| 1. |
- Albinsson, Linda, et al.
(author)
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SKL byter DNA-kit
- 2011
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In: Kriminalteknik. - Linköping : SKL. ; :1, s. 4-5
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Journal article (pop. science, debate, etc.)
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| 2. |
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| 3. |
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| 4. |
- Boiso, Samuel, et al.
(author)
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RapidHIT for the purpose of stain analyses – An interrupted implementation
- 2017
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In: Forensic Science International. - : Elsevier. - 1875-1768 .- 1875-175X. ; 6:Supplement C, s. e589-e590
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Journal article (peer-reviewed)abstract
- Rapid DNA instruments have in recent years been developed, enabling analysis of forensic samples with a minimum of human intervention. Initially intended for fast handling of reference samples, such as samples from suspects in booking suites, attention shifted to include crime scene samples. The aim of this study was to determine whether or not the RapidHIT System (IntegenX) is fit for crime scene samples. The first runs gave very poor results, which was found to be due to an incorrect firmware setting leading to no or just minute amounts of amplicons being injected for electrophoresis. After solving this problem, 28 full runs (seven samples each) applying NGM SElect Express were performed comprising various amounts of blood on cotton swabs. Six of the runs failed completely, four due to cartridge leakage and in two runs the PCR mix was not injected. For 155 samples with 1–5ΌL blood (volumes for which complete DNA profiles are expected), 119 samples (77%) gave complete DNA profiles. Among the most serious failures were incorrect allele calling and leakage of DNA extract or PCR product. Other general issues were failure to export results, anode motor breakdown and broken capillary array. Due to the encountered problems with software, hardware and cartridges, together with the low success rate, it was decided not to continue towards implementation of the RapidHIT System in casework.
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| 5. |
- Borgmästars, Emmy, et al.
(author)
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Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing
- 2017
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In: Food and Environmental Virology. - : Springer Science and Business Media LLC. - 1867-0334 .- 1867-0342. ; 9:4, s. 395-405
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Journal article (peer-reviewed)abstract
- Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with Cq shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.
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| 6. |
- Foreberg, Christina, et al.
(author)
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High-throughput DNA extraction of forensic adhesive tapes
- 2016
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In: Forensic Science International. - : Elsevier. - 1872-4973 .- 1878-0326. ; 24, s. 158-163
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Journal article (peer-reviewed)abstract
- Tape-lifting has since its introduction in the early 2000's become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the "stickiness", rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p=0.13 and p=0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01ng/μL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples.
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| 7. |
- Forsberg, Christina, et al.
(author)
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The need for automation is limited when using a quick and inexpensive one-tube DNA extraction protocol for crime scene samples
- 2019
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In: Forensic Science International: Genetics Supplement Series. - : Elsevier BV. - 1875-1768. ; 7:1, s. 377-378
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Journal article (peer-reviewed)abstract
- Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions. We have developed, validated and implemented a quick, inexpensive and efficient one-tube direct lysis DNA extraction protocol for the majority of our crime scene samples. The method includes one pipetting step, the addition of a lysis buffer based on Chelex beads, Proteinase K and Tween 20. After three incubation steps with vortexing in between, the sample is ready for downstream applications (DNA quantification and STR profiling). The amount of lysis buffer added varies between 200–1000 μL depending on the amount of carrier material in the tube. If needed the extract can be purified or concentrated using a filter device, in our case Amicon Ultra-2. Through in-house validation we show that the method is fit-for-purpose for application in casework for samples containing blood, saliva, shed cells and semen as it provides high DNA yields and high quality STR profiles. The method was implemented at the Swedish National Forensic Centre (NFC) in February 2018. During the first year more than 35,000 crime scene samples were extracted using the method, generating DNA yields equivalent to the previously used method. In conclusion, the need for automation of the DNA extraction process is limited when using a one-tube direct lysis protocol including only the addition of lysis buffer and no transferring steps. In addition it saves costs as there is no need for expensive pipetting robots or service fees and since the reagents used in this direct lysis protocol are relatively inexpensive.
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| 8. |
- Hedell, Ronny, 1985, et al.
(author)
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Determining the optimal forensic DNA analysis procedure following investigation of sample quality
- 2018
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In: International Journal of Legal Medicine. - : Springer Science and Business Media LLC. - 0937-9827 .- 1437-1596. ; 132:4, s. 955-966
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Journal article (peer-reviewed)abstract
- © 2017 Springer-Verlag GmbH Germany Crime scene traces of various types are routinely sent to forensic laboratories for analysis, generally with the aim of addressing questions about the source of the trace. The laboratory may choose to analyse the samples in different ways depending on the type and quality of the sample, the importance of the case and the cost and performance of the available analysis methods. Theoretically well-founded guidelines for the choice of analysis method are, however, lacking in most situations. In this paper, it is shown how such guidelines can be created using Bayesian decision theory. The theory is applied to forensic DNA analysis, showing how the information from the initial qPCR analysis can be utilized. It is assumed the alternatives for analysis are using a standard short tandem repeat (STR) DNA analysis assay, using the standard assay and a complementary assay, or the analysis may be cancelled following quantification. The decision is based on information about the DNA amount and level of DNA degradation of the forensic sample, as well as case circumstances and the cost for analysis. Semi-continuous electropherogram models are used for simulation of DNA profiles and for computation of likelihood ratios. It is shown how tables and graphs, prepared beforehand, can be used to quickly find the optimal decision in forensic casework.
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| 9. |
- Hedell, Ronny, 1985, et al.
(author)
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Enhanced low-template DNA analysis conditions and investigation of allele dropout patterns
- 2015
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In: Forensic Science International: Genetics. - : Elsevier BV. - 1878-0326 .- 1872-4973. ; 14, s. 61-75
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Journal article (peer-reviewed)abstract
- Forensic DNA analysis applying PCR enables profiling of minute biological samples. Enhanced analysis conditions can be applied to further push the limit of detection, coming with the risk of visualising artefacts and allele imbalances. We have evaluated the consecutive increase of PCR cycles from 30 to 35 to investigate the limitations of low-template (LT) DNA analysis, applying the short tandem repeat (STR) analysis kit PowerPlex ESX 16. Mock crime scene DNA extracts of four different quantities (from around 8-84 pg) were tested. All PCR products were analysed using 5, 10 and 20 capillary electrophoresis (CE) injection seconds. Bayesian models describing allele dropout patterns, allele peak heights and heterozygote balance were developed to assess the overall improvements in EPG quality with altered PCR/CE settings. The models were also used to evaluate the impact of amplicon length, STR marker and fluorescent label on the risk for allele dropout. The allele dropout probability decreased for each PCR cycle increment from 30 to 33 PCR cycles. Irrespective of DNA amount, the dropout probability was not affected by further increasing the number of PCR cycles. For the 42 and 84 pg samples, mainly complete DNA profiles were generated applying 32 PCR cycles. For the 8 and 17 pg samples, the allele dropouts decreased from 100% using 30 cycles to about 75% and 20%, respectively. The results for 33, 34 and 35 PCR cycles indicated that heterozygote balance and stutter ratio were mainly affected by DNA amount, and not directly by PCR cycle number and CE injection settings. We found 32 and 33 PCR cycles with 10 CE injection seconds to be optimal, as 34 and 35 PCR cycles did not improve allele detection and also included CE saturation problems. We find allele dropout probability differences between several STR markers. Markers labelled with the fluorescent dyes CXR-ET (red in electropherogram) and TMR-ET (shown as black) generally have higher dropout risks compared with those labelled with JOE (green) and fluorescein (blue). Overall, the marker D10S1248 has the lowest allele dropout probability and D8S1179 the highest. The marker effect is mainly pronounced for 30-32 PCR cycles. Such effects would not be expected if the amplification efficiencies were identical for all markers. Understanding allele dropout risks and the variability in peak heights and balances is important for correct interpretation of forensic DNA profiles.
