All 6 entries tagged Metagenomics
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November 18, 2015
Writing about web page http://biorxiv.org/content/early/2015/11/17/032060
You may recall these two earlier blog posts:
- The story behind the paper: Sedimentary DNA from a submerged site reveals wheat in the British Isles
- Background reading on BBC Horizon's First Britons.
Well, this story has taken an unfortunate turn recently, in that a group from the Max Planck Institutes in Tübingen has contested our finding of wheat in the British Isles 8kya, essentially arguing that the results are too good to be true!
At the heart of their argument is the assumption (almost dogma) that DNA ages in a certain predictable way (through cytosine deamination) and these changes can be used to determine the age of DNA. As they could not detect the signatures of DNA damage in our wheat sequences, they have jumped to the conclusion that the wheat sequences must represent modern contamination.
However,this doesn't take into account the environment in which the DNA has been stored: the submerged sediments have effectively been stored in a refrigerator for the last 8000 years, because the ambient temperature for such sediments is only ~4 ° C. The argument here is a bit like saying that if you bought two loaves of bread and put one at room temperature in the bread bin and the other in the fridge or freezer and then came back a couple of weeks later to find only the loaf in the breadbin was mouldy it was safe to conclude that they couldn't possibly have been bought on the same day!
However, in addition to problems with the substance of the arguments, there have been problems in the way in which they have been made. They have been published in eLife, a fairly new open-access peer-reviewed journal, sponsored by the Max Planck Society:
- Contesting the presence of wheat in the British Isles 8,000 years ago by assessing ancient DNA authenticity from low-coverage data
The fact that the journal is sponsored by the Max Planck Society may or may not mean that authors from Max Planck Centres get an easier ride through peer review: judge for yourself as eLife publishes the reviews and decision letter.
But more problematic is that eLife, despite all its fanfare about being a revolutionary new open-access journal has not given us any right to reply to this publication, even though it is clearly a polemical piece aimed at discrediting our work. Oddly, Science, the journal we published in, also declined to let us publish a response. Luckily, given the old Internet addage that "information wants to be free", we have alternatives!
So, I am pleased to announce the appearance of this manuscript on bioRxiv, the preprint server for biology, and would ask you to read it, comment on it, Tweet it and Like it!
- Thermal age, cytosine deamination and the veracity of 8,000 year old wheat DNA from sediments. Logan Kistler, Oliver Smith, Roselyn Ware, Garry Momber, Richard Bates, Paul Garwood, Simon Fitch, Mark Pallen, Vincent Gaffney, Robin Allaby
- doi: http://dx.doi.org/10.1101/032060
The manuscript goes far beyond a simple rebuttal to encompass an analysisof 148 palaeogenomic data sets to show that the rate of cytosine deamination is a thermally correlated process and that organellar generally shows higher rates of deamination than nuclear DNA in comparable environments. In addition, we argue that the PCR enzyme used in our sedaDNA study would not have had the capability to report 5-prime cytosine deamination, so absence of this feature is to be expected.
Robin Allaby has worked extremely hard to prepare this manuscript and get it up there on bioRxiv. However, I have suggested to him that the work merits eventual publication in a peer-reviewed journal. Who knows, eLife might even take it! Watch this space!! And read and Tweet the manuscript!
March 13, 2015
The story behind the paper: Sedimentary DNA from a submerged site reveals wheat in the British Isles
Writing about web page http://www.sciencemag.org/content/347/6225/998
Late last month, I was proud to be joint last author on a paper in Science on the presence of wheat in the British Isles 8000 years ago. But how does a medical microbiologist come to be involved in a study on the intricacies of the Neolithic transition?
Well, like many of life’s greatest ventures, it all began in a bar…
I have to admit to a weakness for rounding the week off by a Friday evening trip to the bar. This started when I worked in Barts in 1980s and 1990s, where the Robin Brook Centre bar hosted many a lively conversation (and acted as a link to various melodramas, including an alleged murder, hostage taking and a police shoot-out: but that’s another story).
When I arrived at the University of Birmingham in 2001, I was delighted to discover the delights of the Bratby Bar, nestled within the university’s Staff House. During more than a decade of visits, I had the chance to chat to all sorts of people from across the University, from Pro-Vice-Chancellors to post-docs. Fortuitously, John Heath (formerly Head of Biosciences, latterly Birmingham’s PVC for Estates) introduced me to Vince Gaffney, a garrulous landscape archaeologist from Geordieland (below).
