Infectious Disease

Path taken by the ship MV SHANDONG DA ZHI.

Prof. Luana Silva Soares and colleagues from the Department of Virology, Para state, Brazil reported the first instance of a SARS-CoV-2 B.1.617.2 (VoC) outbreak in an Asiatic origin trade ship which traveled from Malaysia and docked in Brazil. Nasopharyngeal swabs from 24 crew members were collected. The laboratory diagnosis was performed using RT-qPCR. Positive samples were used to perform Next Generation Sequencing using CleanPlex® SARS-CoV-2 Research and Surveillance Panel kits. SARS-CoV-2 genomes were obtained and classified as a B.1.617 lineage, confirmed by Pangolin lineage classification. Genetically, the B.1.617 lineage has a particular signature in the Spike protein, characterized by six marker mutations. In addition to those marker mutations, 21 other changes in the Spike protein were also observed in the NGS data. In conclusion, the lab reports the early detection of the SARS-CoV-2 lineage B.1.617 in Brazil. Since there are many factors still unclear about this VoC B.1.617.2, this single outbreak reinforces the necessity of continuous monitoring of travelers to follow the real-time spread of this new SARS-CoV-2 VoC, that could impact public health as well as immunization strategies.

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Genetic variant profiles of the sequenced SARS-CoV-2 relative to the Wuhan-Hu-1 reference genome.
Genetic variant profiles of the sequenced SARS-CoV-2 relative to the Wuhan-Hu-1 reference genome.

Prof. Zimmerman from Case Western University and Prof. Donskey from the Cleveland VA Medical Centre performed a joint study to investigate a cluster of SARS-CoV-2 infections in 6 symptomatic emergency department personnel and 2 control COVID-19 patients. They used the CleanPlex SARS-CoV-2 Panel to perform viral whole genome sequencing to conduct this contact tracing investigation. Their results demonstrated that the suspected 6-person outbreak included 2 distinct transmission clusters and 1 unrelated infection. Their results suggest that SARS-CoV-2 variants initially acquired in the community were subsequently spread among hospital personnel. Hence, they concluded that such SARS-CoV-2 sequencing studies are needed to provide more definitive evidence regarding personnel as a source of transmission.

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Phylogenetic tree

Prof. Rubin and Prof. Li from Case Western University along with their colleagues from Cleveland Clinic Ohio performed a cross-sectional study on 302 patients to examine the association of identified SARS-CoV-2 variants, virus clades, and clade groups with disease severity and patient outcomes. Whole genome sequencing of the virus was performed using the CleanPlex SARS-CoV-2 Panel followed by variant calling. Understanding of SARS-CoV-2 variants that alter disease outcomes are important for clinical risk stratification and provide important clues to the complex virus-host relationship. This study demonstrates a dynamic shift in SARS-CoV-2 clade diversity occurring very early in the pandemic.  These findings suggest that SARS-CoV-2 clade assignment is an important factor that may aid in estimating patient outcomes.

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Phylogenetic tree showing the genomic epidemiology of a Denizli/Turkey focused subsampling of 15 SARS-CoV-2 genomes

Prof. Hakan Akca from Pamukkale University, Turkey, performed whole genome sequencing on 15 COVID-19 positive patients using CleanPlex SARS-CoV-2 Panel . They identified 32 missense, 21 synonymous, and 4 non-coding allele mutations. Out of the missense mutations identified, 11 were rare missense mutations in the virus compared to the reference genome. Phylogenetic analysis revealed that most of the isolates were similar to European sequences. The phylogenetic tree constructed with all the complete SARS-CoV-2 genomes of Turkey showed that the viruses were spread nearly homogenous on eastern and western sides of Turkey.

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Timeline of a reinfection case

Normally, reported SARS-CoV-2 reinfection cases are mainly seen in strains belonging to different clades however Prof. Zurita from Ecuador identified a case of reinfection within the same clade. Both, the infection and reinfection strains, were assigned as Nextstrain 20B, Pangolin lineage B.1.1 and GISAID clade O. These results were obtained by whole genome sequencing of the SARS-CoV-2 virus using the CleanPlex SARS-CoV-2 Panel. These results showed evidence that SARS-CoV-2 reinfection can occur within the same clade and previous infection to the virus cannot render complete immunity in all cases.

