| 1. |
- Allen, Marie, et al.
(författare)
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Author´s response :
- 2006
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Ingår i: Journal of Forensic Sciences. - : Wiley. - 0022-1198 .- 1556-4029. ; 51:4, s. 937-938
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The mitochondrial hypervariable regions I and II have proven to be a useful target for analysis of forensic materials, in which the amount of DNA is limited or highly degraded. Conventional mitochondrial DNA (mtDNA) sequencing can be time-consuming and expensive, limitations that can be minimized using a faster and less expensive typing assay.We have evaluated the exclusion capacity of the linear array mtDNA HVI/HVII region-sequence typing assay (Roche Applied Science) in 16 forensic cases comprising 90 samples. Using the HVI/HVII mtDNA linear array, 56% of the samples were excluded and thus less than half of the samples require further sequencing due to a match or inconclusive results. Of all the samples that were excluded by sequence analysis, 79% could be excluded using the HVI/HVII linear array alone. Using the HVI/HVII mtDNA linear array assay, we demonstrate the potential to decrease sequencing efforts substantially and thereby reduce the cost and the turn-around time in casework analysis.
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| 2. |
- Allen, Marie, et al.
(författare)
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Universal tag arrays in forensic SNP analysis.
- 2005
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Ingår i: Methods in Molecular Biology. - 1064-3745 .- 1940-6029. ; 297, s. 141-154
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Tidskriftsartikel (refereegranskat)abstract
- Microarray-based single nucleotide polymorphism (SNP) genotyping enables simultaneous and rapid detection of a large number of markers and is thus an attractive method for forensic individual acid identification. This assay relies on a one-color detection system and minisequencing in solution before hybridization to universal tag arrays. The minisequencing reaction is based on incorporation of a fluorescent dideoxynucleotide to a primer containing a tag-sequence flanking the position to be interrogated. This one-color system detects C and T polymorphisms in separate reactions on multiple polymerase chain reaction targets with the fluorophore TAMRA coupled to the respective dideoxynucleotide. After incorporation, tagged primer sequences are hybridized through their complementary sequence on the array, and positive signals are detected by a confocal laser-scanner.
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| 3. |
- Alneberg, Johannes, et al.
(författare)
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Genomes from uncultivated prokaryotes : a comparison of metagenome-assembled and single-amplified genomes
- 2018
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Ingår i: Microbiome. - : BioMed Central. - 2049-2618. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Background: Prokaryotes dominate the biosphere and regulate biogeochemical processes essential to all life. Yet, our knowledge about their biology is for the most part limited to the minority that has been successfully cultured. Molecular techniques now allow for obtaining genome sequences of uncultivated prokaryotic taxa, facilitating in-depth analyses that may ultimately improve our understanding of these key organisms. Results: We compared results from two culture-independent strategies for recovering bacterial genomes: single-amplified genomes and metagenome-assembled genomes. Single-amplified genomes were obtained from samples collected at an offshore station in the Baltic Sea Proper and compared to previously obtained metagenome-assembled genomes from a time series at the same station. Among 16 single-amplified genomes analyzed, seven were found to match metagenome-assembled genomes, affiliated with a diverse set of taxa. Notably, genome pairs between the two approaches were nearly identical (average 99.51% sequence identity; range 98.77-99.84%) across overlapping regions (30-80% of each genome). Within matching pairs, the single-amplified genomes were consistently smaller and less complete, whereas the genetic functional profiles were maintained. For the metagenome-assembled genomes, only on average 3.6% of the bases were estimated to be missing from the genomes due to wrongly binned contigs. Conclusions: The strong agreement between the single-amplified and metagenome-assembled genomes emphasizes that both methods generate accurate genome information from uncultivated bacteria. Importantly, this implies that the research questions and the available resources are allowed to determine the selection of genomics approach for microbiome studies.
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| 4. |
- Alneberg, Johannes, et al.
(författare)
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Genomes from uncultivated prokaryotes: a comparison of metagenome-assembled and single-amplified genomes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background: Prokaryotes dominate the biosphere and regulate biogeochemical processes essential to all life. Yet, our knowledge about their biology is for the most part limited to the minority that has been successfully cultured. Molecular techniques now allow for obtaining genome sequences of uncultivated prokaryotic taxa, facilitating in-depth analyses that may ultimately improve our understanding of these key organisms.Results: We compared results from two culture-independent strategies for recovering bacterial genomes: single-amplified genomes and metagenome-assembled genomes. Single-amplified genomes were obtained from samples collected at an offshore station in the Baltic Sea Proper and compared to previously obtained metagenome-assembled genomes from a time series at the same station. Among 16 single-amplified genomes analyzed, seven were found to match metagenome-assembled genomes, affiliated with a diverse set of taxa. Notably, genome pairs between the two approaches were nearly identical (>98.7% identity) across overlapping regions (30-80% of each genome). Within matching pairs, the single-amplified genomes were consistently smaller and less complete, whereas the genetic functional profiles were maintained. For the metagenome-assembled genomes, only on average 3.6% of the bases were estimated to be missing from the genomes due to wrongly binned contigs; the metagenome assembly was found to cause incompleteness to a higher degree than the binning procedure.Conclusions: The strong agreement between the single-amplified and metagenome-assembled genomes emphasizes that both methods generate accurate genome information from uncultivated bacteria. Importantly, this implies that the research questions and the available resources are allowed to determine the selection of genomics approach for microbiome studies.
