Rapid Whole-Genome Sequencing for Detection and Characterization of Microorganisms Directly from Clinical Samples

Abstract Background As a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5’ untranslated region and the Npro protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation. Results To enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19–32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage. Conclusions Further phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.

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SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus

Scientific Reports ◽

10.1038/s41598-019-55144-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Weili Cai ◽

Schyler Nunziata ◽

John Rascoe ◽

Michael J. Stulberg

Keyword(s):

Whole Genome Sequencing ◽

Molecular Characterization ◽

Genome Sequencing ◽

Whole Genome Sequence ◽

Whole Genome ◽

Genome Coverage ◽

Metagenomic Sample ◽

Liberibacter Asiaticus

AbstractHuanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

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Whole genome sequencing of respiratory syncytial (RSV) virus from clinical samples with low viral load v1 (protocols.io.qsedwbe)

protocols.io ◽

10.17504/protocols.io.qsedwbe ◽

2018 ◽

Author(s):

Stephanie Goya ◽

Gabriel L ◽

Mercedes S ◽

Laura E ◽

Alicia S ◽

...

Keyword(s):

Viral Load ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Samples ◽

Whole Genome

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First isolation and characterization of Brucella suis from yak

Genome ◽

10.1139/gen-2019-0101 ◽

2020 ◽

Vol 63 (8) ◽

pp. 397-405

Author(s):

Xiaowen Yang ◽

Ning Wang ◽

Xiaofang Cao ◽

Pengfei Bie ◽

Zhifeng Xing ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Health And Safety ◽

Host Cells ◽

Whole Genome ◽

Genetic Characteristics ◽

Qinghai Province ◽

Isolation And Characterization ◽

Smooth Strain

Brucella spp., facultative intracellular pathogens that can persistently colonize animal host cells and cause zoonosis, affect public health and safety. A Brucella strain was isolated from yak in Qinghai Province. To detect whether this isolate could cause an outbreak of brucellosis and to reveal its genetic characteristics, several typing and whole-genome sequencing methods were applied to identify its species and genetic characteristics. Phylogenetic analysis based on MLVA and whole-genome sequencing revealed the genetic characteristics of the isolated strain. The results showed that the isolated strain is a B. suis biovar 1 smooth strain, and this isolate was named B. suis QH05. The results of comparative genomics and MLVA showed that B. suis QH05 is not a vaccine strain. Comparison with other B. suis strains isolated from humans and animals indicated that B. suis QH05 may be linked to specific animal and human sources. In conclusion, B. suis QH05 does not belong to the Brucella epidemic species in China, and as the first isolation of B. suis from yak, this strain expands the host range of B. suis.

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