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Variant nucleotide positions of the sequences HH-24.I and HH-24.II

Prof. Lütgehetmann from University Medical Center Hamburg-Eppendorf along with his colleagues from German Center for Infection Research performed a study to investigate the SARSCoV2 reinfection despite presence of detectable neutralizing antibodies in a female healthcare worker. Next generation sequencing (NGS) of the viral genome from the pharyngeal swab was performed using the CleanPlex SARS-CoV-2 Panel from the first and second episode of infection and were named #HH-24.I and HH-24.II, respectively. The NGS data revealed that the viral sequences from the initial virus variant in March (HH-24.I) and the variant in December (HH 24.II) belonged to pangoline lineages B.3 and B.1.177, respectively. In total, both sequences differed in 21 positions including two typical variations in spike proteins A222V and D614G. The team concluded that a better understanding about reinfection events may help to identify serological correlates of immunity.

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UPGMA clustering analysis

Prof. Dien Bard and Prof. Gai from Children’s Hospital Los Angeles (CHLA) along with their colleagues from the Keck School of Medicine, USC performed whole genome sequencing (WGS) of 70 SARS-CoV-2 positive individuals using the CleanPlex SARS-CoV-2 NGS panel to evaluate institutional infection surveillance within a 9-month period. The researchers identified 25 potential clusters that warranted further exploration by WGS. Of the 25 suspected clusters analyzed, conclusive results were available in 23 cases from which they confirmed some relatedness in 14 clusters suspected by the contact tracing team including an outbreak within a unit. Importantly, WGS allowed them to rule out 9 highly suspicious clusters within the same units, demonstrating that these were not healthcare-associated infections. WGS analysis of SARS-CoV-2 isolates allowed the research team to monitor disease spread, outbreak dynamics, and infection hot spots within their institution and provided the capability to rule-out suspected clusters and confirm unrelated sources of infection. The team concluded that WGS analysis during the COVID-19 pandemic can be highly effective in a clinical setting as a complement to contact tracing efforts and will become increasingly important in identifying outbreaks.

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SOPHiA Genetics and researchers from 6 different institutes sequenced 200 clinical samples with Paragon Genomics’ CleanPlex SARS-CoV-2 Panel kit to generate what the authors claim as “much-needed recommendations” and guidelines for sequencing and genotyping of SARS-CoV-2 samples. They set criteria for accurate variant detection down to 10%, which enables sample tracing based on geographical origin and time of sampling. Additionally, the researchers identified dissemination patterns of the viral variants 20A.EU1 and 20A.EU2, which were linked to summer travel in 2020 across Europe. The results indicate high specificity with “virtual absence of sequencing reads mapping to the human transcriptome” and high uniformity that are trademarks of Paragon Genomics’ CleanPlex technology. Although more investigation is needed in the global fight against COVID-19, Paragon Genomics is proud to be helping the research community in its quest for better understanding of the SARS-CoV-2 genome.

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Number of hospitalized patients, cases, deaths, in Rio Grande do Sul, April 12,2020-present

Prof. Luis Barth and his colleagues from Federal University of Rio Grande do Sul and Hospital de Clínicas de Porto Alegre performed a study to look at the association of the emergence of the SARS-CoV-2 P.1 lineage and the rapid growth in hospitalization in the northern region of Brazil.  Whole-genome sequencing of 68 patient specimens was performed using the CleanPlex SARS-CoV-2 panel and the data revealed that the previously undetected P.1 lineage was now detected and accounted for 88.9% of specimens collected from patients at a referral COVID-19 hospital. These findings raise concerns regarding a possible association between the lineage P.1 and rapid growth in cases and hospitalizations in the Rio Grande do Sul area.

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Missense3D prediction of the wide type (ILE82, green) and mutant (THR82, red) M proteins.