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| 5. |
- Divne, Anna-Maria, et al.
(författare)
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A DNA microarray system for forensic SNP analysis
- 2005
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Ingår i: Forensic Science International. - : Elsevier BV. - 0379-0738 .- 1872-6283. ; 154:2-3, s. 111-121
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Tidskriftsartikel (refereegranskat)abstract
- Forensic DNA analysis is routinely performed using polymorphic short tandem repeat (STR) markers. However, for degraded or minute DNA samples, analysis of autosomal single nucleotide polymorphisms (SNPs) in short fragments might be more successful. Furthermore, sequencing of mitochondrial DNA (mtDNA) is often performed on highly degraded or scarce samples due to the high copy number of mtDNA in each cell. Due to the increasing number of complete mtDNA genome sequences available, the limited discrimination power of an mtDNA analysis, may be increased by analysis of coding region polymorphisms in addition to the non-coding variation. Since sequence analysis of the coding region would require more material than generally present in forensic samples, an alternative SNP analysis approach is required. We have developed a one-colour microarray-based SNP detection system for limited forensic materials. The method is based on minisequencing in solution prior to hybridisation to universal tag-arrays. In a first outline of a forensic chip, a combination of 12 nuclear and 21 mitochondrial SNP markers are analysed simultaneously. The mitochondrial markers on the chip are polymorphisms within the hypervariable region as well as in the coding region. Even though the number of markers in the current system is limited, it can easily be extended to yield a greater power of discrimination. When fully developed, microarray analysis provides a promising system for efficient sensitive SNP analysis of forensic samples in the future.
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| 6. |
- Divne, Anna-Maria, 1970-
(författare)
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Evaluation of New Technologies for Forensic DNA Analysis
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- DNA samples from crime scenes or mass disasters are often limited and degraded which limits the possibility of successful traditional STR analysis. Moreover, there is a need to decrease the turnaround time in criminal investigations. These circumstances require a wider set of assays and technologies to be investigated for potential use in forensic DNA analysis, which has been explored in this thesis work. DNA analysis can also provide a useful tool in forensic pathology investigations. In a search for mutations involved in The Sudden Infant death Syndrome (SIDS), the entire mitochondrial genome was sequenced in six SIDS infants and shorter mtDNA regions were analysed in paraffin-embedded tissues from an additional 14 SIDS cases. In this sample material no mutations associated with SIDS were found that could explain the death of these infants. To reduce time, cost and effort related to sequencing of the mtDNA HVI/HVII regions in caseworks, a HVI/HVII mtDNA linear array assay was used as a pre-screening for exclusions of suspects or evidence samples. Using this assay, 56% of the samples involved in casework analysis could be excluded before sequencing was undertaken.The possibility to use the new array technology was explored in a SNP assay targeting both mtDNA and nuclear SNPs. The system relies on minisequencing in solution prior to hybridisation to tag arrays. Using this system, we demonstrate a rapid, highly multiplexable and flexible array-format for SNP analysis.The properties of the Pyrosequencing technology being a fast and user-friendly assay was utilised in a study to investigate the possibility to use this method for limited and degraded samples. Ten STR loci, overlapping with standardised kits, were genotyped in 114 Swedish individuals. We found additional variation and higher resolution of repeats at some of these loci that are not detected using standard fragment analysis.
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| 7. |
- Divne, Anna-Maria, et al.
