Advanced molecular surveillance approaches for characterization of blood borne hepatitis viruses

Berg, Michael G.; Olivo, Ana; Forberg, Kenn; Harris, Barbara J.; Yamaguchi, Julie; Shirazi, Rachel; Gozlan, Yael; Sauleda, Sílvia; Kaptué, Lazare; Rodgers, Mary A.; Mor, Orna; Cloherty, Gavin

doi:10.1371/journal.pone.0236046

Cited by 8 publications

(11 citation statements)

References 40 publications

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“…We consistently obtained near-complete or complete genome coverage for specimens below a threshold of C t = 29, corresponding to 10,000 cp/ml or higher. Beyond this cutoff, recovery of the SARS-CoV-2 sequence was highly variable, in agreement with data for other viruses to which we have applied specific xGen target enrichment (18)(19)(20). Specimens that were negative by qPCR were also negative by xGen-NGS.…”

Section: Target Capture Dramatically Improves Genome Coverage Of Low Vl Sars-cov-2 Specimenssupporting

confidence: 82%

“…Long probe lengths readily tolerate the limited number of expected mutations (all known SARS-CoV-2 genomes are >99.8% identical) and amplify targeted libraries from low VL specimens. As demonstrated by our previous work with HIV, Hepatitis B, and Hepatitis Delta viruses, and Bonsall et al's work with Hepatitis C virus, the xGen approach is a highly sensitive tool to maximize viral genome coverage when coupled with Illumina's next-generation sequencing platform (18)(19)(20)(21). Here, we examined SARS-CoV-2 full-genome diversity on specimens collected from sites in the Midwest and Northeast United States from March to June 2020.…”

Section: Introductionmentioning

confidence: 92%

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SNP and Phylogenetic Characterization of Low Viral Load SARS-CoV-2 Specimens by Target Enrichment

et al. 2021

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Background: Surveillance of SARS-CoV-2 across the globe has enabled detection of new variants and informed the public health response. With highly sensitive methods like qPCR widely adopted for diagnosis, the ability to sequence and characterize specimens with low titers needs to keep pace.Methods: Nucleic acids extracted from nasopharyngeal swabs collected from four sites in the United States in early 2020 were converted to NGS libraries to sequence SARS-CoV-2 genomes using metagenomic and xGen target enrichment approaches. Single nucleotide polymorphism (SNP) analysis and phylogeny were used to determine clade assignments and geographic origins of strains.Results: SARS-CoV-2-specific xGen enrichment enabled full genome coverage for 87 specimens with Ct values <29, corresponding to viral loads of >10,000 cp/ml. For samples with viral loads between 103 and 106 cp/ml, the median genome coverage for xGen was 99.1%, sequence depth was 605X, and the “on-target” rate was 57 ± 21%, compared to 13%, 2X and 0.001 ± 0.016%, respectively, for metagenomic sequencing alone. Phylogenetic analysis revealed the presence of most clades that existed at the time of the study, though clade GH dominated in the Midwest.Conclusions: Even as vaccines are being widely distributed, a high case load of SARS-CoV-2 infection persists around the world. Viral genetic surveillance has succeeded in warning the public of new variants in circulation and ensured that diagnostic tools remain resilient to a steadily increasing number of mutations. Target capture offers a means of characterizing low viral load samples which would normally pose a challenge for metagenomic sequencing.

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Section: Target Capture Dramatically Improves Genome Coverage Of Low Vl Sars-cov-2 Specimenssupporting

confidence: 82%

Section: Introductionmentioning

confidence: 92%

SNP and Phylogenetic Characterization of Low Viral Load SARS-CoV-2 Specimens by Target Enrichment

et al. 2021

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“…Probe inefficiency at lower viral loads has also been observed elsewhere 12 . Alternative methods Table 1: Viral load and sequencing output for 20 plasma samples from adults with chronic HBV infection sequenced using an Illumina protocol for full-length HBV genome retrieval (± probe-based enrichment).…”

Section: Discussionsupporting

confidence: 61%

“…nuclease treatment, filtration, ultracentrifugation 5 ), viral amplification (e.g. polymerase chain reaction 6,7 , rolling circle amplification 8,9 ) or viral enrichment (CRISPR cas9-mediated enrichment, probe-based enrichment) [10][11][12][13][14] .…”

Section: Introductionmentioning

confidence: 99%

Pan-genotypic probe-based enrichment to improve efficiency of Hepatitis B virus sequencing

