How many cells are required for successful DNA profiling?

Kanokwongnuwut, Piyamas; Martin, Belinda; Taylor, Duncan; Kirkbride, K. Paul; Linacre, Adrian

doi:10.1016/j.fsigen.2020.102453

Cited by 40 publications

(31 citation statements)

References 35 publications

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“…in which keratinisation stage the cells are in, hence if the DNA within the cells is highly degraded the PCR amplification will naturally fail [25]. 4) The CC method does not detect single molecules of cell-free DNA in the fluorescence microscope at 100X magnification [12,19]. Other studies have shown that cell-free DNA is present in biological material deposited after skin contact [13,26,27], hence this could be a substantial factor to a person's shedder status that is not captured by the applied Diamond TM dye and CC method.…”

Section: Tablementioning

confidence: 99%

“…We compared the cell count method (CC method) to the classical handheld tube method (HH method). For the CC method we used Diamond™ nucleic acid dye which is a fluorescent dye that binds to the grooves of the DNA molecule [18], in both cell-free and nuclear DNA; however cell-free DNA is unlikely to be detected by microscopy using 220X or less magnification [12,19]. The number of cells that appear as bright spots in a fluorescence microscope, within a fingerprint, was counted.…”

Section: Introductionmentioning

confidence: 99%

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Determination of shedder status: A comparison of two methods involving cell counting in fingerprints and the DNA analysis of handheld tubes

Johannessen

Gill

Røseth

et al. 2021

Forensic Science International: Genetics

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The shedder status of an individual may be important to consider in the context of DNA transfer, persistence and recovery and in Bayesian networks where a person's shedder status may have an impact on the outcome. In this study we compared two methods to determine shedder status: the handheld tube (HH) method and a fluorescent cell count (CC) method. A poor association was observed between the numbers of detected cells in a fingerprint using the CC method and the strength of the DNA result with the HH method. The 20 participants were classified into low (25%), medium (50%) and high (25%) shedders based on the HH method. While the low and high shedders showed a good consistency between the replicates, the medium shedders varied more and have to be considered more carefully as they may act as either a high or a low shedder in an event of DNA transfer.

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Section: Tablementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Determination of shedder status: A comparison of two methods involving cell counting in fingerprints and the DNA analysis of handheld tubes

Johannessen

Gill

Røseth

et al. 2021

Forensic Science International: Genetics

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“…Single cells are considered ltDNA (low-template DNA), and handling such material is not uncommon in forensics, where specialists are often faced with low-quantity and low-quality DNA. Since such samples are treated with special lab protocols (e.g., increased number of PCR cycles), it makes them prone to stochastic effects (drop-ins and dropouts) and increased stutter ratios, the interpretation of ltDNA profiles is a common topic of discussion [ 40 , 41 , 42 , 43 , 44 , 45 ]. An alternative to traditional fragment analysis is to sequence the bi-allelic SNPs, which are less prone to artifacts and are characterized by smaller amplicons.…”

Section: Discussionmentioning

confidence: 99%

Pushing the Boundaries: Forensic DNA Phenotyping Challenged by Single-Cell Sequencing

Diepenbroek

Bayer

Anslinger

2021

Genes

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Single-cell sequencing is a fast developing and very promising field; however, it is not commonly used in forensics. The main motivation behind introducing this technology into forensics is to improve mixture deconvolution, especially when a trace consists of the same cell type. Successful studies demonstrate the ability to analyze a mixture by separating single cells and obtaining CE-based STR profiles. This indicates a potential use of the method in other forensic investigations, like forensic DNA phenotyping, in which using mixed traces is not fully recommended. For this study, we collected single-source autopsy blood from which the white cells were first stained and later separated with the DEPArray™ N×T System. Groups of 20, 10, and 5 cells, as well as 20 single cells, were collected and submitted for DNA extraction. Libraries were prepared using the Ion AmpliSeq™ PhenoTrivium Panel, which includes both phenotype (HIrisPlex-S: eye, hair, and skin color) and ancestry-associated SNP-markers. Prior to sequencing, half of the single-cell-based libraries were additionally amplified and purified in order to improve the library concentrations. Ancestry and phenotype analysis resulted in nearly full consensus profiles resulting in correct predictions not only for the cells groups but also for the ten re-amplified single-cell libraries. Our results suggest that sequencing of single cells can be a promising tool used to deconvolute mixed traces submitted for forensic DNA phenotyping.

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“…Genomic DNA analysis was performed on cells sampled from proband and both parents by buccal swab technique [11]. Exomes and flanking splice junctions were captured using the IDT xGen Exome Research Panel v1.0 (Integrated DNA Technologies, Coralville, Iowa USA).…”

Section: Testing Protocolmentioning

confidence: 99%

Phenotype from SAMD9 Mutation at 7p21.1 Appears Attenuated by Novel Compound Heterozygous Variants at RUNX2 and SALL1

Sills¹,

Wood²

2021

Preprint

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Sterile alpha motif domain-containing protein 9 (SAMD9) is a regulatory protein centrally involved in cell proliferation and apoptosis. Mapped to 7p21.1, variants in SAMD9 have been reported in &lt;50 pediatric cases worldwide, typically with early lethality. Germline gain-of-function SAMD9 variants are associated with MIRAGE Syndrome (myelodysplasia, infection, restricted growth, adrenal hypoplasia, genital anomalies, and enteropathy). Spalt like transcription factor 1 (SALL1) is a zinc finger transcriptional repressor located at 16q12.1 where only two transcript variants in SALL1 are known. RUNX2 (6p21.1) encodes a nuclear protein with a Runt DNA-binding domain critical for osteoblastic differentiation, skeletal morphogenesis, and serves as a scaffold for nucleic acids and regulatory factors involved in skeletal gene expression. RUNX2 and SALL1 are thus both ‘master regulators’ of tissue organization and embryo development. Here, we describe exome sequencing and copy number variants in two previously unknown mutations—R824Q in SAMD9, and Q253H in SALL1. A new multiexon 3’ terminal duplication in RUNX2 is also reported. This is the first known phenotype characterization for the intersection of all three variants in a healthy 46,XX adult. Focusing on developmental progress, ultrastructural renal anatomy, and selected reproductive aspects, we describe this unique genotype diagnosed incidentally during Covid-19 illness. Individual disruption in SAMD9, RUNX2, or SALL1 would be expected to give a bleak prognosis. However, the convergence discovered here appears to dampen severe pathology, perhaps by cross-gene silencing of effects normally deleterious when such changes occur alone.

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How many cells are required for successful DNA profiling?

Cited by 40 publications

References 35 publications

Determination of shedder status: A comparison of two methods involving cell counting in fingerprints and the DNA analysis of handheld tubes

Determination of shedder status: A comparison of two methods involving cell counting in fingerprints and the DNA analysis of handheld tubes

Pushing the Boundaries: Forensic DNA Phenotyping Challenged by Single-Cell Sequencing

Phenotype from SAMD9 Mutation at 7p21.1 Appears Attenuated by Novel Compound Heterozygous Variants at RUNX2 and SALL1

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