Development of a capture sequencing assay for enhanced detection and genotyping of tick-borne pathogens

Jain, Komal; Tagliafierro, Teresa; Marques, Adriana; Sánchez-Vicente, Santiago; Gokden, Alper; Fallon, Brian A.; Mishra, Nischay; Briese, Thomas; Kapoor, Vishal; Sameroff, Stephen; Guo, Cheng; Marcos, Luis A.; Hu, Linden T.; Lipkin, W. Ian; Tokarz, Rafal

doi:10.1038/s41598-021-91956-z

Cited by 12 publications

(10 citation statements)

References 49 publications

(51 reference statements)

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“…In a pilot RNA-Seq study using ribodepleted RNA from engorged nymphs infected with WT strain B31, only approximately 6,700 reads mapped to protein coding genes (0.034% of approximately 20 million total raw reads) ( Supplemental Table 1 ; supplemental material available online with this article; https://doi.org/10.1172/JCI166710DS1 ), a value too low to obtain comprehensive transcriptomic data. To overcome this bottleneck, we took advantage of an enrichment strategy, designated TBDCapSeq, developed by Tokarz and colleagues ( 26 , 27 ), which uses hybridization probes to ‘capture’ pathogen-specific amplicons prior to sequencing ( Figure 1 ). Using TBDCapSeq, we compared the transcriptomes of WT and Δ rpoS Bb in fed nymphs and DMCs.…”

Section: Resultsmentioning

confidence: 99%

BosR and PlzA reciprocally regulate RpoS function to sustain Borrelia burgdorferi in ticks and mammals

Grassmann¹,

Tokarz²,

Golino³

et al. 2023

Journal of Clinical Investigation

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The alternative sigma factor RpoS in Borrelia burgdorferi (Bb), the Lyme disease pathogen, is responsible for programmatic positive and negative gene regulation essential for the spirochete's dual-host enzootic cycle. RpoS is expressed during tick-to-mammal transmission and throughout mammalian infection.Although the mammalian-phase RpoS regulon is well described, its counterpart during the transmission blood meal is unknown. Here, we used Bb-specific transcript enrichment by TBDCapSeq to compare the transcriptomes of wild-type and ΔrpoS Bb in engorged nymphs and following mammalian host-adaptation within dialysis membrane chambers. TBDCapSeq revealed dramatic changes in the contours of the RpoS regulon within ticks and mammals and further confirmed that RpoS-mediated repression is specific to the mammalian-phase of Bb's enzootic cycle. We also provide evidence that RpoS-dependent gene regulation, including repression of tick-phase genes, is required for persistence in mice. Comparative transcriptomics of engineered Bb strains revealed that BosR, a non-canonical Fur family regulator, and the c-di-GMP effector PlzA reciprocally regulate the function of RNA polymerase complexed with RpoS. BosR is required for RpoS-mediated transcription activation and repression in addition to its well-defined role promoting RpoN-dependent transcription of rpoS. During transmission, liganded-PlzA antagonizes RpoSmediated repression, presumably acting through BosR.

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Section: Resultsmentioning

confidence: 99%

BosR and PlzA reciprocally regulate RpoS function to sustain Borrelia burgdorferi in ticks and mammals

Grassmann¹,

Tokarz²,

Golino³

et al. 2023

Journal of Clinical Investigation

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“…as we wanted to create a system that could be used with very complex samples, such as air filters. These complex samples contain DNA from many diverse organisms [ 73 , 85 ] that may contain conserved regions of the non-exclusive CDSs also found in Francisellaceae spp., which could lead to non-target enrichment [ 86 ]. However, it is important to point out that this design consideration is context dependent.…”

