Developmental validation of SpeID: A pyrosequencing-based assay for species identification

Ghemrawi, Mirna; Fischinger, Frank; Duncan, George; Dukes, Mary Jones; Guilliano, Mark; McCord, Bruce

doi:10.1016/j.fsigen.2021.102560

Cited by 8 publications

(5 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…DNA was eluted in 40 µL TE. Buffer and extracts were quantified using an in‐house vertebrate‐specific SYBR Green real‐time PCR assay (Rotor‐Gene Q, Qiagen) [23]. DNA was normalized to 10 ng input levels except for chewing gum, which was normalized to 2 ng.…”

Section: Methodsmentioning

confidence: 99%

An examination of differences in epigenetic methylation of saliva type samples based on collection method

Ghemrawi,

Fernandez‐Tejero,

Vaquero

et al. 2024

Electrophoresis

Self Cite

View full text Add to dashboard Cite

In the context of forensic casework, it is imperative to both establish a DNA profile from biological specimens and accurately identify the specific bodily fluid source. To achieve this, DNA methylation markers have been developed for the differentiation of blood, semen, vaginal epithelial secretions, and saliva samples. Saliva, alternatively referred to as oral fluid, is recognized for its heterogeneous cellular composition, characterized by a mixture of epithelial, leukocytic, and bacterial cells. Consequently, our research has revealed variations in methylation percentages that correlate with the method employed for collecting saliva samples. To investigate these concepts, we scrutinized four CpG markers situated within or in proximity to the BCAS4, SLC12A8, SOX2OT, and FAM43A genes. Subsequently, we designed primers based on bioinformatically transformed reference sequences for these markers and rigorously assessed their quality by examining dimer and hairpin formation, melting temperature, and specificity. These loci were identified as saliva markers based on either buccal swabs or spit collection. Yet, there has been minimal or no research conducted to explore the variations in methylation between different collection methods. For this study, buccal, lip, tongue, spit, and nasal swabs were collected from 20 individuals (N = 100). Mock forensic samples, which include chewing gum (N = 10) and cigarettes (N = 10), were also tested. DNA was extracted, bisulfite converted, then amplified using in‐house designed assays, and pyrosequenced. The methylation levels were compared to other body fluids (semen, blood, vaginal epithelia, and menstrual blood [N = 32]). A total of 608 pyrosequencing results demonstrated that sampling location and collection method can greatly influence the level of methylation, highlighting the importance of examining multiple collection/deposition methods for body fluids when developing epigenetic markers.

show abstract

Section: Methodsmentioning

confidence: 99%

An examination of differences in epigenetic methylation of saliva type samples based on collection method

Ghemrawi,

Fernandez‐Tejero,

Vaquero

et al. 2024

Electrophoresis

Self Cite

View full text Add to dashboard Cite

show abstract

“…A multiplex PCR assay was developed to simultaneously identify 22 mammalian species (alpaca, Asiatic black bear, Bactrian camel, brown rat, cat, cow, common raccoon, dog, European rabbit, goat, horse, house mouse, human, Japanese badger, Japanese wild boar, masked palm civet, pig, raccoon dog, red fox, sheep, Siberian weasel, and sika deer) and four poultry species (chicken, domestic turkey, Japanese quail, and mallard) [ 617 ]. A number of other species identification assays have also been reported [ [618] , [619] , [620] ].…”

Section: Emerging Technologies Research Studies and Other Topicsmentioning

confidence: 99%

Recent advances in forensic biology and forensic DNA typing: INTERPOL review 2019–2022

Butler

2023

Forensic Science International: Synergy

View full text Add to dashboard Cite

“…Pyrosequencing has provided a rapid approach to species identification in an NGS format. A variety of mitochondrial loci have been utilized for this purpose, including cytochrome b [28–30], 16S rRNA (the locus used in the Barcode of Life sequences) [31], cytochrome c oxidase [32, 33], 18S rRNA [34], and 12S rRNA genes [35–38] and hypervariable regions HVI/HVII [39]. At present, there are no commercially available kits for species identification via pyrosequencing.…”

Section: Current Forensic Applicationsmentioning

confidence: 99%

“…18S rRNA [34], and 12S rRNA genes [35][36][37][38] and hypervariable regions HVI/HVII [39]. At present, there are no commercially available kits for species identification via pyrosequencing.…”

Section: Species Identificationmentioning

confidence: 99%

“…At present, there are no commercially available kits for species identification via pyrosequencing. However, Ghemrawi et al have developed and validated a pyrosequencing-based species ID assay following SWGDAM guidelines [38]. This methodology can be used to quantify and detect the presence of more than 30 species using a universal primer set, 48 samples can be examined simultaneously within 2 hours [38].…”

Section: Species Identificationmentioning

confidence: 99%

See 1 more Smart Citation

Pyrosequencing: Current forensic methodology and future applications—a review

et al. 2022

Self Cite

View full text Add to dashboard Cite

The recent development of small, single-amplicon-based benchtop systems for pyrosequencing has opened up a host of novel procedures for applications in forensic science. Pyrosequencing is a sequencing by synthesis technique, based on chemiluminescent inorganic pyrophosphate detection. This review explains the pyrosequencing workflow and illustrates the step-by-step chemistry, followed by a description of the assay design and factors to keep in mind for an exemplary assay. Existing and potential forensic applications are highlighted using this technology. Current applications include identifying species, identifying bodily fluids, and determining smoking status. We also review progress in potential applications for the future, including research on distinguishing monozygotic twins, detecting alcohol and drug abuse, and other phenotypic characteristics such as diet and body mass index. Overall, the versatility of the pyrosequencing technologies renders it a useful tool in forensic genomics.

show abstract

Developmental validation of SpeID: A pyrosequencing-based assay for species identification

Cited by 8 publications

References 41 publications

An examination of differences in epigenetic methylation of saliva type samples based on collection method

An examination of differences in epigenetic methylation of saliva type samples based on collection method

Recent advances in forensic biology and forensic DNA typing: INTERPOL review 2019–2022

Pyrosequencing: Current forensic methodology and future applications—a review

Contact Info

Product

Resources

About