):230-43.[5] Thompson RE, Duncan G, McCord BR. An Investigation of PCR Inhibition Using Plexor®-Based Quantitative PCR and Short TandemRepeat Amplification.[6] Kemp BM, Monroe C, Smith DG. Repeat silica extraction: a simple technique for the removal of PCR inhibitors from DNA extracts.Journal of Archaeological Science. 2006;33(12):1680-9.[7] Schmedes S, Marshall P, King J, Budowle B. Effective removal of co-purified inhibitors from extracted DNA samples using synchronouscoefficient of drag alteration (SCODA) technology.[8] Hu Q, Liu Y, Yi S, Huang D. A comparison of four methods for PCR inhibitor removal. Forensic Science International: Genetics.2015;16:94-7.[9] Alaeddini R. Forensic implications of PCR inhibition—A review. Forensic Science International: Genetics. 2012;6(3):297-305.[10] Børsting C, Fordyce SL, Olofsson J, Mogensen HS, Morling N. Evaluation of the Ion Torrent™ HID SNP 169-plex: A SNP typing assaydeveloped for human identification by second generation sequencing. Forensic Science International: Genetics. 2014;12:144-54.[11] Churchill JD, Chang J, Ge J, Rajagopalan N, Wootton SC, Chang C-W, et al.[12] Zeng X, King JL, Stoljarova M, Warshauer DH, LaRue BL, Sajantila A, et al. High sensitivity multiplex short tandem repeat locianalyses with massively parallel sequencing. Forensic Science International: Genetics. 2015;16:38-47.[13] Bottino CG, Chang CW, Wootton S, Rajagopalan N, Langit R, Lagacé RE, et al. STR genotyping using ion torrent PGM and STR 24-plexsystem: Performance and data interpretation. Forensic Science International: Genetics Supplement Series. 2015;5:e325-e6.[14] Eduardoff M, Santos C, de la Puente M, Gross TE, Fondevila M, Strobl C, et al. Inter-laboratory evaluation of SNP-based forensicidentification by massively parallel sequencing using the Ion PGM™. Forensic Science International: Genetics. 2015;17:110-21.[15] Daniel R, Santos C, Phillips C, Fondevila M, van Oorschot RAH, Carracedo Á, et al. A SNaPshot of next generation sequencing forforensic SNP analysis. Forensic Science International: Genetics. 2015;14:50-60.
This work was supported by Award #2015-DN-BX-K066 (National Institute of Justice, Office ofJustice Programs, U.S. Department of Justice). The opinions, findings, and conclusions orrecommendations expressed in this presentation are those of the author(s) and do notnecessarily reflect those of the Department of Justice.
I N T R O D U C T I O N
A C K N O W L E D G E M E N T S
M AT E R I A L S A N D M E T H O D S
M AT E R I A L S A N D M E T H O D S
Sample Preparation DNA samples (N=3) included control DNA 2372Component A Male (NIST, Gaithersburg, MD) and DNA sourced frombuccal swabs provided by two anonymous donors. Samples werecollected in accordance with the Sam Houston State UniversityInstitutional Review Board (#2015-12-26123).
Inhibitor Preparation A range of inhibitor concentrations was used totest the tolerance of STR typing and MPS-based methods for HIDpurposes. Final concentrations for each inhibitor were prepared basedon previously published studies in literature (Table 1). All subsequentworking solutions were made with deionized water. Inhibitors wereadded to the PCR amplification to achieve the desired final inhibitorconcentration in each 25 µL PCR reaction. The concentrations chosen forPCR amplification are shown in Table 1.
STR Amplification Amplification was performed using the GlobalFiler®PCR Amplification Kit (Thermo Fisher Scientific) in a 25 µL reactionvolume as per manufacturer instructions.
Capillary Electrophoresis PCR products were separated and detected viacapillary electrophoresis using the 3500™ Genetic Analyzer (ThermoFisher Scientific). STRs were analyzed using GeneMapper ID-X v. 1.4(Thermo Fisher Scientific).