(författare)
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Forensic analysis of autosomal STR markers using Pyrosequencing
- 2010
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Ingår i: Forensic Science International. - : Elsevier BV. - 1872-4973 .- 1878-0326. ; 4:2, s. 122-129
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Tidskriftsartikel (refereegranskat)abstract
- Short tandem repeats (STRs) are highly variable, and therefore routinely used in forensic investigations for a DNA-based individual identification. The routine assay is commonly performed by size separation using capillary electrophoresis, but alternative technologies can also be used. In this study, a Pyrosequencing assay was developed for analysis of STR markers useful in forensic DNA analysis. The assay was evaluated for 10 different STR loci (CSF1PO, TH01, TPOX, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539 and Penta E) and a total of 114 Swedish individuals were genotyped. This genotyping strategy reveal the actual sequence and variant alleles were seen at several loci, providing additional information compared to fragment size analysis. At the D13S317 locus a T/A SNP located in the last repeat unit was observed in 92% of the genotypes. Moreover, an upstream flanking SNP at locus D7S820, a SNP within the repeats at D3S1358 and D8S1179 and a deletion in the flanking region at locus D5S818 were observed. The Pyrosequencing method was first developed for SNP typing and sequencing of shorter DNA fragments but the method also provides an alternative method for STR analysis of less complex repeats. This assay is suitable for investigation of new markers, a rapid compilation of population data and for confirmation of variant and new alleles.
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| 8. |
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| 9. |
- Divne, Anna-Maria, et al.
(författare)
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Forensic casework analysis using the HVI/HVII mtDNA linear array assay.
- 2005
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Ingår i: Journal of Forensic Sciences. - 0022-1198 .- 1556-4029. ; 50:3, s. 548-554
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Tidskriftsartikel (refereegranskat)abstract
- The mitochondrial hypervariable regions I and II have proven to be a useful target for analysis of forensic materials, in which the amount of DNA is limited or highly degraded. Conventional mitochondrial DNA (mtDNA) sequencing can be time-consuming and expensive, limitations that can be minimized using a faster and less expensive typing assay. We have evaluated the exclusion capacity of the linear array mtDNA HVI/HVII region-sequence typing assay (Roche Applied Science) in 16 forensic cases comprising 90 samples. Using the HVI/HVII mtDNA linear array, 56% of the samples were excluded and thus less than half of the samples require further sequencing due to a match or inconclusive results. Of all the samples that were excluded by sequence analysis, 79% could be excluded using the HVI/HVII linear array alone. Using the HVI/HVII mtDNA linear array assay, we demonstrate the potential to decrease sequencing efforts substantially and thereby reduce the cost and the turn-around time in casework analysis.
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| 10. |
- Florenza, Javier, et al.
(författare)
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De novo assembled single-cell transcriptomes from aquatic phytoflagellates reveal a metabolically distinct cell population
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Single-cell transcriptomics has rapidly become a standard tool for decoding cell identity, fate and interactions in mammalian model organisms. Adopting such techniques to uncover functional dynamics in aquatic single-celled organisms holds huge potential, but evidence of applicability to non-model, poorly understood microeukaryotes remains limited. In the present study, live Ochromonas triangulata cells from fast and slow growth phases were FACS-sorted based on food vacuole staining and chlorophyll fluorescence, and single-cell transcriptomic libraries were prepared following the Smart-seq2 protocol. In total, 744 transcriptomes were Illumina sequenced. Lacking a reference genome, transcriptomes were assembled de novo using Trinity and resulting transcripts were annotated by BLAST using the Swiss-Prot database. Following read mapping, differential gene expression was evaluated using DESeq2 and metabolic maps were generated based on pathways from the KEGG Orthology database. Clustering the read counts revealed the identity of the two expected transcriptional states corresponding to each growth phase as well as a third distinct cluster of cells present in both growth phases. This cryptic group showed extensive downregulation of genes in pathways associated with ribosome-functioning, CO2 fixation and core carbohydrate catabolism such as glycolysis, β oxidation of fatty acids and tricarboxylic acid cycle. Nevertheless, the biological underpinnings of this population, which would have remained unnoticed in an integrated approach, could not be clarified. Additionally, the possibility of using carry-over rRNA reads for taxonomic annotation was tested, verifying the identity of 88% of the O. triangulata cells. In conclusion, we demonstrate the power of single cell transcriptomics for metabolic mapping of microeukaryotes for which reference resources might be limited and thereby highlight its potential as a tool to gain access to microeukaryote dynamics in natural communities.
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| 11. |
- Florenza, Javier, et al.
(författare)
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Fluorescently labelled prey surrogates in combination with FACS successfully discriminate actively feeding mixotrophs in a lake water sample
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Mixotrophic protists are capable of acting both as primary producers and primary consumers at the base of the aquatic food web, thus constituting key organisms in ecosystems where they are abundant. However, their identity, abundance, ecological dynamics and biogeochemical impact in aquatic ecosystems remain understudied in comparison to classically demarcated autotrophs or heterotrophs. In this study, we make use of fluorescently labelled prey surrogates and fluorescence-activated cell sorting to taxonomically identify actively-feeding individual mixotrophic flagellates from lake water. Replicated experiments were carried out to assess the performance of three different fluorescently labelled prey types and a fluorescent dye targeting food vacuoles. In the experiments, water from an oligotrophic lake was exposed independently to each type of reporter and cells were individually sorted based on fluorescent signals for predation and chlorophyll a. A total of 927 individual single cells were successfully recovered, with all fluorescent reporters exhibiting high sensitivity for putative mixotrophic taxa: overall, 87% of the occurrences could be assigned to dictyochophytes, 9% to chrysophytes and 3% to dinoflagellates. As a result, we were able to detect cryptic diversity within pedinellid algae and report a Prorocentrum-like freshwater occurrence. We argue that this procedure can be a valuable tool to uncover relevant and unexpected active mixotrophic species in a wider range of aquatic environments, and could easily be coupled to other techniques to describe the finer details of the trophic status of aquatic microbial communities.
