| 1. |
- Chaitanya, Lakshmi, et al.
(författare)
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Collaborative EDNAP exercise on the IrisPlex system for DNA based prediction of human eye colour
- 2014
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Ingår i: Forensic Science International. - : Elsevier. - 1872-4973 .- 1878-0326. ; 11, s. 241-251
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Tidskriftsartikel (refereegranskat)abstract
- The IrisPlex system is a DNA-based test system for the prediction of human eye colour from biological samples and consists of a single forensically validated multiplex genotyping assay together with a statistical prediction model that is based on genotypes and phenotypes from thousands of individuals. IrisPlex predicts blue and brown human eye colour with, on average, >94% precision accuracy using six of the currently most eye colour informative single nucleotide polymorphisms (HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, SLC45A2 (MATP) rs16891982, TYR rs1393350, and IRF4 rs12203592) according to a previous study, while the accuracy in predicting non-blue and non-brown eye colours is considerably lower. In an effort to vigorously assess the IrisPlex system at the international level, testing was performed by 21 laboratories in the context of a collaborative exercise divided into three tasks and organised by the European DNA Profiling (EDNAP) Group of the International Society of Forensic Genetics (ISFG). Task 1 involved the assessment of 10 blood and saliva samples provided on FTA cards by the organising laboratory together with eye colour phenotypes; 99.4% of the genotypes were correctly reported and 99% of the eye colour phenotypes were correctly predicted. Task 2 involved the assessment of 5 DNA samples extracted by the host laboratory from simulated casework samples, artificially degraded, and provided to the participants in varying DNA concentrations. For this task, 98.7% of the genotypes were correctly determined and 96.2% of eye colour phenotypes were correctly inferred. For Tasks 1 and 2 together, 99.2% (1875) of the 1890 genotypes were correctly generated and of the 15 (0.8%) incorrect genotype calls, only 2 (0.1%) resulted in incorrect eye colour phenotypes. The voluntary Task 3 involved participants choosing their own test subjects for IrisPlex genotyping and eye colour phenotype inference, while eye photographs were provided to the organising laboratory and judged; 96% of the eye colour phenotypes were inferred correctly across 100 samples and 19 laboratories. The high success rates in genotyping and eye colour phenotyping clearly demonstrate the reproducibility and the robustness of the IrisPlex assay as well as the accuracy of the IrisPlex model to predict blue and brown eye colour from DNA. Additionally, this study demonstrates the ease with which the IrisPlex system is implementable and applicable across forensic laboratories around the world with varying pre-existing experiences.
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| 2. |
- Sidstedt, Maja, et al.
(författare)
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The impact of common PCR inhibitors on forensic MPS analysis
- 2019
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Ingår i: Forensic Science International: Genetics. - : Elsevier BV. - 1872-4973. ; 40, s. 182-191
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Tidskriftsartikel (refereegranskat)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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| 3. |
- Sidstedt, Maja, et al.
(författare)
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Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method
- 2024
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Ingår i: Forensic Science International: Genetics. - 1872-4973. ; 71
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Tidskriftsartikel (refereegranskat)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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