Dataset Information

Selective whole genome amplification and sequencing of Coxiella burnetii directly from environmental samples.

ABSTRACT: Whole genome sequencing (WGS) is a widely available, inexpensive means of providing a wealth of information about an organism's diversity and evolution. However, WGS for many pathogenic bacteria remain limited because they are difficult, slow and/or dangerous to culture. To avoid culturing, metagenomic sequencing can be performed directly on samples, but the sequencing effort required to characterize low frequency organisms can be expensive. Recently developed methods for selective whole genome amplification (SWGA) can enrich target DNA to provide efficient sequencing. We amplified Coxiella burnetii (a bacterial select agent and human/livestock pathogen) from 3 three environmental samples that were overwhelmed with host DNA. The 68- to 147-fold enrichment of the bacterial sequences provided enough genome coverage for SNP analyses and phylogenetic placement. SWGA is a valuable tool for the study of difficult-to-culture organisms and has the potential to facilitate high-throughput population characterizations as well as targeted epidemiological or forensic investigations.

SUBMITTER: Cocking JH

PROVIDER: S-EPMC7199880 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Selective whole genome amplification and sequencing of Coxiella burnetii directly from environmental samples.

Cocking Jill Hager JH Deberg Michael M Schupp Jim J Sahl Jason J Wiggins Kristin K Porty Ariel A Hornstra Heidie M HM Hepp Crystal C Jardine Claire C Furstenau Tara N TN Schulte-Hostedde Albrecht A Fofanov Viacheslav Y VY Pearson Talima T

Genomics 20191031 2

Whole genome sequencing (WGS) is a widely available, inexpensive means of providing a wealth of information about an organism's diversity and evolution. However, WGS for many pathogenic bacteria remain limited because they are difficult, slow and/or dangerous to culture. To avoid culturing, metagenomic sequencing can be performed directly on samples, but the sequencing effort required to characterize low frequency organisms can be expensive. Recently developed methods for selective whole genome ...[more]

PMID: 31678592

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Project description:Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ?5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens. IMPORTANCE:Malaria is a disease caused by Plasmodium parasites that caused 214 million symptomatic cases and 438,000 deaths in 2015. Plasmodium vivax is the most widely distributed species, causing the majority of malaria infections outside sub-Saharan Africa. Whole-genome sequencing (WGS) of Plasmodium parasites from clinical samples has revealed important insights into the epidemiology and mechanisms of drug resistance of malaria. However, WGS of P. vivax is challenging due to low parasite levels in humans and the lack of a routine system to culture the parasites. Selective whole-genome amplification (SWGA) preferentially amplifies the genomes of pathogens from mixtures of target and host gDNA. Here, we demonstrate that SWGA is a simple, robust method that can be used to enrich P. vivax genomic DNA (gDNA) from unprocessed human blood samples and dried blood spots for cost-effective, high-quality WGS.

Project description:BackgroundChlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples.MethodsC. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform.ResultsFull length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies.ConclusionsThe combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.

Project description:BackgroundCoxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes.ResultsTo assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples.ConclusionsThis newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.

Project description:Advancement in next generation sequencing offers the possibility of routine use of whole genome sequencing (WGS) for Mycobacterium bovis (M. bovis) genomes in clinical reference laboratories. To date, the M. bovis genome could only be sequenced if the mycobacteria were cultured from tissue. This requirement for culture has been due to the overwhelmingly large amount of host DNA present when DNA is prepared directly from a granuloma. To overcome this formidable hurdle, we evaluated the usefulness of an RNA-based targeted enrichment method to sequence M. bovis DNA directly from tissue samples without culture. Initial spiking experiments for method development were established by spiking DNA extracted from tissue samples with serially diluted M. bovis BCG DNA at the following concentration range: 0.1 ng/μl to 0.1 pg/μl (10-1 to 10-4). Library preparation, hybridization and enrichment was performed using SureSelect custom capture library RNA baits and the SureSelect XT HS2 target enrichment system for Illumina paired-end sequencing. The method validation was then assessed using direct WGS of M. bovis DNA extracted from tissue samples from naturally (n = 6) and experimentally (n = 6) infected animals with variable Ct values. Direct WGS of spiked DNA samples achieved 99.1% mean genome coverage (mean depth of coverage: 108×) and 98.8% mean genome coverage (mean depth of coverage: 26.4×) for tissue samples spiked with BCG DNA at 10-1 (mean Ct value: 20.3) and 10-2 (mean Ct value: 23.4), respectively. The M. bovis genome from the experimentally and naturally infected tissue samples was successfully sequenced with a mean genome coverage of 99.56% and depth of genome coverage ranging from 9.2× to 72.1×. The spoligoyping and M. bovis group assignment derived from sequencing DNA directly from the infected tissue samples matched that of the cultured isolates from the same sample. Our results show that direct sequencing of M. bovis DNA from tissue samples has the potential to provide accurate sequencing of M. bovis genomes significantly faster than WGS from cultures in research and diagnostic settings.

Dataset Information

Selective whole genome amplification and sequencing of Coxiella burnetii directly from environmental samples.

Publications

Selective whole genome amplification and sequencing of Coxiella burnetii directly from environmental samples.

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