Prof. Biegel and Prof. Gai from Children’s Hospital Los Angeles (CHLA) along with their colleagues from the Keck School of Medicine, USC evaluated whole genome sequencing data from a cohort of 143,609 USA SARS-CoV-2 viral genomes to study the recent spike in frequency of the membrane protein (M) gene mutations in the SARS-CoV-2 genome. This cohort included 2,900 patients from CHLA whose whole genome data was generated using the CleanPlex SARS-CoV-2 NGS panel. The group reported a new Variant Of Concern with a signature mutation in the M gene, an otherwise overlooked but significant site of increasing numbers of mutations. In silico analysis revealed that the M protein structure is similar to that of the glucose transporter based upon which the M protein is thought to be involved in rapid viral proliferation, replication, and immune evasion. In the last few months, the incidence of some missense M gene mutations have increased over 100 fold and continue to increase. Hence, the researchers suggest that M gene mutations should be included in ongoing genomic surveillance efforts and warrants further evaluation.

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Prof. Uzzau from University of Sassari along with his colleagues from Sassari University Hospital in Italy performed a retrospective study to investigate the spread of the SARSCoV2 Variant of Concern (VOC) 202012/01 B.1.1.7 in North Italy. They performed whole genome sequencing using the CleanPlex SARS-CoV-2 NGS Panel to confirm inconclusive diagnostic RT-PCR results. According to the Pangolin application, the analyses of this NGS data enabled the researchers to assign these viral genomes to SARS-CoV-2 lineages, confirming the presence of the B.1.1.7 variant. The CleanPlex SARS-CoV-2 NGS panel was able to ID the characteristic mutations along the whole SARS-CoV-2 viral genome including the mutations in the S gene encoding for Spike protein. Such variants represent a serious threat worldwide, thus conclusive and rapid methods are required for their identification.

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Accumulation of SARS-CoV-2 variants in three immunocompromised persistently positive young patients.

Prof. Dien Bard and Prof. Gai from Children’s Hospital Los Angeles (CHLA) along with their colleagues from Stanford and Johns Hopkins performed a study in three patients with impaired humoral immunity who were persistently positive for SARS-CoV-2 infection. Whole-genome sequencing was performed using the CleanPlex SARS-CoV-2 Research and Surveillance NGS Panel and serological studies were performed to measure viral evolution and evidence of immune escape. Their work demonstrates that immunocompromised pediatric and young adult patients are susceptible to prolonged viral infections with continued virus shedding and mutation accumulation some with the potential to escape antibody neutralization. Their results highlight the need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.

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Geographic distribution of SARS-CoV-2 genomes, the lineages detected, and population density among the 12 provinces of Lombardy.

Prof. Fausto Baldanti from Univeristy of Pavia, Italy and Prof. Carlo Federico Perno from Children’s Hospital, IRCCS, Rome, Italy along with their colleagues from University of Milan, Italy and ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy performed a genomic epidemiology study of the SARS-CoV-2 genomes in Lombardy, Italy. In this study, they used an integrated approach comprising of both epidemiological and viral genetic data to reconstruct the pattern of the SARS-CoV-2 spread in this region. Whole-genome sequencing was performed using the CleanPlex SARS-CoV-2 Research and Surveillance NGS Panel for 346 SARS-CoV-2 strains obtained from individuals from various geographical areas in a time span of 2 months. In conclusion, this study contributed to the identification and circulation pattern of seven SARS-CoV-2 lineages in an area highly affected by COVID-19. In addition, they demonstrated the presence of local transmission clusters within three of those lineages, revealing that sustained community transmission was underway before the first COVID-19 case had been detected in Lombardy.

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Relative abundance of circulating Clonal Complexes between pediatrics (≤ 21 years old) and adults.