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| 10. |
- Hedman, Johannes, et al.
(author)
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A fast analysis system for forensic DNA reference samples
- 2008
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In: Forensic Science International: Genetics. - : Elsevier BV. - 1878-0326 .- 1872-4973. ; 2:3, s. 184-189
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Journal article (peer-reviewed)abstract
- On January 1st, 2006, the Swedish legislation on obtaining DNA reference samples from Suspects and the recording of DNA profiles in databases was changed. As a result the number of samples analysed at the Swedish National Laboratory of Forensic Science (SKL) increased from about 4500 in 2005 to more than 25,000 in 2006. To meet this challenge, SKL launched a flew analysis system to create an unbroken chain, from sampling to incorporation of a profile in the national DNA database and subsequent automatic generation of digitally signed hit reports. The system integrates logistics, digital data transfer, new functions in LIMS (ForumDNA Version 4, Ida Infront AB) and laboratory automation. Buccal swab samples are secured on a FTA (R) card attached to an identity form, which is barcoded with a unique sample ID. After sampling, the police officer sends a digital request to SKL. The sample is automatically registered in LIMS and processed on delivery. The resulting DNA profiles are automatically classified according to quality using a custom-made expert system. Building the evaluation around mathematical rules makes it reproducible, standardised and minimises manual work and clerk errors. All samples are run in duplicate and the two profiles are compared within LIMS before incorporation in the database. In the first year of operation, the median time for completion of an analysis was 3 days, measured from delivery of the sample to incorporation of the profile in the national DNA database. In spite of the dramatic increase in the number of reference samples there was no backlog. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
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| 11. |
- Hedman, Johannes, et al.
(author)
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A ranking index for quality assessment of forensic DNA profiles
- 2010
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In: BMC Research Notes. - : BioMed Central Ltd. - 1756-0500. ; 3:290
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Journal article (peer-reviewed)abstract
- BackgroundAssessment of DNA profile quality is vital in forensic DNA analysis, both in order to determine the evidentiary value of DNA results and to compare the performance of different DNA analysis protocols. Generally the quality assessment is performed through manual examination of the DNA profiles based on empirical knowledge, or by comparing the intensities (allelic peak heights) of the capillary electrophoresis electropherograms.ResultsWe recently developed a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles, the forensic DNA profile index (FI) (Hedman et al. Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles, Biotechniques 47 (2009) 951-958). FI uses electropherogram data to combine the intensities of the allelic peaks with the balances within and between loci, using Principal Components Analysis. Here we present the construction of FI. We explain the mathematical and statistical methodologies used and present details about the applied data reduction method. Thereby we show how to adapt the ranking index for any Short Tandem Repeat-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles.ConclusionsThe developed tool provides unbiased quality assessment of forensic DNA profiles. It can be applied for any DNA profiling system based on Short Tandem Repeat markers. Apart from crime related DNA analysis, FI can therefore be used as a quality tool in paternal or familial testing as well as in disaster victim identification.
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| 12. |
- Hedman, Johannes, et al.
(author)
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Crime scene DNA sampling by wet-vacuum applying M-VAC
- 2015
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In: Forensic Science International. - : Elsevier. - 1875-1768 .- 1875-175X. ; 5, s. e89-e90
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Journal article (peer-reviewed)abstract
- A possible alternative to conventional stain recovery by swabbing, taping or cutting, is the M-Vac wet-vacuum instrument (M-Vac Systems Inc.). We have evaluated M-Vac for sampling of dried saliva on porous and non-porous surfaces, shed cells on clothes and touch DNA. M-Vac gave significantly higher DNA yields for dried saliva stains on laminated wood, compared with cotton swabs (average DNA concentrations 1.14 vs. 0.57 ng/μL, p = 0.02). For stains on glass, M-Vac and cotton swabs gave comparable DNA yields. Additionally, M-Vac retrieved three times as much DNA from saliva stains on cotton fabric (T-shirt) compared with saliva on towels (terry cloth), showing that the absorption properties of the surface affect wet-vacuum sampling. M-Vac was also applied for retrieving wearer DNA from clothes, enabling generation of complete DNA profiles from denim jeans, leggings and cotton T-shirt. A mixed DNA profile was retrieved from an “aggressor” pressing a hand against the shoulder area of a worn T-shirt. Since the major component of the obtained mixed DNA profile was from the wearer, M-Vac may not be ideal for touch DNA sampling of clothes. Wet-vacuum sampling requires a fairly large instrument, trained users and DNA extraction procedures handling large sample volumes. The complexity of M-Vac sampling prevents it from being extensively used, but in specific and important cases it can be a valuable sampling tool.
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| 13. |
- Hedman, Johannes
(author)
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DNA Analysis of PCR-Inhibitory Forensic Samples
- 2011
-
Doctoral thesis (other academic/artistic)abstract
- DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, crime scene samples often contain extraneous substances that may interfere with the PCR-based forensic analysis, resulting in partial or negative DNA profiles. Extensive DNA purification may remove inhibitors, but involves the risk of DNA loss. In this work, pre-PCR processing was applied to improve the success rate of forensic DNA analysis of “dirty” samples without interfering with the composition of the samples.An experimental model system was developed to screen for inhibitor-tolerant DNA polymerase-buffer systems. The best-performing polymerases, Bio-X-Act Short, ExTaq HS and PicoMaxx HF, were applied in STR DNA analysis of PCRinhibitory crime scenes samples, i.e. samples that failed to produce complete DNA profiles in routine casework despite containing acceptable levels of DNA. A ranking index, called the forensic DNA profile index (FI), was developed to quantitatively describe DNA profile quality. The application of analysis of variance (ANOVA) to FI values confirmed that the three alternative polymerases significantly improved DNA profile quality for 20 of 32 problematic samples, compared with the standard polymerase AmpliTaq Gold. ExTaq HS and PicoMaxx HF showed complementary inhibitor-relieving properties. A blend of the two polymerases exhibited tolerance to a broader range of extraneous compounds, improving DNA profile quality in 34 of 42 PCR-inhibitory forensic samples. When used separately, ExTaq HS and PicoMaxx HF improved the results of analysis for 26 and 23 samples, respectively. Apart from their complementarity, synergy between the polymerases was mathematically proven by calculating the geometric mean values of FI and applying ANOVA.In November, 2010, the customised DNA polymerase blend was introduced inroutine casework at the Swedish National Laboratory of Forensic Science,increasing the proportion of complete DNA profiles generated from impuresamples from 38% to 87% for saliva, and from 69% to 94% for blood.Many presumed saliva crime scene stains give negative DNA results if presumptive testing is not performed. In this work, amylase activity testing was evaluated as a tool for saliva screening. No direct correlation was found between amylase activity and the DNA content of saliva. However, the sensitivity of the developed swab screening procedure (positive results for 0.5 μL of dried saliva) makes it applicable in cases where the number of DNA analyses is limited due to cost.
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| 14. |
- Hedman, Johannes, et al.
