| 1. |
- 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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| 2. |
- Jansson, Linda, et al.
(author)
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Blending DNA binding dyes to improve detection in real-time PCR
- 2017
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In: Biotechnology Reports. - : Elsevier BV. - 2215-017X. ; 14, s. 34-37
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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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| 3. |
- Junker, Klara, et al.
(author)
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Phenotype prediction accuracy – A Swedish perspective
- 2019
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In: Forensic Science International: Genetics Supplement Series. - : Elsevier BV. - 1875-1768 .- 1875-175X. ; 7:1, s. 384-386
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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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| 4. |
- Müller, Petra, et al.
(author)
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Inter-laboratory study on standardized MPS libraries : evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA
- 2020
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In: International Journal of Legal Medicine. - : Springer Science and Business Media LLC. - 0937-9827 .- 1437-1596. ; 134:1, s. 185-198
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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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| 5. |
- Nedialkova, Lubov Petkova, et al.
(author)
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Temperate phages promote colicin-dependent fitness of Salmonella enterica serovar Typhimurium.
- 2015
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In: Environmental Microbiology. - : Wiley. - 1462-2920 .- 1462-2912. ; 18, s. 1591-1603
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Journal article (peer-reviewed)abstract
- Bacteria employ bacteriocins for interference competition in microbial ecosystems. Colicin Ib (ColIb), a pore-forming bacteriocin, confers a significant fitness benefit to Salmonella enterica serovar Typhimurium (S. Tm) in competition against commensal E. coli in the gut. ColIb is released from S. Tm into the environment, where it kills susceptible competitors. However, colicin-specific release proteins, as they are known for other colicins, have not been identified in case of ColIb. Thus, its release mechanism has remained unclear. In the current study, we have established a new link between ColIb release and lysis activity of temperate, lambdoid phages. By the use of phage-cured S. Tm mutant strains, we show that the presence of temperate phages and their lysis genes is necessary and sufficient for release of active ColIb into the culture supernatant. Furthermore, phage-mediated lysis significantly enhanced S. Tm fitness in competition against a ColIb-susceptible competitor. Finally, transduction with the lambdoid phage 933W rescued the defect of E. coli strain MG1655 with respect to ColIb release. In conclusion, ColIb is released from bacteria in the course of phage lysis. Our data reveal a new mechanism for colicin release and point out a novel function of temperate phages in enhancing colicin-dependent bacterial fitness.
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| 6. |
- Sidstedt, Maja, et al.
(author)
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Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases
- 2017
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:3, s. 1642-1649
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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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| 7. |
- Sidstedt, Maja, et al.
(author)
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Assessing the GeneRead SNP panel for analysis of low-template and PCR-inhibitory samples
- 2017
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In: Forensic Science International: Genetics Supplement Series. - : Elsevier BV. - 1875-1768 .- 1875-175X. ; 6, s. 267-269
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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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| 8. |
- Sidstedt, Maja, et al.
(author)
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Digital PCR inhibition mechanisms using standardized inhibitors representing soil and blood matrices
- 2016
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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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| 9. |
- Sidstedt, Maja, et al.
(author)
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Humic substances cause fluorescence inhibition in real-time PCR.
- 2015
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In: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 487, s. 30-37
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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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| 10. |
- Sidstedt, Maja, et al.
(author)
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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
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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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| 11. |
- Sidstedt, Maja, et al.
(author)
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Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR
- 2018
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In: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 410:10, s. 2569-2583
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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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| 12. |
- Sidstedt, Maja, et al.
(author)
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PCR inhibition in qPCR, dPCR and MPS—mechanisms and solutions
- 2020
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In: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650.
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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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| 13. |
- Sidstedt, Maja
(author)
-
PCR inhibition mechanisms in forensic DNA analysis
- 2019
-
Doctoral thesis (other academic/artistic)abstract
- In forensic DNA analysis, the polymerase chain reaction (PCR) enables DNAanalysis of minute biological crime scene traces. PCR is an enzymatic reaction for amplification of specific DNA fragments involving both biochemical andbiophysical processes. The main analytical challenge is posed by the nature of the samples of interest. Crime scene samples are heterogeneous and often contain background matrices, e.g. PCR inhibitors that impair the limit of detection. The objective of the work described in this Doctoral thesis was to obtain a greater understanding of how relevant PCR inhibitors impact the PCR. The focus was to study PCR inhibition mechanisms using the DNA analysis techniques qPCR, digital PCR (dPCR) and massively parallel sequencing (MPS). The importance of applying a DNA polymerase-buffer system that is compatible with the sample matrix was shown through screening of 16 DNA polymerase-buffer systems for qPCR analysis of environmental samples containing humic substances. In dPCR, the tolerance to humic acid was elevated 48 times when an alternative DNA polymerase-buffer system was used. Also, a statistical Bayesian model was developed to provide an objective dPCR data analysis method. Detailed PCR inhibition mechanisms were elucidated for the main inhibitory molecules in blood and soil matrices. IgG binds to genomic single-stranded DNA, thereby hindering primer annealing and causing delayed amplification. Haemoglobin and its derivative haematin negatively impact the DNA polymerase activity. When studying the PCR inhibition mechanisms of humic acid using a robust DNA polymerase-buffer system, a new bottleneck in the analysis was identified. Namely, that humic acid causes fluorescence quenching, thus hindering amplicon detection. Further studies revealed that not only humic acid, but also haemoglobin causes static quenching of DNA-binding dyes. MPS is finding its way into forensic casework, due to the many opportunities that the technique offers. I could showt hat humic acid and haematin have a considerable negative impact on the multiplex PCR used to prepare the DNA for sequencing. Further, the inhibitor-tolerance of the state-of-the art MPS method is poor, but could be improved through the addition of BSA in the initial PCR. In summary, I have elucidated the PCR inhibition mechanisms of the relevant PCR inhibitors in soil and blood matrices. I have identified and characterized inhibitors affecting DNA polymerase activity, nucleic acids and fluorescence detection. The knowledge gained can be used for developing accurate and reliable DNA analysis methods for forensic DNA analysis as well as for other fields where challenging samples from surfaces or the environment are analysed.
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| 14. |
- 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
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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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| 15. |
- Sidstedt, Maja, et al.
(author)
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Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method
- 2024
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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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| 16. |
- 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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