scholarly journals Evaluation of a Next-Generation Sequencing Metagenomics Assay to Detect and Quantify DNA Viruses in Plasma from Transplant Recipients

2021 ◽  
Author(s):  
Soya S. Sam ◽  
Ralph Rogers ◽  
Fizza S. Gillani ◽  
Gregory J. Tsongalis ◽  
Colleen S. Kraft ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Transplant Recipients ◽  
Next Generation ◽  
Dna Viruses ◽  
Generation Sequencing

scholarly journals An improved experimental pipeline for preparing circular ssDNA viruses for next-generation sequencing

2020 ◽  
Author(s):  
Catherine D. Aimone ◽  
J. Steen Hoyer ◽  
Anna E. Dye ◽  
David O. Deppong ◽  
Siobain Duffy ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rolling Circle Amplification ◽  
Next Generation ◽  
Viral Dna ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Short Read ◽  
Rolling Circle ◽  
Optimized Protocol ◽  
Generation Sequencing

AbstractWe present an optimized protocol for enhanced amplification and enrichment of viral DNA for Next Generation Sequencing of begomovirus genomes. The rapid ability of these viruses to evolve threatens many crops and underscores the importance of using next generation sequencing efficiently to detect and understand the diversity of these viruses. We combined enhanced rolling circle amplification (RCA) with EquiPhi29 polymerase and size selection to generate a cost-effective, short-read sequencing method. This optimized protocol produced short-read sequencing with at least 50% of the reads mapping to the viral reference genome. We provide other insights into common misconceptions about RCA and lessons we have learned from sequencing single-stranded DNA viruses. Our protocol can be used to examine viral DNA as it moves through the entire pathosystem from host to vector, providing valuable information for viral DNA population studies, and would likely work well with other CRESS DNA viruses.HighlightsProtocol for short-read, high throughput sequencing of single-stranded DNA viruses using random primersComparison of the sequencing of total DNA versus size-selected DNAComparison of phi29 and Equiphi29 DNA polymerases for rolling circle amplification of viral single-stranded DNA genomes

scholarly journals 1584. Development and Dynamics of Cytomegalovirus UL97 Ganciclovir Resistance Mutations in Transplant Recipients Detected by Next-generation Sequencing

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S495-S496
Author(s):  
Isabelle Paula Lodding ◽  
Mette Jørgensen ◽  
Marc Bennedbæk ◽  
Nikolai Kirkby ◽  
Klaudia Naegele ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Transplant Recipients ◽  
Next Generation ◽  
Resistance Mutations ◽  
Ganciclovir Resistance ◽  
Generation Sequencing

scholarly journals Enhanced Detection of DNA Viruses in the Cerebrospinal Fluid of Encephalitis Patients Using Metagenomic Next-Generation Sequencing

2020 ◽  
Vol 11 ◽  
Author(s):  
Carmen F. Manso ◽  
David F. Bibby ◽  
Hodan Mohamed ◽  
David W. G. Brown ◽  
Mark Zuckerman ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Dna Viruses ◽  
Generation Sequencing

scholarly journals Complete Genome Sequences, Derived by Next-Generation Sequencing, of JC Polyomavirus Strains Isolated from Vietnamese Renal Transplant Recipients

2020 ◽  
Vol 9 (2) ◽  
Author(s):  
Trang Dinh Van ◽  
Rebecca J. Rockett ◽  
An Phan Hai Ha ◽  
Trung Vu Nguyen ◽  
Dominic E. Dwyer
Keyword(s):  
Next Generation Sequencing ◽  
Renal Transplant ◽  
Complete Genome ◽  
Rolling Circle Amplification ◽  
Renal Transplant Recipients ◽  
Transplant Recipients ◽  
Next Generation ◽  
Genome Sequences ◽  
Jc Polyomavirus ◽  
Generation Sequencing

JC polyomavirus (JCPyV) may cause clinical syndromes such as progressive multifocal leukoencephalopathy in immunocompromised patients. Here, we report seven complete genome sequences of JCPyV genotype 7A, generated directly from urine samples from Vietnamese renal transplant recipients by using rolling-circle amplification and next-generation sequencing.

scholarly journals Diagnosis and analysis of unexplained cases of childhood encephalitis in Australia using metagenomic next-generation sequencing

2021 ◽  
Author(s):  
Ci-Xiu Li ◽  
Rebecca Burrell ◽  
Russell C Dale ◽  
Alison Kesson ◽  
Christopher C Blyth ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Rna Sequencing ◽  
Diagnostic Yield ◽  
Human Infection ◽  
Clinical Samples ◽  
Next Generation ◽  
Dna Viruses ◽  
Immune Mediated ◽  
Generation Sequencing