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| 12. |
- Hildebrandt, Franziska, 1994-, et al.
(författare)
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scDual-Seq of Toxoplasma gondii-infected mouse BMDCs reveals heterogeneity and differential infection dynamics
- 2023
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Dendritic cells and macrophages are integral parts of the innate immune system and gatekeepers against infection. The protozoan pathogen, Toxoplasma gondii, is known to hijack host immune cells and modulate their immune response, making it a compelling model to study host-pathogen interactions. Here we utilize single cell Dual RNA-seq to parse out heterogeneous transcription of mouse bone marrow-derived dendritic cells (BMDCs) infected with two distinct genotypes of T. gondii parasites, over multiple time points post infection. We show that the BMDCs elicit differential responses towards T. gondii infection and that the two parasite lineages distinctly manipulate subpopulations of infected BMDCs. Co-expression networks define host and parasite genes, with implications for modulation of host immunity. Integrative analysis validates previously established immune pathways and additionally, suggests novel candidate genes involved in host-pathogen interactions. Altogether, this study provides a comprehensive resource for characterizing host-pathogen interplay at high-resolution.
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| 13. |
- Nilsson, Martina, et al.
(författare)
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Sensitive forensic analysis using the Pyrosequencing technology
- 2006
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Ingår i: Progress in Forensic Genetics. - : Elsevier BV. ; :1288, s. 625-627
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Tidskriftsartikel (refereegranskat)abstract
- In forensic casework analysis, rapid and flexible systems for detection of variation found in nuclear or mitochondrial genomes are valuable. We have developed several different typing systems based on the Pyrosequencing technology to allow sensitive analysis of mtDNA as well as autosomal and Y-chromosome SNPs or STRs in short amplicons.
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| 14. |
- Styrman, Hanna, et al.
(författare)
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STR sequence variants revealed by Pyrosequencing technology
- 2006
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Ingår i: Excerpta Medica. - : Elsevier BV. - 0531-5131 .- 1873-6157. ; :1288, s. 669-671
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Tidskriftsartikel (refereegranskat)abstract
- Pyrosequencing is a fast, real-time, non-electrophoretic sequencing-by-synthesis method that can be used to genotype short tandem repeat (STRs) markers. In this study, a total of 18 Y-chromosome and autosomal STRs have been successfully analyzed using Pyrosequencing technology. Several sequence variants were detected, demonstrating that additional information besides the fragment length can be provided in a forensic DNA investigation by these assays.
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| 15. |
- Troell, Karin, et al.
(författare)
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Cryptosporidium as a testbed for single cell genome characterization of unicellular eukaryotes
- 2016
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- Background: Infectious disease involving multiple genetically distinct populations of pathogens is frequently concurrent, but difficult to detect or describe with current routine methodology. Cryptosporidium sp. is a widespread gastrointestinal protozoan of global significance in both animals and humans. It cannot be easily maintained in culture and infections of multiple strains have been reported. To explore the potential use of single cell genomics methodology for revealing genome-level variation in clinical samples from Cryptosporidium-infected hosts, we sorted individual oocysts for subsequent genome amplification and full-genome sequencing. Results: Cells were identified with fluorescent antibodies with an 80 % success rate for the entire single cell genomics workflow, demonstrating that the methodology can be applied directly to purified fecal samples. Ten amplified genomes from sorted single cells were selected for genome sequencing and compared both to the original population and a reference genome in order to evaluate the accuracy and performance of the method. Single cell genome coverage was on average 81 % even with a moderate sequencing effort and by combining the 10 single cell genomes, the full genome was accounted for. By a comparison to the original sample, biological variation could be distinguished and separated from noise introduced in the amplification. Conclusions: As a proof of principle, we have demonstrated the power of applying single cell genomics to dissect infectious disease caused by closely related parasite species or subtypes. The workflow can easily be expanded and adapted to target other protozoans, and potential applications include mapping genome-encoded traits, virulence, pathogenicity, host specificity and resistance at the level of cells as truly meaningful biological units.
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