Prof. Paul Planet from Children’s Hospital of Philadelphia (CHOP) and Prof. Dien Bard and Prof. Gai from Children’s Hospital Los Angeles (CHLA) along with their colleagues from USC and UPenn performed a study in a pediatric population to track the molecular epidemiology of the SARS-CoV-2 virus locally in Philadelphia. They used the CleanPlex SARS-CoV-2 Research and Surveillance NGS Panel to perform whole genome sequencing followed by novel genotyping tools to quantifying the viral diversity on 169 SARS-CoV-2 genomes (83 genomes <21 years old) collected over three months. They show that the early pandemic and peak in Philadelphia were characterized by multiple, diverse, circulating viral variants, especially amongst children. Their analysis uncovered 13 major lineages that changed in relative abundance as cases peaked in mid-April. They conclude that no clear differences in clinical presentation were associated with the different virus lineages.

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Map of SARS-CoV-2 variations across the viral genome

Prof. Derly Andrade Molina from Espiritu Santo University and Prof. Ricardo Soto Rifo from University of Chile along with their colleagues performed an epidemiological surveillance and reported two sequences in South America of the new SARS-CoV-2 variant, B.1.526, harboring the immune escape-associated mutation E484K. These viral sequences of the new variant were obtained from two individuals with a negative RT-PCR test but were analyzed further by whole genome sequencing using the CleanPlex ® SARS-CoV-2 FLEX Panel.  From all the mutations identified in this new variant, mutation E484K is located in the receptor binding domain of the spike protein hence being considered a variant of concern. Such variants can be transmitted faster and could be implicated in increased mortality and decreased vaccine efficiency. Therefore, the researchers conclude that genomic and immune surveillance are critical to determining the impact of this new variant in SARS-CoV-2 transmission and immunity dynamics.

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Researchers at 59th Medical Wing, Clinical Investigations & Research Support Laboratory presented a pipeline for molecular epidemiological surveillance enabling public health officials to track viral evolution and outbreaks. The entire workflow including RNA extraction, library preparation with Paragon Genomics’ CleanPlex SARS-CoV-2 Panel, and data visualization plus analysis can be completed in less than one week. The research team concluded that “in the event that actionable sequencing data is required, tiling library prep is the most efficient and effective method”, in comparison to shotgun and hybrid capture methods.

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Prof. Jennifer Dien Bard and Prof. Xiaowu Gai along with their colleagues from Children’s Hospital Los Angeles and Keck School of Medicine at USC,  performed a study in pediatric patients infected with COVID-19 to study the genetic diversity of the SARS-CoV-2 viral genome. Whole genome sequencing was performed on extracted viral RNA from respiratory specimens collected on all SARS-CoV-2 positive children (n=141) using Paragon Genomics CleanPlex SARS-CoV-2 Research and Surveillance NGS Panel. They analyzed the relationships between viral genetic variants and clinical characteristics in children. Genomic evaluation demonstrated greater than expected genetic diversity, presence of the D614G mutation in the viral spike protein, increased mutation rate, and evidence of multiple introductions of SARSCoV-2 into Southern California. Their findings suggest a possible association of phylogenetic clade 20C with severe disease, but small sample size precludes a definitive conclusion. The researchers suggest a further multi-institutional genomic evaluation is needed and can have implications on infection control practices.

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PCORI Health Care Horizon Scanning System

The PCORI Health Care Horizon Scanning System has recently made a notable mention of Paragon Genomics to the likes of companies such as Illumina and Helix to be instrumental in developing several NGS assays for COVID-19 testing. The Horizon Scanning System identifies technology and systems innovations that could disrupt or cause significant shifts in health care. The Horizon scanning system can identify new (and new uses of existing) lab tests and procedures, public health and health promotion activities, and therapeutic interventions. They have issued a high impact report this September 2020 reporting several companies like Illumina, Paragon Genomics, Helix and others that have developed several NGS assays that can employ deep sequencing and computational methods to detect nucleic acids of the coronavirus. Such tests can amplify and detect sequences from the whole viral genome or several genomic regions. In their words ”NGS assays purportedly offer high throughput, enabling 2000 to 3000 samples to be processed in about 12 hours, with accuracy comparable to that of standard PCR tests and these assays might also be used in epidemiology studies to track the spread of coronavirus or to screen individuals who are planning to return to work or school.”

