High-throughput sequencing and clinical microbiology: progress, opportunities and challenges

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High-throughput sequencing is sweeping through clinical microbiology, transforming our discipline in its wake. It is already providing an enhanced view of pathogen biology through rapid and inexpensive whole-genome sequencing and more sophisticated applications such as RNA-seq. It also promises to deliver high-resolution genomic epidemiology as the ultimate typing method for bacteria. However, the most revolutionary effect of this ‘disruptive technology’ is likely to be creation of a novel sequence-based, culture-independent diagnostic microbiology that incorporates microbial community profiling, metagenomics and single-cell genomics. We should prepare for the coming ‘technological singularity’ in sequencing, when this technology becomes so fast and so cheap that it threatens to out-compete existing diagnostic and typing methods in microbiology.

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.

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