Lumley

Jennings

Waddilove

et al. 2023

Preprint

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Hepatitis B Virus (HBV) genome sequencing can be used to provide more complete genetic information at the population and individual level to shed light on the limitations of current interventions, and inform new strategies for elimination. HBV sequencing is challenging due to the partially dsDNA genome, high diversity, low viral loads and presence of large amounts of host genetic material in clinical samples. Here we describe the design and use of a pan-genotypic panel of 74 HBV specific capture-probes and nuclease treatment in improving sequencing efficiency. We processed 20 plasma samples (viral loads 1.98 to 4.07 log10, genotypes A-E) and three positive controls (human total brain RNA and bacteriophage lambda DNA) in triplicate to compare DNAse vs. RNAse vs. no nuclease treatment. We prepared libraries using the Takara Bio SMARTer Stranded Total RNA-Seq Kit v3, split the library in two, enriching half with the custom-designed probe panel and xGen Hybridization and Wash Kit (IDT), the other half was not enriched. Both libraries were sequenced on the NovaSeq6000 platform with 2x150nt paired-end reads. Capture resulted in a 47,970 fold increase in the number of reads mapped to the HBV genome in the no nuclease arm (243 HBV reads per million reads sequenced in the capture pool vs. 5x10-3 reads per million in the no-capture pool). Out of 20 samples, only 1 without capture generated HBV reads (viral load 3.89 log10 IU/ml) vs. 19 samples with capture. HBV sequence yield was increased in the capture arm and resulted in 2.30 log10 (95% confidence interval 1.99 - 2.48 log10) increase in HBV reads (per million reads sequenced) per log10 increase in viral load. The proportion of HBV reads increased a median of 12 fold with RNAse treatment. We developed a targeted pan-genotypic sequencing method using a custom panel of biotinylated oligos that increases the sequencing efficacy of HBV. This method will allow us to gain a better insight into HBV diversity.

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“…NGS sequencing was conducted at both Abbott Laboratories, Abbott Park, IL USA and Nacional University, Medellin, Colombia. Random-primed libraries derived from plasma specimens were sequenced by metagenomic and target enrichment approaches as described [ 15 ]. Metagenomic libraries were sequenced on a Novaseq at Novagene, Inc. (Sacramento, CA) and “target enriched” using Twist Bioscience’s Comprehensive Viral Research Panel (CVRP) probes followed by sequencing on a MiSeq (Illumina, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Oropouche virus as an emerging cause of acute febrile illness in Colombia

Čiuoderis

Berg

Pérez

et al. 2022

Emerging Microbes & Infections

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Arbovirus infections are frequent causes of acute febrile illness (AFI) in tropical countries. We conducted health facility-based AFI surveillance at four sites in Colombia (Cucuta, Cali, Villavicencio, Leticia) during 2019-2022. Demographic, clinical and risk factor data were collected from persons with AFI that consented to participate in the study ( n = 2,967). Serologic specimens were obtained and tested for multiple pathogens by RT–PCR and rapid test (Antigen/IgM), with 20.7% identified as dengue positive from combined testing. Oropouche virus (OROV) was initially detected in serum by metagenomic next-generation sequencing (mNGS) and virus target capture in a patient from Cúcuta. Three additional infections from Leticia were confirmed by conventional PCR, sequenced, and isolated in tissue culture. Phylogenetic analysis determined there have been at least two independent OROV introductions into Colombia. To assess OROV spread, a RT-qPCR dual-target assay was developed which identified 87/791 (10.9%) viremic cases in AFI specimens from Cali (3/53), Cucuta (3/19), Villavicencio (38/566), and Leticia (43/153). In parallel, an automated anti-nucleocapsid antibody assay detected IgM in 27/503 (5.4%) and IgG in 92/568 (16.2%) patients screened, for which 24/68 (35.3%) of PCR positives had antibodies. Dengue was found primarily in people aged <18 years and linked to several clinical manifestations (weakness, skin rash and petechiae), whereas Oropouche cases were associated with the location, climate phase, and odynophagia symptom. Our results confirm OROV as an emerging pathogen and recommend increased surveillance to determine its burden as a cause of AFI in Colombia.

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Advanced molecular surveillance approaches for characterization of blood borne hepatitis viruses

Cited by 8 publications

References 40 publications

SNP and Phylogenetic Characterization of Low Viral Load SARS-CoV-2 Specimens by Target Enrichment

SNP and Phylogenetic Characterization of Low Viral Load SARS-CoV-2 Specimens by Target Enrichment

Pan-genotypic probe-based enrichment to improve efficiency of Hepatitis B virus sequencing

Oropouche virus as an emerging cause of acute febrile illness in Colombia

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