(author)
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Enhanced forensic DNA recovery with appropriate swabs and optimized swabbing technique
- 2021
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In: Forensic Science International: Genetics. - : Elsevier BV. - 1872-4973 .- 1878-0326. ; 53
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Journal article (peer-reviewed)abstract
- Efficient sampling with swabs is crucial for optimal forensic DNA analysis. The DNA recovery is determined by the skill of the practitioner and the compatibility between the applied swab and the surface. Here we investigate the impact of swabbing technique and swab type on the DNA yield. Thirteen different swabs from four categories (cotton, flocked nylon, small foam and large foam) provided equal DNA yields for smooth/non-absorbing surfaces. Large foam swabs gave higher DNA recovery for an absorbing wood surface. Factorial design of experiments and ANOVA was applied to study swabbing techniques for cotton swabs. Two key factors for efficient sampling were found to be 1) holding the swab with an approximate 60° angle against the surface and 2) to rotate the swab during sampling. For absorbing wood, it was beneficial to wet the swab heavily. The results of the factorial experiments were used to develop swabbing protocols for different surfaces. When ten experienced practitioners sampled according to these protocols, the DNA yield was increased for ridged plastic (around 1.25 times more DNA) and absorbing wood (2.2–6.2 times more DNA). For window glass, representing a smooth/non-absorbing surface, sampling according to the protocol gave DNA yields equivalent to applying individual sampling techniques. The protocol lowered person-to-person variation for ridged plastic. In conclusion, we have developed instructive protocols for cotton swab sampling on three types of surfaces: smooth/non-absorbing, ridged/non-absorbing and smooth/absorbing. We believe that such swabbing protocols will streamline and simplify the training of new practitioners and improve sampling efficiency for invisible DNA residues in casework.
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| 15. |
- Hedman, Johannes, et al.
(author)
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Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs
- 2011
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In: FORENSIC SCIENCE INTERNATIONAL-GENETICS. - : ELSEVIER IRELAND LTD, ELSEVIER HOUSE, BROOKVALE PLAZA, EAST PARK SHANNON, CO, CLARE, 00000, IRELAND. - 1872-4973 .- 1878-0326. ; 5:3, s. 194-198
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Journal article (peer-reviewed)abstract
- Amylase testing has been used as a presumptive test for crime scene saliva for over three decades, mainly to locate saliva stains on surfaces. We have developed a saliva screening application for crime scene trace swabs, utilising an amylase sensitive paper (Phadebas (R) Forensic Press test). Positive results were obtained for all tested dried saliva stains (0.5-32 mu L) with high or intermediate amylase activity (840 and 290 kU/L). Results were typically obtained within 5 min, and all samples that produced DNA profiles were positive. However, salivary amylase activities, as well as DNA concentrations, vary significantly between individuals. We show that there is no correlation between amylase activity and amount of DNA in fresh saliva. Even so, a positive amylase result indicates presence of saliva, and thereby presence of DNA. Amylase testing may be useful for screening in investigations where the number of DNA analyses is limited due to cost, e. g., in volume crime.
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| 16. |
- Hedman, Johannes, et al.
(author)
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Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles
- 2009
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In: BIOTECHNIQUES. - : Eaton Publishing. - 0736-6205 .- 1940-9818. ; 47:5, s. 951-958
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Journal article (peer-reviewed)abstract
- DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, DNA samples from crime scenes often contain PCR-inhibitory substances, which may generate blank or incomplete DNA profiles. Extensive DNA purification can be required to rid the sample of these inhibitors, although these procedures increase the risk of DNA loss. Most forensic laboratories use commercial DNA amplification kits (e.g., AmpFlSTR SGM Plus) with the DNA polymerase AmpliTaq Gold as the gold standard. Here, we show that alternative DNA polymerase-buffer systems can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition in crime scene samples, without additional sample preparation. DNA profiles from 20 of 32 totally or partially inhibited crime scene saliva samples were significantly improved using Bio-X-Act Short, ExTaq Hot Start, or PicoMaxx High Fidelity instead of AmpliTaq Gold. A statistical model for unbiased quality control of forensic DNA profiles was developed to quantify the results. Our study demonstrates the importance of adjusting the chemistry of the PCR to enhance forensic DNA analysis and diagnostic PCR, providing an alternative to laborious sample preparation protocols.
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| 17. |
- Hedman, Johannes, et al.
(author)
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Pre-PCR processing in bioterrorism preparedness : improved diagnostic capabilities for laboratory response networks
- 2013
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In: Biosecurity and bioterrorism. - : Mary Ann Liebert. - 1538-7135 .- 1557-850X. ; 11:S1, s. S87-S101
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Journal article (peer-reviewed)abstract
- Diagnostic DNA analysis using polymerase chain reaction (PCR) has become a valuable tool for rapid detection of biothreat agents. However, analysis is often challenging because of the limited size, quality, and purity of the biological target. Pre-PCR processing is an integrated concept in which the issues of analytical limit of detection and simplicity for automation are addressed in all steps leading up to PCR amplification—that is, sampling, sample treatment, and the chemical composition of PCR. The sampling method should maximize target uptake and minimize uptake of extraneous substances that could impair the analysis—so-called PCR inhibitors. In sample treatment, there is a trade-off between yield and purity, as extensive purification leads to DNA loss. A cornerstone of pre-PCR processing is to apply DNA polymerase-buffer systems that are tolerant to specific sample impurities, thereby lowering the need for expensive purification steps and maximizing DNA recovery. Improved awareness among Laboratory Response Networks (LRNs) regarding pre-PCR processing is important, as ineffective sample processing leads to increased cost and possibly false-negative or ambiguous results, hindering the decision-making process in a bioterrorism crisis. This article covers the nature and mechanisms of PCR-inhibitory substances relevant for agroterrorism and bioterrorism preparedness, methods for quality control of PCR reactions, and applications of pre-PCR processing to optimize and simplify the analysis of various biothreat agents. Knowledge about pre-PCR processing will improve diagnostic capabilities of LRNs involved in the response to bioterrorism incidents.
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| 18. |
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| 19. |
- Hedman, Johannes, et al.
(author)
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Samägd marktjänstutrustning på Stockholm Arlanda Airport
- 2018
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Other publicationabstract
- Flygindustrin har sett en stadig ökning de senaste åren och under 2017 ökade luftrumsrörelserna i Sverige med 6% från året innan. Den stadiga ökningen sätter stor press på det komplexa systemet som är på en flygplats då det är många parter som ska samarbeta. Därför krävs det att resurserna på flygplatsen utnyttjas optimalt för att få en flygplats som verkar effektivt. En av dessa parter som ofta hamnar i kläm är marktjänstbolagen, som både måste hålla låga priser för att flygbolagen ska kunna ha ett brett utbud till konkurrenskraftiga priser. Samtidigt som de måste betala de avgifter till flygplatsen som ökar i takt med trafiken.I nuläget tillhandahåller alla marktjänstbolag på Stockholm Arlanda Airport sin egen marktjänstutrustning, vilket gör att de måste optimera mängden utrustning efter sina egna toppar istället för hur helheten på flygplatsen ser ut. Något som leder till att det cirkulerar för mycket marktjänstutrustning på flygplatsen i nuläget. Därför undersöker detta examensarbetet om Stockholm Arlanda Airport kan få ett bättre resursutnyttjande med gemensam marktjänstutrustning och hur detta skulle påverka flygplatsens avgifter, samt vilka övriga effekter det kan medföra.Examensarbetet utgår från principen med förändringsarbete och modellen “Stegen i ett förbättringsarbete” används. Genom intervjuer med anställda på Swedavia och med information från marktjänstbolagen har nuläget kartlagts, vilket är det första steget i förbättringsarbetet. Författarna har även genom intervjuerna identifierat olika möjligheter och problem som kan uppstå med gemensam marktjänstutrustning. Mängden marktjänstutrustning som behövs med gemensam marktjänstutrustning beräknades genom intervallfärgning. Genom en totalkostnadsanalys har sedan tre olika förslag tagits fram på vilken typ av marktjänstutrustning som Swedavia borde äga för att det ska vara lönsamt för både Swedavia och marktjänstbolagen. De ekonomiska analyserna har tillsammans med de övriga effekterna legat till grund för de rekommendationer som författarna har givit till Swedavia.Författarna till rapporten har kommit fram till resultatet att med gemensam marktjänstutrustning finns ekonomiska besparingar att göraför både Swedavia och marktjänstbolagen. Andra effekter Stockholm Arlanda Airport kan vänta sig av förändringen är bland annat:Ökad flexibilitetGenom att marktjänstbolagen kan använda all marktjänstutrustningGenom att det alltid finns marktjänstutrustning på platsMindre mängd marktjänstutrustningSkapar fler fria ytor då det krävs mindre förvaringsytorBättre arbetsmiljöDå den ekonomiska pressen på marktjänstbolagen minskarMiljöpåverkanMindre utsläpp då Swedavia har större möjlighet att påverka vilken utrustning som används på flygplatsen
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| 20. |
- Hedman, Johannes, et al.
