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September 23, 2014

Sequence the sputum: using metagenomics to diagnose tuberculosis

Writing about web page https://peerj.com/articles/585/

Laboratory diagnosis of tuberculosis (TB) using conventional approaches is a long drawn-out process, which takes weeks or months—plus, relying on laboratory culture means using techniques that date back to the 1880s!

In a report published today in the peer-reviewed journal PeerJ, we describe a new approach to the diagnosis of TB that relies on metagenomics—that is direct sequencing of DNA extracted from sputum—to detect and characterize the bacteria that cause TB without the need for time-consuming culture in the laboratory. Using the latest high-throughput sequencing technologies and some smart bioinformatics, we can now obtain sequences from the bacteria that cause TB in just a few days straight from clinical samples and gain insights into their genome sequences and the lineages they belong to, all without having to culture cells or capture or amplify DNA.

In this study, first-year PhD student Emma Doughty (https://twitter.com/EmmaDoughty6) and bioinformatician Dr Martin Sergeant, both working at Warwick Medical School, have worked with African scientists Dr Martin Antonio and Dr Ifedayo Adetifaworking at the MRC Unit in The Gambia to develop and exploit novel sequencing and analytic approaches. They detected sequences from the TB bacteria in all eight sputum samples they investigated and were able to assign the bacteria to a known lineage in seven of the samples. Two samples were found to contain sequences from Mycobacterium africanum, a variety of the TB bacterium that is particular to West Africa.

This is part of a connected programme of research in the Pallen group, where we have been using metagenomics to detect bacterial pathogens in contemporary and historical human material. Last year, we used metagenomics to obtain an outbreak strain genome from stool samples from an E. coli outbreak and to recover TB genomes from ~200-year-old Hungarian mummies. Earlier this year, we recovered the genome of Brucella melitensis, which causes an infection called brucellosis in livestock and humans, from a 700-year-old skeleton from Sardinia, Italy.

We now aim to work on a larger number of sputum samples, perhaps looking at a hundred consecutive samples in the fullness of time. But, before then, we need to spend a bit more time optimising our DNA extraction protocols. We were pleasantly surprised that the protocol we used worked “out of the box”, but we are confident that we can improve things so we get fewer human DNA sequences and more mycobacterial sequences from each sample. If we can increase coverage of the TB genomes, we may soon be able to detect mutations associated with drug-resistance directly from the sputum.

The final goal, shimmering on the horizon, is that we might one day be able to extract information from all the macromolecules in a sample (DNA, RNA, proteins) so that we get a read-out of what pathogens are there, what virulence or resistance genes are being expressed, what host responses are switched on and also maybe detect cancerous or pre-cancerous changes in the patient’s genome. This is probably going to rely on a new kind of approach: nanopore sequencing—to learn more about this, watch the recent Bioinformatics and Balti session on YouTube. The future is looking very exciting!

PS: we have been very impressed with the service offered by PeerJ, with just two weeks from submission to acceptance!

Professor Mark Pallen, Professor of Microbial Genomics and Head of the Microbiology and Infection Unit,

Warwick Medical School

http://www2.warwick.ac.uk/fac/med/research/microinfect/staff/pallen/
http://twitter.com/mjpallen
https://www.youtube.com/user/pallenm
http://scholar.google.co.uk/citations?hl=en&user=gg8yViYAAAAJ


July 18, 2013

Tuberculosis genomes recovered from 200–year old Hungarian mummy


Press release on our recent publication in the New England Journal of Medicine

Metagenomic Analysis of Tuberculosis in a Mummy
N Engl J Med 2013; 369:289-290July 18, 2013DOI: 10.1056/NEJMc1302295
Jacqueline Z.-M. Chan, Martin J. Sergeant, Oona Y.-C. Lee, David E. Minnikin, Gurdyal S. Besra, Ilidkó Pap, Mark Spigelman, Helen D. Donoghue, Mark J. Pallen



Researchers at the University of Warwick have recovered tuberculosis (TB) genomes from the lung tissue of a 215-year old mummy using a technique known as metagenomics.

The team, led by Professor Mark Pallen, Professor of Microbial Genomics at Warwick Medical School, working with Helen Donoghue at University College London and collaborators in Birmingham and Budapest, sought to use the technique to identify TB DNA in a historical specimen.

The term ‘metagenomics’ is used to describe the open-ended sequencing of DNA from samples without the need for culture or target-specific amplification or enrichment. This approach avoids the complex and unreliable workflows associated with culture of bacteria or amplification of DNA and draws on the remarkable throughput and ease of use of modern sequencing approaches.

The sample came from a Hungarian woman, Terézia Hausmann, who died aged 28 on 25 December 1797. Her mummified remains were recovered from a crypt in the town of Vác, Hungary. When the crypt was opened in 1994, it was found to contain the naturally mummified bodies of 242 people. Molecular analyses of the chest sample in a previous study confirmed the diagnosis of tuberculosis and hinted that TB DNA was extremely well preserved in her body.

Professor Pallen explained the importance of the breakthrough,

“Most other attempts to recover DNA sequences from historical or ancient samples have suffered from the risk of contamination, because they rely on amplification of DNA in the laboratory, plus they have required onerous optimisation of target-specific assays. The beauty of metagenomics is that it provides a simple but highly informative, assumption-free, one-size-fits-all approach that works in a wide variety of contexts. A few months ago we showed that metagenomics allowed us to identify an E. coli outbreak strains from faecal samples and a few weeks ago a similar approach was shown by another group to deliver a leprosy genome from historical material”.

The research, published this week in the New England Journal of Medicine, showed that Terézia Hausmann suffered from a mixed infection with two different strains of the TB bacterium. This information, combined with work on contemporary tuberculosis, highlights the significance of mixed-strain infections, particularly when tuberculosis is highly prevalent.

Professor Pallen added,

“It was fascinating to see the similarities between the TB genome sequences we recovered and the genome of a recent outbreak strain in Germany. It shows once more that using metagenomics can be remarkably effective in tracking the evolution and spread of microbes without the need for culture—in this case, metagenomes revealed that some strain lineages have been circulating in Europe for more than two centuries.”

Notes for editors

For more information, or to arrange interviews with author Mark J. Pallen, M.A., M.D., Ph.D., email m.pallen@warwick.ac.uk.

Alternatively, contact Warwick Medical School Press Officer Luke Harrison on +44 (0) 2476 574255/150483 or +44 (0) 7920531221 luke.harrison@warwick.ac.uk


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