A Draft Genome of \u3ci\u3eYersinia Pestis\u3c/i\u3e From Victims of the Black Death

Technological advances in DNA recovery and sequencing have drastically expanded the scope of genetic analyses of ancient specimens to the extent that full genomic investigations are now feasible and are quickly becoming standard1. This trend has important implications for infectious disease research because genomic data from ancient microbes may help to elucidate mechanisms of pathogen evolution and adaptation for emerging and re-emerging infections. Here we report a reconstructed ancient genome of Yersinia pestis at 30-fold average coverage from Black Death victims securely dated to episodes of pestilence-associated mortality in London, England, 1348–1350. Genetic architecture and phylogenetic analysis indicate that the ancient organism is ancestral to most extant strains and sits very close to the ancestral node of all Y. pestis commonly associated with human infection. Temporal estimates suggest that the Black Death of 1347–1351 was the main historical event responsible for the introduction and widespread dissemination of the ancestor to all currently circulating Y. pestis strains pathogenic to humans, and further indicates that contemporary Y. pestis epidemics have their origins in the medieval era. Comparisons against modern genomes reveal no unique derived positions in the medieval organism, indicating that the perceived increased virulence of the disease during the Black Death may not have been due to bacterial phenotype. These findings support the notion that factors other than microbial genetics, such as environment, vector dynamics and host susceptibility, should be at the forefront of epidemiological discussions regarding emerging Y. pestis infections

Development of an amplicon-based sequencing approach in response to the global emergence of mpox

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.This publication was made possible by CTSA Grant Number UL1 TR001863 from the National Center for Advancing Translational Science (NCATS), a component of the National Institutes of Health (NIH) awarded to CBFV. INSA was partially funded by the HERA project (Grant/ 2021/PHF/23776) supported by the European Commission through the European Centre for Disease Control (to VB).info:eu-repo/semantics/publishedVersio

Early Emergence Phase of SARS-CoV-2 Delta Variant in Florida, US

SARS-CoV-2, the causative agent of COVID-19, emerged in late 2019. The highly contagious B.1.617.2 (Delta) variant of concern (VOC) was first identified in October 2020 in India and subsequently disseminated worldwide, later becoming the dominant lineage in the US. Understanding the local transmission dynamics of early SARS-CoV-2 introductions may inform actionable mitigation efforts during subsequent pandemic waves. Yet, despite considerable genomic analysis of SARS-CoV-2 in the US, several gaps remain. Here, we explore the early emergence of the Delta variant in Florida, US using phylogenetic analysis of representative Florida and globally sampled genomes. We find multiple independent introductions into Florida primarily from North America and Europe, with a minority originating from Asia. These introductions led to three distinct clades that demonstrated varying relative rates of transmission and possessed five distinct substitutions that were 3–21 times more prevalent in the Florida sample as compared to the global sample. Our results underscore the benefits of routine viral genomic surveillance to monitor epidemic spread and support the need for more comprehensive genomic epidemiology studies of emerging variants. In addition, we provide a model of epidemic spread of newly emerging VOCs that can inform future public health responses

ddPCR Reveals SARS-CoV-2 Variants in Florida Wastewater

Wastewater was screened for the presence of functionally significant mutations in SARS-CoV-2 associated with emerging variants of concern (VOC) by ddPCR, and results accorded with sequencing of clinical samples from the same region. We propose that PCR-based screening of wastewater can provide a powerful tool for rapid and inexpensive screening of large population segments for VOC-associated mutations and can hone complementary sampling and sequencing of direct (human) test material to track emerging VOC

Targeted deep amplicon sequencing of antimalarial resistance markers in Plasmodium falciparum isolates from Cameroon

BACKGROUND: Recent studies show the first emergence of the R561H artemisinin-associated resistance marker in Africa, which highlights the importance of continued molecular surveillance to assess the selection and spread of this and other drug resistance markers in the region.METHOD: In this study, we used targeted deep amplicon sequencing (TADS) of 116 isolates collected in two areas of Cameroon to genotype the major drug resistance genes k13, crt, mdr1, dhfr, dhps, and the cytochrome b (cytb) in P. falciparum.RESULTS: No confirmed or associated artemisinin resistance markers were observed in Pfk13. In comparison, both major and minor alleles associated with drug resistance were found in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps. Notably, a high frequency of other non-synonymous mutations was observed across all the genes, except Pfcytb, suggesting continued selection pressure.CONCLUSIONS: The results from this study support the continued use of artemisinin combination therapy (ACT) for treatment and administration of sulphadoxine-pyrimethamine for intermittent preventive therapy in pregnant women and for seasonal chemoprevention in these study sites in Cameroon

A Comparison and Integration of MiSeq and MinION Platforms for Sequencing Single Source and Mixed Mitochondrial Genomes

<div><p>Single source and multiple donor (mixed) samples of human mitochondrial DNA were analyzed and compared using the MinION and the MiSeq platforms. A generalized variant detection strategy was employed to provide a cursory framework for evaluating the reliability and accuracy of mitochondrial sequences produced by the MinION. The feasibility of long-read phasing was investigated to establish its efficacy in quantitatively distinguishing and deconvolving individuals in a mixture. Finally, a proof-of-concept was demonstrated by integrating both platforms in a hybrid assembly that leverages solely mixture data to accurately reconstruct full mitochondrial genomes.</p></div

Assembly Statistics.

<p>The two assemblies, 005 (blue) and 047 (red), are depicted in each plot where the x-axis is percent of contigs and the y-axis is size in kilobases (kb). A) Plot of the Nx where the dotted line is the N50. B) Plot of the NGAx where the dotted line is NGA50.</p

Single Source Sample Concordance by VAF.

<p>Each single source sample (004, 005, and 047) is characterized by a heatmap, which compares SNP call sets between the two platforms. SNP call sets are plotted by VAF for both the MinION and MiSeq data from 0.05 to 0.95 using increments of 0.05. The MinION VAF is on the x-axis and the MiSeq VAF is across the y-axis. Concordance is determined by calculating the F1-Score using the MiSeq calls as the ground truth. The value of each F1-Score comparison is shown as increasingly darker shades of blue for higher values. The highest F1-Scores are shown for each of the source samples 004, 005, and 047 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167600#pone.0167600.g003" target="_blank">Fig 3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167600#pone.0167600.s003" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167600#pone.0167600.s004" target="_blank">S3</a> Figs, respectively.</p

Coverage and Concordance Circos for 004.

<p>The coverage depth per base is shown for the MiSeq (orange) and MinION (blue) shown on the outer ring using a log10 scale. The inner ring shows concordance at each SNP using a MiSeq VAF of 0.90 and a MinION VAF of 0.65. Text color denotes the categorization of each SNP under the VAF combination providing the highest concordance (F1-Score). Green text indicates true positives and black text is for false negatives present in the MinION call sets. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167600#pone.0167600.s003" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167600#pone.0167600.s004" target="_blank">S3</a> Figs for similar plots of 005 and 047, respectively. Black arrows indicate the locations and orientations of the primers used for amplification.</p