(author)
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Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis
- 2010
-
In: Analytical Biochemistry. - - : Elsevier Inc.. - 0003-2697 .- 1096-0309. ; 405, s. 192-200
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Journal article (peer-reviewed)abstract
- The success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substancespresent in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene salivastains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNApolymerase–buffer systems (Hedman et al., BioTechniques 47 (2009) 951–958). Here we show thatblending inhibitor-resistant DNA polymerase–buffer systems further increases the success rate of PCRfor various types of real crime scene samples showing inhibition. For 34 of 42 ‘‘inhibited” crime scenestains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq HotStart and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmedby analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerasesused separately for all tested sample types. When used separately, the performance of the DNApolymerases varied depending on the nature of the sample. The superiority of the blend is discussed interms of complementary effects and synergy between the DNA polymerase–buffer systems.
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| 21. |
- Hedman, Johannes, et al.
(author)
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The double-swab technique versus single swabs for human DNA recovery from various surfaces
- 2020
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In: Forensic Science International: Genetics. - : Elsevier BV. - 1872-4973 .- 1878-0326. ; 46
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Journal article (peer-reviewed)abstract
- Most crime scene DNA evidence is retrieved using cotton swabs. Since the late 90’s, the double-swab technique has been favoured by many practitioners throughout the world. However, the superiority of double-swabbing over applying single wet swabs has not been broadly verified. Here we set out to evaluate the need for the second dry swab for various surfaces, aiming at mimicking the range of surfaces encountered at crime scenes: flat and ridged, absorbing and non-absorbing. For the tested non-absorbing surfaces, i.e., window glass, steel, brass, synthetic leather and ridged plastic, the first wet swabs gave at least 16 times higher DNA yields compared to the second dry swabs. In addition, second wet swabs gave more DNA than second dry ones, opposing the common notion that the purpose of the second swab is to absorb excess liquid. When ten experienced staff members sampled saliva stains on a window glass surface the variation between persons was considerable, with mean DNA yields for the first wet swabs ranging from 0.045 ± 0.022 to 0.13 ± 0.024 ng/μL. The first wet swabs gave 4–162 times more DNA than the second dry swabs, with higher DNA amounts on second swabs coinciding with lower amounts for first swabs. We show that for non-absorbing surfaces, the first wet swab takes up most of the cells in dried stains, making it less valuable to apply a second dry swab. The differences in DNA recovery between first and second swabs were notable also for absorbing surfaces. Double-swabbing may be preferable for some complex surfaces, but focusing on efficient sampling technique with single wet swabs is likely a better general approach.
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| 22. |
- Hedman, Johannes, et al.
(author)
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Using the new Phadebas® Forensic Press test to find crime scene saliva stains suitable for DNA analysis
- 2008
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In: Forensic Science International: Genetics Supplement Series. - : Elsevier BV. - 1875-1768. ; 1:1, s. 430-432
-
Journal article (peer-reviewed)abstract
- The Phadebas® Forensic Press test is a new product that detects saliva stains by reacting with amylase. When the paper is pressed against a saliva stain a blue spot occurs. To test the sensitivity of the paper, a set of dilution series of saliva down to 1:500 was prepared on cotton fabric. Blue spots could be seen for dilutions of 1:100 when incubated at room temperature, and 1:200 in 37 °C. However, incubation at 37 °C did not provide acceptable reproducibility. The Phadebas® test was compared to four different lightsources for the ability to detect saliva on different carrier materials. Cotton fabric (T-shirt), denim, suede, leather, painted wood and untreated wood were tested. On denim, no stains could be detected with the lightsources, but Phadebas® showed all stains for pure saliva and dilution 1:5. DNA analysis was performed on detected stains and corresponding spots on the Phadebas® paper. Complete DNA profiles were produced for stains from pure saliva and dilution 1:5.
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| 23. |
- Hedman, Johannes, et al.
(author)
-
Validation guidelines for PCR workflows in bioterrorism preparedness, food safety and forensics
- 2018
-
In: Accreditation and Quality Assurance. - : Springer Science and Business Media LLC. - 0949-1775 .- 1432-0517. ; 23:3, s. 133-144
-
Journal article (peer-reviewed)abstract
- The polymerase chain reaction (PCR) is the backbone of contemporary DNA/RNA analysis, ideally enabling detection of one or just a few target molecules. However, when analysing food or forensic samples the analytical procedure is often challenged by low amounts of poor quality template molecules and complex matrices. Applying optimised and validated methods in all steps of the analysis workflow, i.e. sampling, sample treatment, DNA/RNA extraction and PCR (including reverse transcription for RNA analysis), is thus necessary to ensure the reliability of analysis. In this paper, we describe how in-house validation can be performed for the different modules of the diagnostic PCR process, providing practical examples as tools for laboratories in their planning of validation studies. The focus is analysis of heterogeneous samples with interfering matrices, with relevance in food testing, forensic DNA analysis, bioterrorism preparedness and veterinary medicine. Our objective is to enable rational in-house validation for reliable and swift quality assurance when results are urgent, for example in the event of a crisis such as a foodborne outbreak or a crime requiring the analysis of a large number of diverse samples. To that end, we explain the performance characteristics associated with method validation from a PCR and biological sample matrix perspective and suggest which characteristics to investigate depending on the type of method to be validated. Also, we include a modular approach to validation within the PCR workflow, aiming at efficient validation and a flexible use of methods.
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| 24. |
- Jansson, Linda, et al.