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Prof. Hadi M. Yassine from Qatar University in Doha, Qatar in collaboration with other researchers investigated the within-host SARS-CoV-2 virus diversity of 19 and 27 COVID-19 patients with mild and severe clinical manifestations respectively. Using our CleanPlex® SARS-CoV-2 Panel kit they performed targeted virus genome sequencing and reported patients with severe symptoms exhibited a significantly higher number of variants in coding and non-coding regions compared to mild cases. Their analysis also revealed higher prevalence of certain variants among severe cases only. Most importantly, severe cases exhibited significantly higher within-host diversity compared to mild cases. These observations helped them conclude that within-host diversity of the virus might play a role in the development of severe disease outcomes in COVID-19 patients.

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Imaging of the SARS-Cov-2 Ank-1 isolate using environmental SEM

Prof. Aykut Özkul from Ankara University in Turkey performed a study to isolate SARS-CoV-2 viruses circulating in Anatolia, and to investigate virus propagation in frequently-used cells and experimental animals. Using our CleanPlex® SARS-CoV-2 panel for their amplicon-based next generation sequencing, they identified two local SARS-CoV-2 virus isolates, and investigated in vitro growth dynamics in frequently-used Vero E6 cells and identified IFNAR−/− mice as a potential animal model for SARS-CoV-2 experiments.

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Children's Hospital Los Angeles logo

Prof. Xiaowu Gai and Prof. Jennifer Dien Bard from Children’s Hospital Los Angeles (CHLA) recently published a paper exploring clinical features and SARS-CoV-2 genetic variation in children presenting with COVID-19. They observed diverse clinical presentations and identified association between disease severity, viral load and age. By using CleanPlex SARS-CoV-2 Research and Surveillance NGS Panel, they were able to conduct highly sensitive and full-genome interrogation of the SARS-CoV-2 virus using amplicon sequencing on Illumina platforms.

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Rendering of a T Cell

Prof. Bryan Greenhouse, Dr. Sofonias Tessema and fellow researchers from Chan Zuckerberg Biohub, University of San Francisco, USA in collaboration with researchers from University of Massachusetts, Brown University, Clinton Health Access Initiative, and Centro de Investigação em Saúde de Manhiça (CISM) of Mozambique demonstrated that targeted NGS sequencing with a custom CleanPlex Panel is a flexible, low cost, “promising tool for studying the transmission dynamics of  P. falciparum” infections (Malaria), without the need for WGA, describing that in fact, their CleanPlex approach “outperformed whole genome sequences obtained from 50 times more total reads”. They further go on to note that their custom CleanPlex Panel “demonstrated high specificity regardless of the proportions of strains in the mixture (true positive rate = 99.8)… [and] sensitivity of the method was high across a wide range of parasite densities.”

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The origin of SARS-CoV-2 in Istanbul: Sequencing findings from the epicenter of the pandemic in Turkey

Prof. Dinler-Doganay’s Lab at Istanbul Technical University, Prof. Doganay and his colleagues at University of Health Sciences, Istanbul, Turkey and Dr. Ilker Karacan from Science and Advanced Technology Research Center, Istanbul, Turkey reported the SARS-CoV-2 viral genomes circulating in Istanbul for the first time. By using Paragon Genomics’ CleanPlex SARS-CoV-2 Research and Surveillance Panel, amplicon-based next-generation sequencing approach was successfully applied to generate near-complete genomes with an average depth of 2616. All three viral genomes carried the D614G variant (G clade according to GISAID classification) with implications for the origin of a spread.

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Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing

Prof. Xiaowu Gai and Prof. Jennifer Dien Bard from Children’s Hospital Los Angeles (CHLA), Paragon Genomics R&D teams led by Dr. Zhitong Liu, Prof. Alexander Urban from Stanford University and scientists from MGI / BGI-ShenZhen collaborated on sequencing COVID-19 patient samples using Paragon Genomics’s CleanPlex SARS-CoV-2 Panel for highly sensitive and full-genome interrogation of the SARS-CoV-2 virus using multiplexed PCR enrichment followed by next-generation sequencing.

