No abstract

HighlightsDeep sequencing has potential as an improved adventitious virus screening method.15 laboratories sequenced a common reagent containing 25 target viruses.6 viruses were detected by all lab, the remainder were detected by 4–14 labs.A wide range of sample preparation and bioinformatics methods is currently used.A common reference material is essential to enable results to be compared.

High-throughput sequencing (HTS) has demonstrated capabilities for broad virus detection based upon discovery of known and novel viruses in a variety of samples, including clinical, environmental, and biological. An important goal for HTS applications in biologics is to establish parameter settings that can afford adequate sensitivity at an acceptable computational cost (computation time, computer memory, storage, expense or/and efficiency), at critical steps in the bioinformatics pipeline, including initial data quality assessment, trimming/cleaning, and assembly (to reduce data volume and increase likelihood of appropriate sequence identification). Additionally, the quality and reliability of the results depend on the availability of a complete and curated viral database for obtaining accurate results; selection of sequence alignment programs and their configuration, that retains specificity for broad virus detection with reduced false-positive signals; removal of host sequences without loss of endogenous viral sequences of interest; and use of a meaningful reporting format, which can retain critical information of the analysis for presentation of readily interpretable data and actionable results. Furthermore, after alignment, both automated and manual evaluation may be needed to verify the results and help assign a potential risk level to residual, unmapped reads. We hope that the collective considerations discussed in this paper aid toward optimization of data analysis pipelines for virus detection by HTS.

The aim of the present study is to develop an assay for the specific identification of meat from Capreolus capreolus, Cervus elaphus, Capra ibex, Rupicapra rupicapra, targeting sequences of the cytochrome b (cyt b) gene of mitochondrial DNA. The assay is also intended to enable differentiation between meat from these wild species as well as Ovis aries, Capra hircus, Bubalus bubalis, Bos taurus and Sus scrofa domestic species.The primers used in the preliminary PCR were designed in well conserved regions upstream and downstream of the diagnosis sites. They successfully amplified a conserved 232 bp region from the cyt b gene of all the species taken into consideration. The sites of diagnosis have been interrogated using a minisequencing reaction and capillary electrophoresis. All the results of the multiplex PER (primer extension reaction) test were confirmed by fragment sequencing. The assay offers the possibility of discriminating nine species at the same time.

A polymerase chain reaction (PCR) method based on the RNA polymerase alpha subunit (rpoA) gene was developed for the detection of the Vibrio genus. The specific primers were designed aligning the rpoA gene sequences available in GenBank of all Vibrio species. The specificity of the primers was tested against 35 Vibrio species. In addition, 12 species phylogenetically related to the Vibrio genus were used as negative control. Moreover, in order to eliminate any false-negative results, bacteriumspecific primers targeting the 16S rRNA gene were introduced in the test as a noncompetitive internal amplification control. The rpoA primers correctly amplified all the Vibrio species considered. No cross-reaction was observed when tested against closely related species. To estimate the applicability of this method, 336 Vibrio wildtype strains isolated from Italian aquaculture products and from imported seafoods were tested. The sensitivity, tested using serial dilutions of different pure cultures of certified strains, resulted to 10 3 colony-forming units per milliliter. The assay proved to be specific, rapid, and reliable. It can be proposed as a routine screening technique for the confirmation of Vibrio genus in isolated colonies.