(author)
-
Assessing the consistency of shedder status under various experimental conditions
- 2024
-
In: Forensic Science International: Genetics. - : ELSEVIER IRELAND LTD. - 1872-4973 .- 1878-0326. ; 69
-
Journal article (peer-reviewed)abstract
- Shedder status is defined as the propensity of an individual to leave DNA behind on touched items or surfaces and has been suggested as one of the major factors influencing DNA transfer. However, little is known about whether shedder status is a constant property of an individual across multiple measurements or when the environmental conditions are changed. We have assessed DNA depositions of six males on 20 occasions to acquire a reference data set and to classify the participants into high, intermediate, or low shedders. This data set was also used to investigate how the probability of a correct shedder status classification changed when the number of DNA deposition measurements increased. Individual sweat rates were measured with a VapoMeter and data regarding hygiene routines were collected through a questionnaire on each sampling occasion. Next, we investigated how changes in the experimental conditions such as seasonal variation, hygiene routines, the temperature of the touched object, and repeated handling of an object influenced the DNA shedding. Additionally, we assessed DNA collected from the face and from T-shirts worn by the six participants to explore whether shedder status may be associated with the relative amount of DNA obtained from other body parts. Our results indicate that shedder status is a stable property across different seasons and different temperatures of handled objects. The relative DNA amounts obtained from repeatedly handled tubes, worn T-shirts, and from faces reflected the shedder status of the participants. We suggest that an individual's shedder status is highly influenced by the DNA levels on other body parts than hands, accumulating on the palms by frequently touching e.g., the face or previously handled items harboring self-DNA. Assessing physiological differences between the participants revealed that there were no associations between DNA shedding and individual sweat rates.
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| 25. |
- Jansson, Linda, et al.
(author)
-
Blending DNA binding dyes to improve detection in real-time PCR
- 2017
-
In: Biotechnology Reports. - : Elsevier BV. - 2215-017X. ; 14, s. 34-37
-
Journal article (peer-reviewed)abstract
- The success of real-time PCR (qPCR) analysis is partly limited by the presence of inhibitory compounds in the nucleic acid samples. For example, humic acid (HA) from soil and aqueous sediment interferes with amplification and also quenches the fluorescence of double-stranded (ds) DNA binding dyes, thus hindering amplicon detection. We aimed to counteract the HA fluorescence quenching effect by blending complementary dsDNA binding dyes, thereby elevating the dye saturation levels and increasing the fluorescence signals. A blend of the four dyes EvaGreen, ResoLight, SYBR Green and SYTO9 gave significantly higher fluorescence intensities in the presence and absence of HA, compared with the dyes applied separately and two-dye blends. We propose blending of dyes as a generally applicable means for elevating qPCR fluorescence signals and thus enabling detection in the presence of quenching substances.
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| 26. |
- Jansson, Linda, et al.
(author)
-
Challenging the proposed causes of the PCR plateau phase
- 2019
-
In: Biomolecular Detection and Quantification. - : Elsevier BV. - 2214-7535. ; 17
-
Journal article (peer-reviewed)abstract
- Despite the wide-spread use of the polymerase chain reaction (PCR) in various life-science applications, the causes of arrested amplicon generation in late cycles have not been confidently identified. This so-called plateau phase has been attributed to depletion or thermal break-down of primers or nucleotides, thermal inactivation of the DNA polymerase, and product accumulation resulting in competition between primer annealing and product re-hybridization as well as blocking of DNA polymerase by double-stranded amplicons. In the current study, we experimentally investigate the proposed limiting factors of PCR product formation. By applying robust and validated qPCR assays, we elucidate the impact of adding non-target and target amplicons to the reactions, mimicking the high amount of products in late PCR cycles. Further, the impact of increased primer concentrations and thermal stability of reagents are explored. Our results show that high amounts of non-target amplicons inhibit amplification by binding to the DNA polymerase, but that this effect is counteracted by addition of more DNA polymerase or prolonged annealing/extension times. Adding high amounts of target amplicons that also act as templates in the reaction is far less inhibitory to amplification, although a decrease in amplification rate is seen. When primer concentrations are increased, both amplification rates and end-product yields are elevated. Taken together, our results suggest that the main cause of PCR plateau formation is primer depletion and not product accumulation or degradation of reagents. We stress that a PCR plateau caused by primer depletion is assay-dependent, i.e. dependent on the primer design and primer characteristics such as the probability of primer-dimer formation. Our findings contribute to an improved understanding of the major parameters controlling the PCR dynamics at later cycles and the limitations of continued product formation, which in the end can facilitate PCR optimization.
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| 27. |
- Jansson, Linda, et al.
(author)
-
Evaluation and modification of lanthanum-based flocculation for isolation of bacteria from water samples
- 2018
-
In: Biotechnology Reports. - : Elsevier BV. - 2215-017X. ; 19
-
Journal article (peer-reviewed)abstract
- Molecular detection of pathogenic microorganisms in drinking and natural water is often challenged by low concentrations of the sought-after agents. Convenient methods to concentrate bacteria from water samples ranging from 1-10 L are highly warranted. Here we account for the evaluation of a lanthanum-based flocculation method to concentrate bacteria from water samples, applying four different bacterial species in tap water as well as river water. Our results show that the success of lanthanum-based flocculation is determined by both the bacterial species and the nature of the water sample. For tap water, satisfying flocculation efficiencies (above 60 %) were only reached for autoclaved water samples. However, the performance of the lanthanum-based flocculation method for non-autoclaved water was markedly improved by the addition of 20 mM bicarbonate to increase alkalinity. Our modified flocculation protocol may be applied as an alternative concentration method for bacteria in water samples of one liter or more.
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| 28. |
- Jansson, Linda, et al.
(author)
-
Factors affecting DNA recovery from cartridge cases
- 2020
-
In: Forensic Science International: Genetics. - : Elsevier BV. - 1872-4973 .- 1878-0326. ; 48
-
Journal article (peer-reviewed)abstract
- Cartridge cases are often the sole items left behind after a shooting incident and DNA traces from these can identify persons connected to the shooting. However, the chance of retrieving usable DNA profiles from cartridge cases is limited, due to the low amounts of deposited DNA and subsequent DNA loss associated with the firing process. In the current study, we set out to increase the DNA recovery from cartridge cases and cartridges by evaluating different swab types and detergents used for trace collection. A protocol applying nylon-flocked swabs instead of cotton swabs was implemented in casework at the Swedish National Forensic Centre (NFC), increasing DNA yield. The number of samples providing a DNA concentration ≥ 0.001 ng/μL (the in-house cut-off for processing low-template samples) increased from 11.1 to 28.6 % for cartridge cases and from 16.0 to 43.3 % for cartridges. There was also a substantial increase in mixed STR profiles, too complex to use for comparisons. Thus, it was not possible to take the full advantage of the elevated DNA yield provided by nylon-flocked swabs. The number of usable STR profiles increased from 5.0 to 8.0 % for cartridge cases and remained unchanged for cartridges. Controlled studies were performed to assess the impact on the DNA recovery from different persons handling the ammunition, different material and size of the cartridge cases, and the type of firearm used. These studies reflected an ideal situation, where all cartridges were extensively handled and loaded without gloves, thus providing a higher expected DNA yield compared to most casework samples. The total peak height differed by up to a factor of ∼50 when 20 different persons handled cartridges prior to shooting. By evaluating eleven combinations of different firearms and ammunition, it was found that the casing material and type of firearm also have a substantial impact on DNA yield.
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| 29. |
- Jansson, Linda, et al.