• STR chemistry (GlobalFiler®) was more tolerant to inhibitors with 1 ngDNA than 0.1 ng.
• GlobalFiler® was more tolerant to the PCR inhibitors tested in thisstudy compared to the Ion PGM sequencing chemistry for the HID-IonAmpliSeqTM Library Kit and Identity Panel.
• With MPS the inhibitors most vulnerable were hematin, humic acid,and melanin, but was more tolerant to samples spiked with collagenand calcium than STR analysis.
• Samples inhibited with collagen and calcium behaved similarly, whilehumic acid, melanin, and hematin showed similar patterns ofinhibition and amplification success.
Massively Parallel Sequencing For this preliminary study, MPS wasperformed with a DNA template amount of 1 ng. Samples wereprepared for amplification using the HID-Ion AmpliSeqTM Library Kit andIdentity Panel (Thermo Fisher Scientific). After amplification, primersequences were partially digested. Following the partial digestion of theprimer sequences barcode adapters were ligated to the amplicons (adifferent barcode for every sample).
After the ligation of the barcode adapters, Agencourt® AMPure® XPReagent was added to each library and placed on a magnetic rack.Ethanol (70% solution) was added to the libraries to purify the DNAadhered to the beads. After the ethanol was removed from the libraries,Low TE was added to elute the DNA from the beads.
Following library purification, the samples were quantified using theIon Library TaqMan® Quantitation Assay (Thermo Fisher Scientific) usingthe 7500 Real-Time PCR System (Thermo Fisher Scientific).
After quantification, libraries were diluted to 20 pM and pooledtogether. The pooled library was added to the Ion Chef™ System(Thermo Fisher Scientific) and loaded onto 316 barcoded semiconductorchips. Once the Ion Chef™ run was complete, the chips were placed onthe Ion PGM™ to be sequenced.
Data analysis including ISP loading, percent enrichment, percentlibrary, and total usable reads was completed using Torrent SuiteSoftware v4.6. SNP calls, coverage, and number of reads were analyzedusing HID_SNP_Genotyper v4.3 plugin.
• STR success decreased as the concentration of each of the 5 inhibitors increased at both 1 and 0.1 ng of DNA input (Fig. 1).• Peak heights decreased as the concentration of inhibitor increased for both 1 and 0.1 ng of DNA input. Heterozygote peak height ratios did not
appear to notably decrease as locus size increased (data not shown).
• STR analysis (GlobalFiler® kit) was most vulnerable to samples inhibited with high levels of humic acid and hematin.
MPS Inhibitor Tolerance
Mass disasters can be categorized as environmental, natural disasters,medical, vehicle, industrial, and terrorist attacks (1-3). Missing personscases can also be referred to as mass disasters taking place over a longerperiod of time (4).
Mass disasters, missing persons, and forensic cases may present withfragmented and highly decomposed or skeletonized human remains,comingled, contaminated with environmental elements, and/or intenseheat damage (1). DNA in these samples may be highly degraded,damaged, and/or inhibited because remains may be exposed to adverseconditions, which can also increase the rate of decomposition (3).
Highly degraded, damaged or environmentally effected samples canbe problematic and may reduce the success of downstream DNA typingfor identification purposes. Inhibitors are chemical or biological matrixinterferences that can affect DNA extraction and/or PCR amplificationprocesses during DNA analysis (5). Common co-extracted inhibitorsinclude humic acid, hematin, collagen, calcium, melanin, indigo, bilesalt, and urea and have different mechanisms by which they inhibit DNAamplification (5-9). This study will focus on PCR inhibitors mostcommonly encountered in skeletal and decomposed remains.
Human Identification (HID) is traditionally performed using capillaryelectrophoresis-based STR typing and mitochondrial DNA analyses.Massively parallel sequencing (MPS) has offered an alternative methodto Sanger sequencing for mitochondrial genome sequencing, STR, andSNP typing for HID purposes (10). With MPS, DNA molecules aresequenced in parallel, which increases throughput and can also providemore genetic information for each sample than conventional STR typing,including but not limited to, sequence variations (or SNPs) within STRs,ancestral, and phenotypic information (11-15).
