BackgroundNewcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs.MethodsAn amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow.ResultsFor all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 101 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq.ConclusionThe depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV.Electronic supplementary materialThe online version of this article (10.1186/s12985-018-1077-5) contains supplementary material, which is available to authorized users.

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BackgroundNewcastle disease viruses (NDV) are highly contagious and cause disease in both wild birds and poultry. A pigeon-adapted variant of genotype VI NDV, often termed pigeon paramyxovirus 1, is commonly isolated from columbids in the United States and worldwide. Complete genomic characterization of these genotype VI viruses circulating in wild columbids in the United States is limited, and due to the genetic variability of the virus, failure of rapid diagnostic detection has been reported. Therefore, in this study, formalin-fixed paraffin-embedded (FFPE) samples were subjected to next-generation sequencing (NGS) to identify and characterize these circulating viruses, providing valuable genetic information. NGS enables multiple samples to be deep-sequenced in parallel. When used on FFPE samples, this methodology allows for retrospective studies of infectious organisms.MethodsFFPE wild pigeon tissue samples (kidney, liver and spleen) from 10 mortality events in the U.S. between 2010 and 2016 were analyzed using NGS to detect and sequence NDV genomes from randomly amplified total RNA. Results were compared to the previously published immunohistochemistry (IHC) results conducted on the same samples. Additionally, phylogenetic analyses were conducted on the complete and partial fusion gene and complete genome coding sequences.ResultsTwenty-three out of 29 IHC-positive FFPE pigeon samples were identified as positive for NDV by NGS. Positive samples produced an average genome coverage of 99.6% and an average median depth of 199. A previously described sub-genotype (VIa) and a novel sub-genotype (VIn) of NDV were identified as the causative agent of 10 pigeon mortality events in the U.S. from 2010 to 2016. The distribution of these viruses from the North American lineages match the distribution of the Eurasian collared-doves and rock pigeons in the U.S.ConclusionsThis work reports the first successful evolutionary study using deep sequencing of complete NDV genomes from FFPE samples of wild bird origin. There are at least two distinct U.S. lineages of genotype VI NDV maintained in wild pigeons that are continuously evolving independently from each other and have no evident epidemiological connections to viruses circulating abroad. These findings support the hypothesis that columbids are serving as reservoirs of virulent NDV in the U.S.Electronic supplementary materialThe online version of this article (10.1186/s12985-017-0914-2) contains supplementary material, which is available to authorized users.

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U.S. public health agencies have employed next-generation sequencing (NGS) as a tool to quickly identify foodborne pathogens during outbreaks. Although established short-read NGS technologies are known to provide highly accurate data, long-read sequencing is still needed to resolve highly-repetitive genomic regions and genomic arrangement, and to close the sequences of bacterial chromosomes and plasmids. Here, we report the use of long-read nanopore sequencing to simultaneously sequence the entire chromosome and plasmid of Salmonella enterica subsp. enterica serovar Bareilly and Escherichia coli O157:H7. We developed a rapid and random sequencing approach coupled with de novo genome assembly within a customized data analysis workflow that uses publicly-available tools. In sequencing runs as short as four hours, using the MinION instrument, we obtained full-length genomes with an average identity of 99.87% for Salmonella Bareilly and 99.89% for E. coli in comparison to the respective MiSeq references. These nanopore-only assemblies provided readily available information on serotype, virulence factors, and antimicrobial resistance genes. We also demonstrate the potential of nanopore sequencing assemblies for rapid preliminary phylogenetic inference. Nanopore sequencing provides additional advantages as very low capital investment and footprint, and shorter (10 hours library preparation and sequencing) turnaround time compared to other NGS technologies.

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18United States public health agencies are focusing on next-generation sequencing (NGS) to 19 quickly identify and characterize foodborne pathogens. Here, the MinION nanopore, long-read 20 sequencer was used to simultaneously sequence the entire chromosome and plasmids of Salmonella 21 enterica subsp. enterica serovar Bareilly and Escherichia coli O157:H7. A rapid, random sequencing 22 approach, coupled with de novo genome assembly within a customized data analysis workflow, that can 23 resolve highly-repetitive genomic regions, was developed. In sequencing runs, as short as four hours, 24 using nanopore data alone, full-length genomes were obtained with an average identity of 99.87% for 25Salmonella Bareilly and 99.89% for E. coli in comparison to the respective MiSeq references. These long-26 read assemblies provided information on serotype, virulence factors, and antimicrobial resistance genes. 27Using a custom-developed, SNP-selection workflow, the potential of the nanopore-only assemblies 28 (after only 30 minutes of sequencing) for rapid phylogenetic inference, with identical topology 29 compared to the published dataset, was demonstrated. To achieve maximum quality assemblies, the 30 developed bioinformatics workflow employed additional polishing steps to correct the systematic errors 31 produced by the nanopore-only assemblies. Nanopore sequencing provided a shorter (10 hours library 32 preparation and sequencing) turnaround time compared to other NGS technologies. 33 34 Keywords: MinION, complete genome sequencing, plasmids, foodborne pathogen, long-reads, assembly 35 workflow, tracing. 36 basecalled using Albacore (ONT v 2.0.2b) and subsampled for assembly using Filtlong (v.0.2.0) 24 to a 132

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