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Oncology

This plot shows the coverage uniformity (%amplicons > 0.2*mean) in each library prepared using ETCs from 5 selected patients.

Prof. Lowe from Stanford University along with colleagues from RareCyte, Inc performed a study to identify and characterize Effusion Tumor Cells (ETCs) from malignant pleural effusions in patients with metastatic lung adenocarcinoma. Pleural effusion specimens have not yet been routinely used for molecular testing because of their frequent low tumor fraction and sparse cellularity. In this study, researchers modified and applied a Circulating Tumor Cells testing method to examine malignancies from pleural effusion specimens. To obtain high-quality  ETC libraries, they selected 5 representative patient samples each consisting of either a single cell or a small cell cluster. To generate Next-Generation Sequencing libraries, cell lysates were used as template with the CleanPlex OncoZoom Cancer Hotspot NGS Panel. Data Analysis and ETC Pool Variant Analysis was also performed. They retrieved high-quality sequencing results with great coverage uniformity. In this study, the authors developed a novel ETC-testing assay that detected epithelial malignancies in pleural effusions with 89.5% sensitivity and 100% specificity. Molecular profiling of ETCs showed promising concordance with their respective clinical molecular findings.

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CRISPR Regulatory T Cells Gene Network

Prof. Alexander Marson from University of California, San Francisco (UCSF), CA, USA in collaboration with researchers from University of California, Berkeley, CA, USA, Technische Universität München (TUM), Munich, Germany and multiple renowned Cancer Immunotherapy institutes in San Francisco published findings in Nature Immunology showing CRISPR–Cas9 ribonucleoprotein (RNP) technology allows dissection of complex gene modules in primary human Treg cells through targeted gene perturbation studies. Using our CleanPlex custom panels, researchers were able to perform CRISPR editing efficiency check and show pooled Cas9 RNP screens link indels with phenotypic changes in human Treg cells. The overall study resulted in functional network maps which may help to guide future development of drug targets and design of Treg cell– based immunotherapies.

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Rendering of a T Cell

Dr. Satu Mustjoki and fellow researchers from University of Helsinki, Finland, in collaboration with the University Hospital and Computer Science Department of Aalto University School of Science, report an increase in somatic mutations in CD4+ and CD8+ T cells of patients with immunodeficiency. The team used a custom CleanPlex Panel to validate the low VAF somatic mutations to gain higher confidence in their findings.

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Prof. Elliot Stieglitz‘s Lab at the UCSF Benioff Children’s Hospital developed a NGS assay powered by Paragon Genomics’ CleanPlex technology to measure the molecular burden of juvenile myelomonocytic leukemia (JMML). The researchers demonstrated that molecular testing can be helpful to distinguish between responders and nonresponders and should become an integral part of clinical care.

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Inherited Disease

The role of genetic mutations in intrahepatic cholestasis of pregnancy

Dr. Gültekin Adanaş Aydın, Dr. Gülten Özgen, and Dr. Orhan Görükmez from Bursa Yuksek Ihtisas Training and Research Hospital, Bursa, Turkey published a paper about the role of genetic mutations in intrahepatic cholestasis of pregnancy using a CleanPlex custom panel.

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Rendering of a T Cell

Prof. José A.Sánchez-Alcázar and fellow researchers from University of Pablo de Olavide, Spain collaborated with researchers from Department of Neurology, Uniklinikum C. G. Carus, Dresden, Germany and Instituto de Salud Carlos III, Spain and used CleanPlex Mitochondrial Disease Panel to study parkin-mediated mitophagy and autophagy flux disruption in cellular models of MERRF syndrome.

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Agrigenomics

Dr. Scott Geib and fellow researchers at the USDA-Agricultural Research Service developed new approach for building phylogenomic data sets using highly multiplexed amplicon sequencing powered by Paragon Genomics’ CleanPlex technology. The work provided unparalleled resolution of phylogenetic relationships in fruit flies.

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