High-throughput sequencing and clinical microbiology: progress, opportunities and challenges
Introduction
Genome sequencing has already transformed the study of microbial pathogens. Three years ago, one of the authors reviewed a decade or more of progress in ‘bacterial pathogenomics’ in a book and a review article of the same name [1, 2]. Both publications seem like the closing chapters of a bygone era, as high-throughput (or ‘next-generation’) sequencing sweeps through our discipline. For the first time, sequencing on a genomic scale now falls within the technical capability of the average university department and within the financial envelope of a modest research grant. Now that bacterial genomes can be sequenced in days or weeks rather than months or years, microbial genomics is at last poised to make a direct impact in clinical diagnostics, epidemiology and infection control. High-throughput sequencing also stands to revolutionise our view of the host response to infection and vaccination [3, 4], but our focus here will be on the pathogens themselves, rather than their hosts.
Section snippets
What is high-throughput sequencing?
High-throughput sequencing is an umbrella term applied to new sequencing technologies that deliver sequence data hundreds or thousands times more cheaply and speedily than traditional approaches [5••, 6••]. Three technologies have achieved widespread market penetration: the Roche 454 platform, the Solexa/Illumina platform and Life Technologies’ SOLiD platform. The 454 platform currently provides much longer read lengths (≥500 bp) than the other two platforms (∼100 bp for Illumina; 50–75 bp for
Culture-dependent applications: pathogen biology
High-throughput sequencing can be applied to organisms isolated in pure culture to improve our understanding of pathogen biology or provide high-resolution diagnostic or epidemiological information. One elegant application in pathogen biology, hit upon independently by four different research groups, is the use of high-throughput sequencing to screen transposon libraries to identify genes and pathways that contribute to fitness in various environments [9•, 10•, 11•, 12•].
RNA-seq, unbiased
Bacterial genomic epidemiology
Bacterial whole-genome sequencing represents the last word in epidemiological typing—a portable, digital, one-size-fits-all typing method capable of resolving a single base change between two genomes. However, although genome sequencing played a key role in solving the high-profile Amerithrax case, [29••], it is only now, thanks to high-throughput sequencing, that it can be applied to more routine epidemiological investigations. So far, such studies have concentrated on the identification and
Pathogen evolution
High-throughput sequencing has been used to look at the evolution of bacterial pathogens in the short term and over longer time scales. He et al. combined 454 and Sanger sequencing to look at large-scale changes in the genomes of eight diverse Clostridium difficile isolates. They then used Illumina technology to probe the evolutionary dynamics of 21 isolates from a hypervirulent lineage over a much shorter time scale [36•]. Several other studies have used whole-genome sequencing to track the
Culture-independent microbiology
Isolation of bacteria on laboratory media is a 19th-century technology, familiar even to the first generation of microbiologists. However, obtaining organisms in pure culture and identifying them using traditional phenotypic approaches is not only labour-intensive but also error-prone (e.g. in failing to discriminate between closely related genomospecies) and insensitive (common bacteria swamp rare ones and many species resist in vitro culture). High-throughput sequencing promises to bring
Community profiling
For the past quarter-century, sequencing of libraries of molecular bar codes, such as 16S rRNA genes, has provided a molecular culture-free approach to profiling the inhabitants of complex microbial communities, including those associated with the human body (the ‘human microbiome’). High-throughput sequencing breathes new life into these old approaches, by avoiding the need for cloning and delivering ultra-deep coverage [40]. Relman and co-workers performed early studies in this area,
Clinical metagenomics and pathogen discovery
Metagenomics is a term applied to the sequencing en masse of DNA extracted from a complex microbial community, without sub-culture or fractionation. The most impressive application of metagenomics to the human microbiome stemmed from a collaboration between European and Chinese researchers, using Illumina-based sequencing of bacterial DNA to survey the human facecal metagenome. After generating and assembling nearly 600 gigabases of sequence, the researchers were able to identify over three
Single-cell microbiology
Single-cell genomics is an innovative culture-independent approach, which provides access to the genome sequence of an individual bacterial cell via a genome amplification approach known as multiple displacement amplification or MDA [66, 67]. A recent study exploited MDA to recover 95–99.6% of a cyanobacterial genome starting from a single cell [68••]. In another paper provocatively named ‘one bacterial cell, one complete genome’, Woyke et al. recovered a complete genome sequence from an
Conclusions
High-throughput sequencing is already transforming the research landscape in microbiology. However, it is only a matter of time before it will also transform the practice of clinical microbiology in the reference and routine laboratory setting. High-throughput sequencing technologies are already obeying Moore's law—with a year-on-year exponential increase in performance—and a range of ‘next-next generation’ technologies are on the horizon, with the promise of read-lengths and run times ideal
Conflicts of interest
None identified.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgement
We thank the British Biotechnology and Biological Science Research Council for supporting NLJ via grant BBE0111791 and the Hospital Infection Society for supporting the study by Lewis et al.