The Ion Personal Genome Machine (PGM) is a MPS platform that canbe used in combination with commercial library preparation kits forSNP-based HID panels, ancestry panels, and whole genomemitochondrial analysis for forensic and investigative purposes. The aimof this study was to determine the comparative tolerance to inhibitorsfor the chemistries used for the SNP-based HID panel via MPS andconventional STR analysis.
Figure 1: Percentage of STR alleles reported with 1 ng (left) and 0.1 ng (right) of DNA input, at 5 different concentrations with 5 inhibitors (Table 1). Concentration 0 = no inhibitor. Data presented as average ± SD (N = 3).
Table 2. Percentage of STRs vs SNP alleles reported for five PCR inhibitors at five concentrations.
• In general, the HID-Ion AmpliSeqTM Library Kit and Identity Panel chemistrywas less tolerant to the PCR inhibitors most relevant to skeletal samples thanSTR analysis using the GlobalFiler® PCR Amplification kit.
• HID-Ion AmpliSeqTM Library and Identity Panel chemistries were the mostsusceptible to humic acid, melanin, and hematin (Table 2 and Fig. 2 B).
• SNP typing with the HID-Ion AmpliSeqTM Library Kit and Identity Panelchemistry via MPS was more tolerant than STR analysis with collagen andcalcium (Table 2).
• Overall STR analysis produced more balanced amplification than the SNPmarkers as measured by heterozygote peak height ratios (Fig. 2 A & B).
• Samples inhibited with hematin, humic acid, and melanin resulted in theleast balanced SNP typing results. However, the HID-Ion AmpliSeqTM IdentityPanel continued to produce balanced amplification for collagen and calciumsamples as the level of inhibition increased (Fig. 2 B).
STR Inhibitor Tolerance
Humic Acidng/µL 5 7 10 17 25STRs 100.0% 100.0% 100.0% 100.0% 100.0%SNPs 94.0% 59.3% 33.0% 10.7% 14.3% 100%
70 - 99 %
Melaninng/µL 4 5 7 10 12 40 - 69 %STRs 100.0% 100.0% 100.0% 100.0% 100.0% < 39 %SNPs 96.3% 81.7% 91.0% 31.3% 65.0%
HematinµM 1 3 5 7 10
STRs 100.0% 100.0% 100.0% 100.0% 100.0%SNPs 99.0% 39.7% 7.7% 7.7% 20.3%
Collagenng/µL 50 100 113 130 160STRs 100.0% 100.0% 100.0% 97.8% 48.8%SNPs 100.0% 100.0% 100.0% 100.0% 100.0%
CalciumµM 250 350 500 650 850
STRs 100.0% 100.0% 87.2% 47.4% 36.6%SNPs 100.0% 100.0% 100.0% 83.0% 63.7%
Inhibitor UnitsInhibitor Concentrations
0 1 2 3 4 5
Humic Acid ng/µL 0 50 100 200 225 250
Melanin ng/µL 0 25 35 40 45 50
Hematin µM 0 300 500 1000 1050 1100
Collagen ng/µL 0 50 100 112.5 130 160
Calcium µM 0 250 350 500 650 850
Table 1. The various concentrations of the five PCR inhibitors tested in this study.
0
10
20
30
40
50
60
70
80
90
100
Humic Acid Melanin Hematin Collagen Calcium
No.
Alle
les (
%)
0
1
2
3
4
5
0
10
20
30
40
50
60
70
80
90
100
Humic Acid Melanin Hematin Collagen Calcium
No.
Ale
lles (
%)
Heterozygote Allele Balance
Figure 2: Heterozygote allele balance, with 1 ng DNA input for five inhibitors at five different concentrations using the A) STRs (GlobalFiler®), and B) SNPs (AmpliSeqTM
Identity Panel) . Concentration 0 = no inhibitor.
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5
PHR
(%)
A
B
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5
PHR
(%)
Humic Acid Melanin Hematin Collagen Calcium
Slide Number 1