(author)
-
Impact of swab material on microbial surface sampling
- 2020
-
In: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012. ; 176
-
Journal article (peer-reviewed)abstract
- Efficient microbial sampling from surfaces for subsequent detection and quantification is crucial in fields such as food safety and hygiene monitoring. Cotton swabs are traditionally used for sample collection, but today there are numerous swabs of alternative material and different sizes available. Recovery efficiencies of swabs for different applications have been compared in several studies. However, the results are often contradictory. We have compared 15 different swabs made of cotton (n = 5), flocked nylon (n = 3) and foam (n = 7), for sampling of Listeria monocytogenes and mengovirus on small (4 cm2) and large (100 cm2) areas of window glass, ridged plastic and absorbing wood. Molecular quantification methods (qPCR and RT-qPCR) were applied, and all sampling and sample processing were standardized. Specific swabs gave higher DNA/RNA yields than others, depending on both the surface characteristics and the collected target. The highest DNA yields were achieved by applying Selefa or Puritan cotton swabs for Listeria sampling on 4 cm2 areas of window glass and ridged plastic. Certain foam swabs (Critical swab with medium head and Macrofoam) gave the highest yields when sampling Listeria on 4 cm2 areas of wood and on 100 cm2 areas of ridged plastic and wood. Most foam swabs, and especially Sigma Virocult, were advantageous for virus sampling, regardless of surface. Nylon-flocked swabs showed poor recovery regardless of surface characteristics. The recovery varied substantially between swabs made of the same material, suggesting that a single swab may not be representative for a certain swab material.
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| 30. |
- Jansson, Linda, et al.
(author)
-
Individual shedder status and the origin of touch DNA
- 2022
-
In: Forensic Science International: Genetics. - : Elsevier BV. - 1872-4973 .- 1878-0326. ; 56
-
Journal article (peer-reviewed)abstract
- Due to improved laboratory techniques, touched surfaces and items are increasingly employed as sources of forensic DNA evidence. This has urged a need to better understand the mechanisms of DNA transfer between individuals. Shedder status (i.e. the propensity to leave DNA behind) has been identified as one major factor regulating DNA transfer. It is known that some individuals tend to shed more DNA than others, but the mechanisms behind shedder status are largely unknown. By comparing the amounts of DNA deposited from active hands (i.e. used “as usual”) and inactive hands (i.e. not allowed to touch anything), we show that some of the self-DNA deposited from hands is likely to have accumulated on hands from other parts of the body or previously handled items (active hands: 2.1 ± 2.7 ng, inactive hands: 0.83 ± 1.1 ng, paired t-test: p = 0.014, n = 27 pairs of hands). Further investigation showed that individual levels of deposited DNA are highly associated with the level of DNA accumulation on the skin of the face (Pearson's correlation: r = 0.90, p < 0.00001 and Spearman's ranked correlation: rs = 0.56, p = 0.0016, n = 29). We hypothesized that individual differences in sebum secretion levels could influence the amount of DNA accumulation in facial areas, but no such correlation was seen (Pearson's correlation: r = − 0.13, p = 0.66, n = 14). Neither was there any correlation between DNA levels on hands or forehead and the time since hand or face wash. We propose that the amount of self-DNA deposited from hands is highly influenced by the individual levels of accumulated facial DNA, and that cells/DNA is often transferred to hands by touching or rubbing one's face.
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| 31. |
- Junker, Klara, et al.
(author)
-
Phenotype prediction accuracy – A Swedish perspective
- 2019
-
In: Forensic Science International: Genetics Supplement Series. - : Elsevier BV. - 1875-1768 .- 1875-175X. ; 7:1, s. 384-386
-
Journal article (peer-reviewed)abstract
- Methods for SNP-based phenotype prediction have recently been developed, but prediction accuracy data for several populations and regions are missing. We analysed the accuracy of hair and eye colour predictions for 111 individuals residing in Sweden, using the ForenSeq system and the MiSeq FGx instrument (Verogen). Observed colours were compared to predicted colours, using the colour with the highest probability value for each prediction. Overall, 80% of eye colour predictions were correct, but the system failed to predict intermediate/green eye colour in our cohort. For hair colour, 58% of predictions were correct, and the majority of incorrect predictions were related to brown hair. To assess if prediction accuracy could be improved by the exclusion of predictions with low probabilities, we applied a threshold of ≥0.7. The threshold improved eye colour prediction, from 80% to 85% correct predictions, whereas hair colour prediction accuracy was virtually unaffected (58% versus 57% correct predictions). In summary, the phenotype prediction accuracy was acceptable in our cohort and the use of a threshold was only useful for eye colour predictions.
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| 32. |
- Müller, Petra, et al.
(author)
-
Inter-laboratory study on standardized MPS libraries : evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA
- 2020
-
In: International Journal of Legal Medicine. - : Springer Science and Business Media LLC. - 0937-9827 .- 1437-1596. ; 134:1, s. 185-198
-
Journal article (peer-reviewed)abstract
- We present results from an inter-laboratory massively parallel sequencing (MPS) study in the framework of the SeqForSTRs project to evaluate forensically relevant parameters, such as performance, concordance, and sensitivity, using a standardized sequencing library including reference material, mixtures, and ancient DNA samples. The standardized library was prepared using the ForenSeq DNA Signature Prep Kit (primer mix A). The library was shared between eight European laboratories located in Austria, France, Germany, The Netherlands, and Sweden to perform MPS on their particular MiSeq FGx sequencers. Despite variation in performance between sequencing runs, all laboratories obtained quality metrics that fell within the manufacturer’s recommended ranges. Furthermore, differences in locus coverage did not inevitably adversely affect heterozygous balance. Inter-laboratory concordance showed 100% concordant genotypes for the included autosomal and Y-STRs, and still, X-STR concordance exceeded 83%. The exclusive reasons for X-STR discordances were drop-outs at DXS10103. Sensitivity experiments demonstrated that correct allele calling varied between sequencing instruments in particular for lower DNA amounts (≤ 125 pg). The analysis of compromised DNA samples showed the drop-out of one sample (FA10013B01A) while for the remaining three degraded DNA samples MPS was able to successfully type ≥ 87% of all aSTRs, ≥ 78% of all Y-STRs, ≥ 68% of all X-STRs, and ≥ 92% of all iSNPs demonstrating that MPS is a promising tool for human identity testing, which in return, has to undergo rigorous in-house validation before it can be implemented into forensic routine casework.
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| 33. |
- Nordgaard, Anders, 1962-, et al.
(author)
-
The forensic DNA profile index - a tool for comparison of electropherograms
- 2010
-
In: English Speaking Working Group (ESWG), International Society of Forensic Genetics (ISFG) Stockholm, Sweden.
-
Conference paper (other academic/artistic)abstract
- Background Assessment of DNA profile quality is vital in forensic DNA analysis, both in order to determine the evidentiary value of DNA results and to compare the performances of different DNA analysis protocols. Generally the quality assessment is performed through manual examination of the DNA profiles based on empirical knowledge, or by comparing the intensities (allelic peak heights) of the capillary electrophoresis electropherograms. We recently1 developed a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles, the forensic DNA profile index (FI). Core of presentation FI uses electropherogram data to combine the intensities of the allelic peaks with the balances within and between loci, using Principal Components Analysis. Here we present the construction of FI. We explain the mathematical and statistical methodologies used and present details about the applied data reduction method. Thereby we show how to adapt the ranking index for any STR-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles. 1 Hedman J, Nordgaard A, Rasmusson B, Ansell R, Rådström P (2009), Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modelling of DNA profiles, Biotechniques 47, 351-358.
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| 34. |
- Sanga, Malin, et al.
