A semi-automated protocol for NGS metabarcoding and fungal analysis in forensic

Giampaoli, Saverio; Vittori, Elisabetta De; Frajese, Giovanni Vanni; Paytuví, A.; Sanseverino, Walter; Anselmo, Anna; Barni, Filippo; Berti, Andrea

doi:10.1016/j.forsciint.2019.110052

Cited by 5 publications

(6 citation statements)

References 45 publications

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“…Libraries were normalized by Qubit 2.0 Fluorometer, pooled and sequenced on MiSeq using paired 300‐bp reads, and MiSeq v3 reagents (Illumina). Sequence reads were analyzed in the cloud bioinformatics platform GAIA ( https://metagenomics.sequentiabiotech.com ) (Giampaoli et al 2020 ). Sequencing quality was assessed using FastQC (bioinformatics.babraham.ac.uk/projects/fastqc/) and BBduk (jgi.doe.gov/data-and-tools/bbtools/), setting a minim length of 35 bp and a minimum Phred-quality score of 25.…”

Section: Methodsmentioning

confidence: 99%

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Study of the variation of the Malassezia load in the interdigital fold of dogs with pododermatitis

et al. 2022

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The yeast Malassezia pachydermatis is a common inhabitant of the skin and mucosae of dogs. However, under certain circumstances this yeast can overgrow and act as an opportunistic pathogen causing otitis and dermatitis in dogs. Canine pododermatitis is a common disorder in dogs in which M. pachydermatis acts as an opportunistic pathogen. In the present study, the presence of Malassezia yeasts was assessed and quantified in samples collected from the interdigital space of dogs with pododermatitis before and after treatment, and from healthy dogs. The samples were subjected to two different cytological examinations, culture on Sabouraud glucose agar and modified Dixon’s agar and a quantitative PCR targeting the internal transcribed spacer (ITS) genomic region. A selection of samples was analyzed by next generation sequencing (NGS) using the D1D2 domain of the large subunit of the ribosomal DNA as target. The pododermatitis samples before treatment showed higher cell counts, colony-forming units and ITS copies than the rest of samples. The NGS analysis revealed that Ascomycota was the main phylum in the healthy and post-treatment samples. However, Basidiomycota and M. pachydermatis was more abundant in the pododermatitis samples before treatment. These results support M. pachydermatis as an opportunistic agent in canine pododermatitis by a variety of methods, and demonstrate the correlation between cytologic and molecular methods for quantification.

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Section: Methodsmentioning

confidence: 99%

“…seque ntiab iotech. com) (Giampaoli et al 2020). Sequencing quality was assessed using FastQC (bioinformatics.babraham.ac.uk/projects/fastqc/) and BBduk (jgi.…”

Section: Ngs and Data Analysismentioning

confidence: 99%

Study of the variation of the Malassezia load in the interdigital fold of dogs with pododermatitis

et al. 2022

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“…Specifically, DNA from the microbial community is isolated, an internal genomic sequence (barcode) is amplified using a polymerase chain reaction (PCR), and this is then sequenced using high-throughput next-generation sequencing (NGS) and analyzed with subsequent bioinformatical tools, which are used to sort the DNA barcodes and compare them to a reference database such as GenBank ( ), or UNITE database [ 45 ] for species identification. This method provides information on complex microbiomes [ 46 , 47 , 48 , 49 , 50 ] and allows for estimates of the richness and diversity of microbial communities.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, the diversity of fungal communities revealed by the metabarcoding approach has allowed researchers to discriminate the sites of cadaver decomposition where the cadaver was placed or from where it was moved [ 54 ]. The forensic science community would benefit from standardized, specific guidelines for metabarcoding data in legal disputations with regard to environmental conditions and geographic location [ 48 ], as the dynamics of fungal succession during the decomposition of animal bodies are still not fully understood [ 55 ]. While work on this area shows great promise, little is currently known about geographic variation on broad spatial scales (e.g., inter-continental differences) in how fungal communities assemble or what major taxonomic groups differ across space and time.…”

Section: Introductionmentioning

confidence: 99%

Assessment of Fungal Succession in Decomposing Swine Carcasses (Sus scrofa L.) Using DNA Metabarcoding

Gemmellaro,

Lorusso,

Domke

et al. 2023

JoF

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The decomposition of animal bodies is a process defined by specific stages, described by the state of the body and participation of certain guilds of invertebrates and microorganisms. While the participation of invertebrates in decomposing is well-studied and actively used in crime scene investigations, information on bacteria and fungi from the scene is rarely collected or used in the identification of important factors such as estimated time of death. Modern molecular techniques such as DNA metabarcoding allow the identification and quantification of the composition of microbial communities. In this study, we used DNA metabarcoding to monitor fungal succession during the decomposition of juvenile pigs in grasslands of New Jersey, USA. Our findings show that decomposition stages differ in a diversity of fungal communities. In particular, we noted increased fungal species richness in the more advanced stages of decomposition (e.g., bloat and decay stages), with unique fungal taxa becoming active with the progression of decay. Overall, our findings improve knowledge of how fungi contribute to forensically relevant decomposition and could help with the assessment of crime scenes.

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“…This type of sequencing allows the simultaneous sequencing of multiple DNA barcodes from diverse organisms in a mixed sample. Metabarcoding provides a comprehensive overview of biodiversity within an ecosystem (Giampaoli, 2020).…”

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confidence: 99%

Application of the Next Generation Sequencing in Biology and Medicine

Gerilovych,

Sushko,

Mandyhra

et al. 2024

OneHealthJournal

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Next-Generation Sequencing (NGS), also known as high-throughput sequencing, refers to a set of modern DNA sequencing technologies that have revolutionized the field of genomics. Advantages of NGS techniques involving high speed (parallel sequencing is faster than traditional methods, allowing researchers to obtain results more quickly), cost-effectiveness (ability to sequence multiple fragments simultaneously reduces the cost per base compared to traditional sequencing), and scalability (platforms can be scaled to accommodate varying levels of throughput depending on experimental needs). NGS has significantly accelerated genomics research, enabling breakthroughs in fields such as personalized medicine, cancer genomics, and evolutionary biology. However, challenges such as data analysis complexity, error rates, and cost still exist and are areas of ongoing research and improvement within the field of sequencing technologies. Paper contains the brief explanation of the current NGS platforms and their features. NGS biomedical application is described with the main advantages and abilities of the analysed tools.

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A semi-automated protocol for NGS metabarcoding and fungal analysis in forensic

Cited by 5 publications

References 45 publications

Study of the variation of the Malassezia load in the interdigital fold of dogs with pododermatitis

Study of the variation of the Malassezia load in the interdigital fold of dogs with pododermatitis

Assessment of Fungal Succession in Decomposing Swine Carcasses (Sus scrofa L.) Using DNA Metabarcoding

Application of the Next Generation Sequencing in Biology and Medicine

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