Encephalitis is most often caused by a variety of infectious agents, the identity of which is commonly determined through diagnostic tests utilising cerebrospinal fluid (CSF). Immune-mediated disorders are also a differential in encephalitis cases. We investigated the clinical characteristics and potential aetiological agents of unexplained encephalitis through metagenomic next-generation sequencing of residual clinical samples of multiple tissue types and independent clinical review. A total of 43 specimens, from both sterile and non-sterile sites, were collected from 18 encephalitis cases with no cause identified by the Australian Childhood Encephalitis study. Samples were subjected to total RNA sequencing to determine the presence and abundance of potential pathogens, to reveal mixed infections, pathogen genotypes, and epidemiological origins, and to describe the possible aetiologies of unexplained encephalitis. From this, we identified five RNA and two DNA viruses associated with human infection from both non-sterile (nasopharyngeal aspirates, nose/throat swabs, urine, stool rectal swab) and sterile (cerebrospinal fluid, blood) sites. These comprised two human rhinoviruses, two human seasonal coronaviruses, two polyomaviruses and one picobirnavirus. With the exception of picobirnavirus all have been previously associated with respiratory disease. Human rhinovirus and seasonal coronaviruses may be responsible for five of the encephalitis cases reported here. Immune-mediated encephalitis was considered clinically likely in six cases and RNA sequencing did not identify a possible pathogen in these cases. The aetiology remained unknown in nine cases. Our study emphasises the importance of respiratory viruses in the aetiology of unexplained child encephalitis and suggests that the routine inclusion of non-CNS sampling in encephalitis clinical guidelines/protocols could improve the diagnostic yield.

scholarly journals 645. Rapid Diagnosis of Disseminated Mycobacterium kansasii infection in Renal Transplant Recipients Using Plasma Microbial Cell Free DNA Next Generation Sequencing

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S424-S425
Author(s):  
Tosin Ogunsiakan ◽  
Kristen D Fajgenbaum ◽  
Gautam Phadke ◽  
Thomas Montgomery ◽  
Kiran Gajurel
Keyword(s):  
Next Generation Sequencing ◽  
Transplant Recipient ◽  
Kidney Transplant ◽  
Kidney Transplant Recipient ◽  
Rapid Diagnosis ◽  
Mycobacterium Kansasii ◽  
Transplant Recipients ◽  
Next Generation ◽  
Free Dna ◽  
Generation Sequencing

Abstract Background Disseminated Mycobacterium kansasii infection is rare in kidney transplant recipients. The diagnosis may not be suspected readily due to non-specific clinical presentation. The diagnosis and treatment can be further delayed due to poor sensitivity of culture (especially of extra-pulmonary sites) and slow growth in culture media. Accurate and rapid diagnosis of disseminated M. kansasii infections in transplant recipients is important for antimicrobial management. Methods Two cases of disseminated M. kansasii infections with unusual presentation in which rapid diagnosis was made using the Karius test (KT) are presented. The KT is a CLIA certified/CAP-accredited next-generation sequencing (NGS) plasma test that detects microbial cell-free DNA (mcfDNA). After mcfDNA is extracted and NGS performed, human reads are removed, and remaining sequences are aligned to a curated database of >1400 organisms. Organisms present above a statistical threshold are reported. Results Case 1: A 31-year female kidney transplant recipient presented with a thyroglossal duct cyst, as well as swelling of her right metacarpophalangeal joint and left 3rd finger. AFB culture of the thyroglossal cyst aspiration done on post admission day (PAD) 2 took 27 days to be identified as M. kansasii (on PAD 29) whereas plasma sent for KT on PAD 5 reported a positive test for M. kansasii at 284 molecules/microliter (MPM) in 4 days (on PAD 9). Case 2: A 59-year male kidney transplant recipient presented with generalized weakness, arthralgia, pericardial effusion, cytopenia, weight loss and intermittent fevers. Plasma sent for KT on PAD 12 was reported positive for M. kansasii at 1314 MPM in 3 days (on PAD 15). PET CT done simultaneously was consistent with an infection of an old AV graft in the left upper extremity. The AFB culture of the resected graft was confirmed as M. kansasii in 22 days on PAD 36. After the KT was available (before confirmation of M. kansasii on culture), the first patient underwent modification of empiric treatment and the second patient was started on specific treatment for M. kansasii. Table of M. kansasii cases Rapid diagnosis of disseminated M. kansasii infection Conclusion Open-ended NGS plasma testing for mcfDNA identified disseminated M kansasii infection much earlier than standard microbiology and thus helped in initiation and modification of pathogen directed treatment. Disclosures All Authors: No reported disclosures

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