(author)
-
A panel of PCR-inhibitory reference materials for quality evaluation of multiplex STR analysis kits
- 2015
-
In: Forensic Science International. - : Elsevier. - 1875-1768 .- 1875-175X. ; 5, s. e317-319
-
Journal article (peer-reviewed)abstract
- PCR inhibition is a critical parameter in forensic DNA analysis. Substances interfering with amplification of short tandem repeats (STRs) may generate partial and/or ambiguous DNA profiles. We present a strategy for developing a broad panel of PCR-inhibitory reference materials (RMs), representing common casework samples. Our panel, including solutions prepared from for example cigarettes, chewing gum and soil, is a tool for in-house validation and lot testing of STR systems. PowerPlex ESX 16 Fast System tolerated high levels of some RMs, but several substances caused amplification problems. Humic acid had a negative effect on amelogenin, moist snuff hindered amplification of longer fragments, and chewing gum caused generally lowered allele peak heights. Applying a broad panel of RMs ensures that a wide range of inhibitory substances are tested, giving an improved understanding of inhibitor tolerance and effects.
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| 35. |
- Sidstedt, Maja, et al.
(author)
-
Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases
- 2017
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:3, s. 1642-1649
-
Journal article (peer-reviewed)abstract
- Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to S U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.
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| 36. |
- Sidstedt, Maja, et al.
(author)
-
Assessing the GeneRead SNP panel for analysis of low-template and PCR-inhibitory samples
- 2017
-
In: Forensic Science International: Genetics Supplement Series. - : Elsevier BV. - 1875-1768 .- 1875-175X. ; 6, s. 267-269
-
Journal article (peer-reviewed)abstract
- Massive parallel sequencing (MPS) is increasingly used for human identification purposes in forensic DNA laboratories. Forensic DNA samples are by nature heterogeneous and of varying quality, both concerning DNA integrity and matrices, creating a need for assays that can handle low amounts of DNA as well as impurities. Commercial short tandem repeat (STR) analysis kits for capillary electrophoresis-based separation have evolved drastically over the past years to handle low-template samples and high amounts of various PCR inhibitors. If MPS is to be used extensively in forensic laboratories there is a need to ascertain a similar performance. We have evaluated the GeneRead Individual Identity SNP panel (Qiagen) that includes 140 SNP markers, following the GeneRead DNAseq Targeted Panels V2 handbook for library preparation, applying low levels of DNA and relevant impurities. Analysis of down to 0.1 ng DNA generated SNP profiles with at least 85% called SNPs, after increasing the number of PCR cycles in the initial PCR from 20 to 24. The SNP assay handled extracts from four different DNA extraction methods, including Chelex with blood and saliva, without detrimental effects. Further, the assay was shown to tolerate relevant amounts of inhibitor solutions from soil, cigarettes, snuff and chewing gum. In conclusion, the performance of the SNP panel was satisfactory for casework-like samples.
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| 37. |
- Sidstedt, Maja, et al.
(author)
-
Digital PCR inhibition mechanisms using standardized inhibitors representing soil and blood matrices
- 2016
-
Conference paper (other academic/artistic)abstract
- Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentrationis determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrices such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. Here, we present how certain inhibitors disturb dPCR quantification and suggest solutions to these problems. Furthermore, we use real-time PCR, dPCR and isothermal titration calorimetry as tools to elucidate the mechanisms underlying the PCR inhibition. The impact of impurities on dPCR quantification was studied using humic acid as a model inhibitor. We show that the inhibitor-tolerance differs greatly for three different DNA polymerases, illustrating the importance of choosing a DNA polymerase-buffer system that is compatible with the samples to be analysed. Various inhibitory-substances from blood were found to disturb the system in different ways. For example, hemoglobin was found to cause quenching of fluorescence and a dramatic decrease of the number of positive reactions, leading to an underestimation of DNA quantity. IgG caused an increased number of late-starters. The system was more susceptible to inhibition by IgG when single-stranded DNA was used as template, compared with double-stranded DNA. By understanding more about the mechanisms of PCR inhibitors it will be possible to design more optimal PCR chemistries, improving dPCR detection and quantification.
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| 38. |
- Sidstedt, Maja, et al.
(author)
-
Humic substances cause fluorescence inhibition in real-time PCR.
- 2015
-
In: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 487, s. 30-37
-
Journal article (peer-reviewed)abstract
- Real-time PCR (qPCR) is the cornerstone of DNA analysis, enabling detection and quantification of minute nucleic acid amounts. However, PCR-based analysis is limited, in part, by the presence of inhibitors in the samples. PCR inhibition has been viewed solely as failure to efficiently generate amplicons, i.e. amplification inhibition. Humic substances (HS) are well known inhibitors of PCR amplification. Here we show that HS from environmental samples, specifically humic acid (HA), are very potent detection inhibitors, i.e. quench the fluorescence signal of dsDNA binding dyes. HA quenched the fluorescence of the commonly used qPCR dyes EvaGreen, ResoLight, SYBR Green I and SYTO 82, generating lowered amplification plots although amplicon production was unaffected. For EvaGreen, 500 ng HA quenched almost all fluorescence, whereas 1000 ng HA completely inhibited amplification when applying Immolase DNA polymerase with BSA. Fluorescence spectroscopy measurements showed that HA quenching was either static or collisional, and indicated that HA bound directly to the dye. Fulvic acid did not act as a qPCR detection inhibitor, but inhibited amplification similarly to HA. Hydrolysis probe fluorescence was not quenched by HA. Detection inhibition is an overlooked phenomenon that needs to be considered to allow for development of optimal qPCR assays.
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| 39. |
- Sidstedt, Maja, et al.
(author)
-
In-house validation of MPS-based methods in a forensic laboratory
- 2019
-
In: Forensic Science International: Genetics Supplement Series. - : Elsevier BV. - 1875-1768. ; 7:1, s. 635-636
-
Journal article (peer-reviewed)abstract
- Massively parallel sequencing (MPS) methods are increasingly applied in forensic casework. However, adequate validation guidelines are lacking. In this work, we describe our in-house validation of the ForenSeq DNA Signature Prep Kit (Verogen) for analysis of ancestry- and phenotype-informative SNPs. We also discuss in-house validation of MPS assays in general terms. When validating the SNP assay, we focused on the reliability of SNP genotype calls and the compatibility with commonly analysed sample types. Other issues, for example analytical thresholds and accuracy of the data prediction model were considered to be covered by the developmental validation of the kit. Our study included determination of (1) concordance, (2) limit of detection, (3) matrix effects, (4) repeatability, and (5) contamination risk. In conclusion, the MPS-based SNP assay showed overall adequate performance for single-source samples, with correct genotype calls. We welcome a broad discussion on how to perform in-house validation of MPS-based methods, as this is vital to ensure timely implementation of reliable assays in forensic laboratories.
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| 40. |
- Sidstedt, Maja, et al.
(author)
-
Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR
- 2018
-
In: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 410:10, s. 2569-2583
-
Journal article (peer-reviewed)abstract
- Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. [Figure not available: see fulltext.]
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| 41. |
- Sidstedt, Maja, et al.
(author)
-
PCR inhibition in qPCR, dPCR and MPS—mechanisms and solutions
- 2020
-
In: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650.
