The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% Ϯ 15.6%. PV genomes covering Ͼ99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance.
Background Haemophilus influenzae (Hi) can cause invasive diseases such as meningitis, pneumonia, or sepsis. Typeable Hi includes six serotypes (a through f), each expressing a unique capsular polysaccharide. The capsule, encoded by the genes within the capsule locus, is a major virulence factor of typeable Hi. Non-typeable (NTHi) does not express capsule and is associated with invasive and non-invasive diseases. Methods A total of 395 typeable and 293 NTHi isolates were characterized by whole genome sequencing (WGS). Phylogenetic analysis and multilocus sequence typing were used to characterize the overall genetic diversity. Pair-wise comparisons were used to evaluate the capsule loci. A WGS serotyping method was developed to predict the Hi serotype. WGS serotyping results were compared to slide agglutination (SAST) or real-time PCR (rt-PCR) serotyping. Results Isolates of each Hi serotype clustered into one or two subclades, with each subclade being associated with a distinct sequence type (ST). NTHi isolates were genetically diverse, with seven subclades and 125 STs being detected. Regions I and III of the capsule locus were conserved among the six serotypes (≥82% nucleotide identity). In contrast, genes in Region II were less conserved, with only six gene pairs from all serotypes showing ≥56% nucleotide identity. The WGS serotyping method was 99.9% concordant with SAST and 100% concordant with rt-PCR in determining the Hi serotype. Conclusions Genomic analysis revealed a higher degree of genetic diversity among NTHi compared to typeable Hi. The WGS serotyping method accurately predicted the Hi capsule type and can serve as an alternative method for Hi serotyping.
Invasive meningococcal disease is mainly caused by Neisseria meningitidis serogroups A, B, C, X, W, and Y. The serogroup is typically determined by slide agglutination serogrouping (SASG) and real-time PCR (RT-PCR). We describe a whole-genome sequencing (WGS)-based method to characterize the capsule polysaccharide synthesis (cps) locus, classify N. meningitidis serogroups, and identify mechanisms for nongroupability using 453 isolates from a global strain collection. We identified novel genomic organizations within functional cps loci, consisting of insertion sequence (IS) elements in unique positions that did not disrupt the coding sequence. Genetic mutations (partial gene deletion, missing genes, IS insertion, internal stop, and phase-variable off) that led to nongroupability were identified. The results of WGS and SASG were in 91% to 100% agreement for all serogroups, while the results of WGS and RT-PCR showed 99% to 100% agreement. Among isolates determined to be nongroupable by WGS (31 of 453), the results of all three methods agreed 100% for those without a capsule polymerase gene. However, 61% (WGS versus SASG) and 36% (WGS versus RT-PCR) agreements were observed for the isolates, particularly those with phase variations or internal stops in cps loci, which warrant further characterization by additional tests. Our WGS-based serogrouping method provides comprehensive characterization of the N. meningitidis capsule, which is critical for meningococcal surveillance and outbreak investigations.
Computational infrastructure and bioinformatics analysis are bottlenecks in the application of NGS to viral pathogens. As of September 2021, VPipe has been used by the U.S.
The production of a recombinant protein, named Gluconacin, opens new avenues for the agro-biotechnology application in agriculture, mainly with regard to the sugarcane crop.
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