Section: Resultsmentioning

confidence: 99%

Genomic characterization of Francisella tularensis and other diverse Francisella species from complex samples

et al. 2022

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Francisella tularensis, the bacterium that causes the zoonosis tularemia, and its genetic near neighbor species, can be difficult or impossible to cultivate from complex samples. Thus, there is a lack of genomic information for these species that has, among other things, limited the development of robust detection assays for F. tularensis that are both specific and sensitive. The objective of this study was to develop and validate approaches to capture, enrich, sequence, and analyze Francisella DNA present in DNA extracts generated from complex samples. RNA capture probes were designed based upon the known pan genome of F. tularensis and other diverse species in the family Francisellaceae. Probes that targeted genomic regions also present in non-Francisellaceae species were excluded, and probes specific to particular Francisella species or phylogenetic clades were identified. The capture-enrichment system was then applied to diverse, complex DNA extracts containing low-level Francisella DNA, including human clinical tularemia samples, environmental samples (i.e., animal tissue and air filters), and whole ticks/tick cell lines, which was followed by sequencing of the enriched samples. Analysis of the resulting data facilitated rigorous and unambiguous confirmation of the detection of F. tularensis or other Francisella species in complex samples, identification of mixtures of different Francisella species in the same sample, analysis of gene content (e.g., known virulence and antimicrobial resistance loci), and high-resolution whole genome-based genotyping. The benefits of this capture-enrichment system include: even very low target DNA can be amplified; it is culture-independent, reducing exposure for research and/or clinical personnel and allowing genomic information to be obtained from samples that do not yield isolates; and the resulting comprehensive data not only provide robust means to confirm the presence of a target species in a sample, but also can provide data useful for source attribution, which is important from a genomic epidemiology perspective.

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“…We employed an updated TBDCapSeq probe set that included oligos targeting the complete genomes of 14 primary tick-borne agents found in the United States. The targets included B. burgdorferi , Borrelia mayonii , Borrelia miyamotoi , Babesia microti , Anaplasma phagocytophilum , Ehrlichia muris eauclaireensis , Ehrlichia chaffeensis , Rickettsia parkeri , Rickettsia rickettsii , and Francisella tularensis , Powassan virus, Heartland virus, Colorado tick fever virus, and Bourbon virus ( Jain et al, 2021 ). For B. burgdorferi , we targeted the genomes of three distinct genotypes (strains B31, N40, and 297).…”

Section: Methodsmentioning

confidence: 99%

“…Capture sequencing results in a substantial increase in sensitivity surpassing quantitative PCR. The utility of this approach has been documented for viral and bacterial agents, and with the recent development of the TBD Capture Sequencing Assay (TBDCapSeq), we have demonstrated its potential for detection of tick-borne pathogens ( Jain et al, 2021 ). In this study, we examined the utility of TBDCapSeq as a tool for molecular detection of tick-borne agents in human blood with a focus on specimens from patients with early localized Lyme disease.…”

Section: Introductionmentioning

confidence: 99%

Capture Sequencing Enables Sensitive Detection of Tick-Borne Agents in Human Blood

et al. 2022

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Assay sensitivity can be a limiting factor in the use of PCR as a tool for the detection of tick-borne pathogens in blood. We evaluated the performance of Tick-borne disease Capture Sequencing Assay (TBDCapSeq), a capture sequencing assay targeting tick-borne agents, to test 158 whole blood specimens obtained from the Lyme Disease Biobank. These included samples from 98 individuals with signs and symptoms of acute Lyme disease, 25 healthy individuals residing in Lyme disease endemic areas, and 35 samples collected from patients admitted to the Massachusetts General Hospital or referred to the infectious disease clinic. Compared to PCR, TBDCapSeq had better sensitivity and could identify infections with a wider range of tick-borne agents. TBDCapSeq identified a higher rate of samples positive for Borrelia burgdorferi (8 vs. 1 by PCR) and Babesia microti (26 vs. 15 by PCR). TBDCapSeq also identified previously unknown infections with Borrelia miyamotoi, Ehrlichia, and Rickettsia species. Overall, TBDCapSeq identified a pathogen in 43 samples vs. 23 using PCR, with four co-infections detected versus zero by PCR. We conclude that capture sequencing enables superior detection of tick-borne agents relative to PCR.

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Development of a capture sequencing assay for enhanced detection and genotyping of tick-borne pathogens

Cited by 12 publications

References 49 publications

BosR and PlzA reciprocally regulate RpoS function to sustain Borrelia burgdorferi in ticks and mammals

BosR and PlzA reciprocally regulate RpoS function to sustain Borrelia burgdorferi in ticks and mammals

Genomic characterization of Francisella tularensis and other diverse Francisella species from complex samples

Capture Sequencing Enables Sensitive Detection of Tick-Borne Agents in Human Blood

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