-
Research review (peer-reviewed)abstract
- DNA analysis has seen an incredible development in terms of instrumentation, assays and applications over the last years. Massively parallel sequencing (MPS) and digital PCR are now broadly applied in research and diagnostics, and quantitative PCR is used for more and more practises. All these techniques are based on in vitro DNA polymerization and fluorescence measurements. A major limitation for successful analysis is the various sample-related substances that interfere with the analysis, i.e. PCR inhibitors. PCR inhibition affects library preparation in MPS analysis and skews quantification in qPCR, and some inhibitors have been found to quench the fluorescence of the applied fluorophores. Here, we provide a deeper understanding of mechanisms of specific PCR inhibitors and how these impact specific analytical techniques. This background knowledge is necessary in order to take full advantage of modern DNA analysis techniques, specifically for analysis of samples with low amounts of template and high amounts of background material. The classical solution to handle PCR inhibition is to purify or dilute DNA extracts, which leads to DNA loss. Applying inhibitor-tolerant DNA polymerases, either single enzymes or blends, provides a more straightforward and powerful solution. This review includes mechanisms of specific PCR inhibitors as well as solutions to the inhibition problem in relation to cutting-edge DNA analysis.
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| 42. |
- Sidstedt, Maja, et al.
(author)
-
The impact of common PCR inhibitors on forensic MPS analysis
- 2019
-
In: Forensic Science International: Genetics. - : Elsevier BV. - 1872-4973. ; 40, s. 182-191
-
Journal article (peer-reviewed)abstract
- Massively parallel sequencing holds great promise for new possibilities in the field of forensic genetics, enabling simultaneous analysis of multiple markers as well as offering enhanced short tandem repeat allele resolution. A challenge in forensic DNA analysis is that the samples often contain low amounts of DNA in a background that may interfere with downstream analysis. PCR inhibition mechanisms of some relevant molecules have been studied applying e.g. real-time PCR and digital PCR. However, a detailed understanding of the effects of inhibitory molecules on forensic MPS, including mechanisms and ways to relieve inhibition, is missing. In this study, the effects of two well-characterized PCR inhibitors, humic acid and hematin, have been studied using the ForenSeq DNA Signature Prep kit. Humic acid and hematin resulted in lowered read numbers as well as specific negative effects on certain markers. Quality control of libraries with Fragment analyzer showed that increasing amounts of inhibitors caused a lowered amplicon quantity and that the larger amplicons were more likely to drop out. Further, the inhibitor tolerance could be improved 5–10 times by addition of bovine serum albumin in the initial PCR. On the contrary to the samples with inhibitors, low-template samples resulted in lowered read numbers for all markers. This difference strengthened the conclusion that the inhibitors have a negative effect on the DNA polymerase activity in the initial PCR. Additionally, a common capillary gel electrophoresis-based STR kit was shown to handle at least 200 times more inhibitors than the ForenSeq DNA Signature Prep kit. This suggests that there is room for improvement of the PCR components to ensure analytical success for challenging samples, which is needed for a broad application of MPS for forensic STR analysis.
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| 43. |
- Sidstedt, Maja, et al.
(author)
-
Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method
- 2024
-
In: Forensic Science International: Genetics. - : Elsevier Ireland Ltd. - 1872-4973 .- 1878-0326. ; 71
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Journal article (peer-reviewed)abstract
- Massively parallel sequencing (MPS) is increasingly applied in forensic short tandem repeat (STR) analysis. The presence of stutter artefacts and other PCR or sequencing errors in the MPS-STR data partly limits the detection of low DNA amounts, e.g., in complex mixtures. Unique molecular identifiers (UMIs) have been applied in several scientific fields to reduce noise in sequencing. UMIs consist of a stretch of random nucleotides, a unique barcode for each starting DNA molecule, that is incorporated in the DNA template using either ligation or PCR. The barcode is used to generate consensus reads, thus removing errors. The SiMSen-Seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing) method relies on PCR-based introduction of UMIs and includes a sophisticated hairpin design to reduce unspecific primer binding as well as PCR protocol adjustments to further optimize the reaction. In this study, SiMSen-Seq is applied to develop a proof-of-concept seven STR multiplex for MPS library preparation and an associated bioinformatics pipeline. Additionally, machine learning (ML) models were evaluated to further improve UMI allele calling. Overall, the seven STR multiplex resulted in complete detection and concordant alleles for 47 single-source samples at 1 ng input DNA as well as for low-template samples at 62.5 pg input DNA. For twelve challenging mixtures with minor contributions of 10 pg to 150 pg and ratios of 1–15% relative to the major donor, 99.2% of the expected alleles were detected by applying the UMIs in combination with an ML filter. The main impact of UMIs was a substantially lowered number of artefacts as well as reduced stutter ratios, which were generally below 5% of the parental allele. In conclusion, UMI-based STR sequencing opens new means for improved analysis of challenging crime scene samples including complex mixtures.
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| 44. |
- Staadig, Adam, et al.
(author)
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Applying Unique Molecular Indices with an Extensive All-in-One Forensic SNP Panel for Improved Genotype Accuracy and Sensitivity
- 2023
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In: Genes. - : MDPI AG. - 2073-4425. ; 14:4
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Journal article (peer-reviewed)abstract
- One of the major challenges in forensic genetics is being able to detect very small amounts of DNA. Massively parallel sequencing (MPS) enables sensitive detection; however, genotype errors may exist and could interfere with the interpretation. Common errors in MPS-based analysis are often induced during PCR or sequencing. Unique molecular indices (UMIs) are short random nucleotide sequences ligated to each template molecule prior to amplification. Applying UMIs can improve the limit of detection by enabling accurate counting of initial template molecules and removal of erroneous data. In this study, we applied the FORCE panel, which includes ~5500 SNPs, with a QIAseq Targeted DNA Custom Panel (Qiagen), including UMIs. Our main objective was to investigate whether UMIs can enhance the sensitivity and accuracy of forensic genotyping and to evaluate the overall assay performance. We analyzed the data both with and without the UMI information, and the results showed that both genotype accuracy and sensitivity were improved when applying UMIs. The results showed very high genotype accuracies (>99%) for both reference DNA and challenging samples, down to 125 pg. To conclude, we show successful assay performance for several forensic applications and improvements in forensic genotyping when applying UMIs.
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| 45. |
- Xavier, Catarina, et al.
(author)
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Evaluation of the VISAGE basic tool for appearance and ancestry inference using ForenSeq® chemistry on the MiSeq FGx® system
- 2022
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In: Forensic Science International: Genetics. - : Elsevier BV. - 1872-4973. ; 58
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Journal article (peer-reviewed)abstract
- The possibility of providing investigative leads when conventional DNA identification methods fail to solve a case can be of extreme relevance to law enforcement. Therefore, the forensic genetics community has focused research towards the broadened use of DNA, particularly for prediction of appearance traits, bio-geographical ancestry and age. The VISible Attributes through GEnomics (VISAGE) Consortium expanded the use of DNA phenotyping by developing new molecular and statistical tools for appearance, age and ancestry prediction. The VISAGE basic tool for appearance (EVC) and ancestry (BGA) prediction was initially developed using Ampliseq chemistry, but here is being evaluated using ForenSeq chemistry. The VISAGE basic tool offers a total of 41 EVC and 115 BGA SNPs and thus provides more predictions, i.e., skin color, than achieved with the ForenSeq DNA Signature Prep kit that is based on 24 EVC and 56 BGA SNPs. Five VISAGE laboratories participated in collaborative experiments to provide foreground for developmental validation of the assay. Assessment of assay performance and quality metrics, reproducibility, sensitivity, inhibitor tolerance and species specificity are described. Furthermore, the assay was tested using challenging samples such as mock casework samples and artificially degraded DNA. Two different analysis strategies were applied for this study and output on genotype calls and read depth was compared. Overall, inter-laboratory, inter-method and concordance with publicly available data were analysed and compared. Finally, the results showed a reliable and robust tool, which can be easily applied for laboratories already using a MiSeq FGx with ForenSeq reagents.
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