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Genome reconstruction of white spot syndrome virus (WSSV) from archival Davidson's-fixed paraffin embedded shrimp (Penaeus vannamei) tissue.

ABSTRACT: Formalin-fixed paraffin-embedded (FFPE) tissues are a priceless resource for diagnostic laboratories worldwide. However, DNA extracted from these tissues is often not optimal for most downstream molecular analysis due to fragmentation and chemical modification. In this study, the complete genome of white spot syndrome virus (WSSV) was reconstructed from?~?2-year-old archived Davidson's-fixed paraffin-embedded (DFPE) shrimp tissue using Next Generation Sequencing (NGS). A histological analysis was performed on archived DFPE shrimp tissue and a sample showing a high level of WSSV infection was selected for molecular analysis. The viral infection was further confirmed by molecular methods. DNA isolated from DFPE and fresh frozen (FF) tissues were sequenced by NGS. The complete genome reconstruction of WSSV (~?305 kbp) was achieved from both DFPE and FF tissue. Single nucleotide polymorphisms, insertion and deletions were compared between the genomes. Thirty-eight mutations were identified in the WSSV genomes from the DFPE and FF that differed from the reference genome. This is the first study that has successfully sequenced the complete genome of a virus of over 300 kbp from archival DFPE tissue. These findings demonstrate that DFPE shrimp tissue represents an invaluable resource for prospective and retrospective studies, evolutionary studies and opens avenues for pathogen discovery.

SUBMITTER: Cruz-Flores R

PROVIDER: S-EPMC7417530 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Genome reconstruction of white spot syndrome virus (WSSV) from archival Davidson's-fixed paraffin embedded shrimp (Penaeus vannamei) tissue.

Cruz-Flores Roberto R Mai Hung N HN Kanrar Siddhartha S Aranguren Caro Luis Fernando LF Dhar Arun K AK

Scientific reports 20200810 1

Formalin-fixed paraffin-embedded (FFPE) tissues are a priceless resource for diagnostic laboratories worldwide. However, DNA extracted from these tissues is often not optimal for most downstream molecular analysis due to fragmentation and chemical modification. In this study, the complete genome of white spot syndrome virus (WSSV) was reconstructed from ~ 2-year-old archived Davidson's-fixed paraffin-embedded (DFPE) shrimp tissue using Next Generation Sequencing (NGS). A histological analysis wa ...[more]

PMID: 32778727

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Project description:There is increasing acceptance of the critical importance of correlating the morphologic features of tissue with the data obtained from various molecular analytic techniques. Access to archived formalin-fixed and paraffin-embedded (FFPE) tissue specimens via shotgun-based proteomic analyses may, therefore, open new avenues for both prospective and retrospective translational research. However, one of the remaining issues in performing comparative proteomic measurements among FFPE tissues relates to potential variability in protein composition and retrieval based on length of storage periods. Optimized protein extraction and digestion procedures for handling FFPE tissues are coupled with the capillary isotachophoresis-based proteome technology to evaluate the effects of length of storage period on archival tissue proteome analysis across 10 archived uterine mesenchymal tumor tissue blocks, including 9 uterine leiomyomas dating from 1990 to 2002 and a single case of alveolar soft part sarcoma (ASPS) from 1980. Several statistical measures, including the Pearson correlation coefficient, coefficient of variance, k-means clustering, and ANOVA, are employed to evaluate the possibility of an archival effect on individual proteins or groups of proteins within nine leiomyomas. Low abundance proteins may be more susceptible to the long-term storage as these proteins are more difficult to be retrieved and extracted as the tissue block ages in paraffin. Despite using tissue blocks stored for as many as 28 years, high confidence and comparative proteome analysis between the leiomyomas and the sarcoma is achieved. Though sharing over 1800 common proteins in a core set, a total of 80 proteins unique to the sarcoma are identified distinguishing the ASPS from the leiomyomas. Vacuolar proton translocating ATPase 116 kDa subunit isoform a3, one of the unique proteins expressed in the ASPS, is further validated by immunohistochemistry (IHC). Although IHC is highly sensitive and provides the subcellular resolution, mass spectrometry-based proteome profiling enables global identification and quantification of thousands of proteins without a priori knowledge of individual proteins being analyzed or the need of validated antibodies.

Project description:BACKGROUND: The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE) surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. METHODS: A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc) were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB) of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status). Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. RESULTS: Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated with poor outcomes in breast cancer patients. CONCLUSION: We demonstrated that FFPE specimens retained important prognostic information that could be identified using a recent gene profiling technology. Our study supports the use of FFPE specimens for the development and refinement of prognostic gene signatures for breast cancer. Clinical applications of such prognostic gene profiles await future large-scale validation studies.

Dataset Information

Genome reconstruction of white spot syndrome virus (WSSV) from archival Davidson's-fixed paraffin embedded shrimp (Penaeus vannamei) tissue.

Publications

Genome reconstruction of white spot syndrome virus (WSSV) from archival Davidson's-fixed paraffin embedded shrimp (Penaeus vannamei) tissue.

Similar Datasets

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