References (74)
- et al.
Virus-host interactions: from systems biology to translational research
Curr Opin Microbiol
(2009) - et al.
Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung
Proc Natl Acad Sci U S A
(2009) - et al.
Exploring microbial diversity and taxonomy using SSU rRNA hypervariable tag sequencing
PLoS Genet
(2008) - et al.
Bacterial diversity in surgical site infections: not just aerobic cocci any more
J Wound Care
(2009) - et al.
A new arenavirus in a cluster of fatal transplant-associated diseases
N Engl J Med
(2008) - et al.
One bacterial cell, one complete genome
PLoS One
(2010) - et al.
Bacterial pathogenomics
Nature
(2007) - et al.
Next-generation sequencing: a transformative tool for vaccinology
Expert Rev Vaccines
(2009) - et al.
Microbiology in the post-genomic era
Nat Rev Microbiol
(2008)
Sequencing technologies – the next generation
Nat Rev Genet
Comparative genome analysis provides insights into the evolution and adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum
PLoS One
A draft genome sequence and functional screen reveals the repertoire of type III secreted proteins of Pseudomonas syringae pathovar tabaci 11528
BMC Genomics
Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms
Nat Methods
Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants
Genome Res
Identifying genetic determinants needed to establish a human gut symbiont in its habitat
Cell Host Microbe
Prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity
Nat Rev Genet
Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing
Proc Natl Acad Sci U S A
Structure and complexity of a bacterial transcriptome
J Bacteriol
Bacillus anthracis genome organization in light of whole transcriptome sequencing
BMC Bioinformatics
Molecular mechanisms of ethanol-induced pathogenesis revealed by RNA-sequencing
PLoS Pathog
Deep sequencing-based discovery of the Chlamydia trachomatis transcriptome
Nucleic Acids Res
The primary transcriptome of the major human pathogen Helicobacter pylori
Nature
A strand-specific RNA-Seq analysis of the transcriptome of the typhoid bacillus Salmonella typhi
PLoS Genet
A new technique for obtaining whole pathogen transcriptomes from infected host tissues
Biotechniques
High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach
PLoS One
A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis
Science
Genome sequencing of linezolid-resistant Streptococcus pneumoniae mutants reveals novel mechanisms of resistance
Genome Res
Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance
Genome Res
Viral population estimation using pyrosequencing
PLoS Comput Biol
Low-abundance HIV drug-resistant viral variants in treatment-experienced persons correlate with historical antiretroviral use
PLoS One
Low-abundance drug-resistant viral variants in chronically HIV-infected, antiretroviral treatment-naive patients significantly impact treatment outcomes
J Infect Dis
Evolution of MRSA during hospital transmission and intercontinental spread
Science
Molecular complexity of successive bacterial epidemics deconvoluted by comparative pathogenomics
Proc Natl Acad Sci U S A
High-throughput genome sequencing of two Listeria monocytogenes clinical isolates during a large foodborne outbreak
BMC Genomics
Genomic diversity and evolution of Mycobacterium ulcerans revealed by next-generation sequencing
PLoS Pathog
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