Having recently set up a next-generation sequencing service and also having picked up on the excitement of ancient DNA research, at intervals I suggested to Vince that he should let us have some archaeological material to play with, to see if we could get any sequences out of it. Imagining we could tread in the footsteps of Schliemann or Carter, I had in mind something glamorous like a mummified hand or a skeleton from a ritual burial. Instead, we ended up with some mud! But mud of a highly precious and productive sort.
Vince was interested in understanding how the Neolithic transition (the spread of farming after the domestication of plants and animals) arrived in northwest Europe. The arrival of farming in this part of the world coincided with rising sea levels following the end of the last Ice Age. Vince had a track record in studying the landscapes that were inundated during this time and he was convinced the earliest clues to the arrival of the Neolithic in this part of the world would be found in these now-submerged sites.
Vince pointed me in the direction of some pioneering studies on sedimentary ancient DNA, which had established that DNA from macroscopic plants and animals could be detected in sediments even in the absence of macrofossils and could be used to reconstruct past environments. Two studies in particular stood out: one on the Viking settlements in Greenland and the other on the detection of sheep and moa DNA from outside a cave in New Zealand. It struck me that this was an exciting emerging field, fertile with opportunity.
Vince suggested that we try to detect signs of Neolithisation by searching submerged sediments for DNA from domesticated species that had no natural relatives in North Western Europe. That ruled out cows (wild relative: the aurochs) and pigs (related to wild boar), but made sheep and goats an attractive target. I pointed out to Vince that although we had the wherewithal to do the high-throughput sequencing and bioinformatics, it would be a rather fraught process trying to devise and implement protocols for target-specific amplification of ancient DNA. Instead, buoyed up by recent success with metagenomics on human faecal samples, I suggested that we try simple shotgun metagenomics—in other words we just extract DNA from the sediment cores and sequence it directly without any attempt at target-specific amplification or capture.
And then a period of turbulence descended on our academic lives…
I was headhunted and recruited to a new position at the University of Warwick in April 2013, while Vince was preparing to leave the University of Birmingham and eventually ended up at the University of Bradford. This could have signalled the end of the proposed research, but Vince and I were determined to continue with the work.
In fact, as luck would have it, my move to Warwick breathed new life into the project, as I hooked up with Robin Allaby from Warwick’s School of Life Sciences. Robin, seen here in the guise of a modern-day Jesus of the barley field, not only had a track record in the evolution of domesticated species, particularly plants, but had also established a dedicated ancient DNA laboratory at Warwick, ideal for performing DNA extractions from sediment cores.
I quickly persuaded Robin of the merits of the project and, as I was preoccupied with establishing a new Division of Microbiology and Infection, passed over to him day-to-day supervision of the work. Fortunately, Robin was able to recruit his recently graduated PhD student, Oliver Smith to the study. Oliver was an ideal candidate in having experience with ancient DNA studies, while also being between projects. Funding for the work came from my start-up package from Warwick Medical School, which paid for a sequencing instrument (an Illumina MiSeq), sequencing reagents and a salary for Oliver for nine-ten months.
By the middle of 2013, Vince had tracked down the perfect samples for the project—some 8000-year-old submerged sediment cores that had been collected from the Solent by an maritime archaeologist Gary Momber. Oliver extracted DNA from four samples of sediment in the ancient DNA lab and then sequenced them on our MiSeq. He and Robin then analysed the metagenomic sequences. Robin soon recognised that naïve use of existing metagenomics analysis pipelines was likely to turn up spurious results because of biases in what was represented in the databases (see recent Ed Yong's blog post on “discovery” of platypus DNA in Virginia and plague on New York subway), so he devised an improved method that avoided the problem.
Contrary to our initial hopes, Robin and Oliver did not discover any sheep or goat DNA. Instead, they discovered sequences from wheat, a domesticated plant that originated in the Middle East, with no close wild relatives in Northern Europe. This represented a triumph for metagenomics in an ancient DNA research, confirming two advantages of this approach over target-specific assays:
- It is open-ended, not just targeting what you expect to find, but also revealing the unsuspected.
- It is probably more sensitive than target-based amplification in garnering relevant information from billions of base pairs of unamplified DNA rather than amplified copies of just a few hundred base pairs of a sequence barcode.
After that, Robin played a key role in co-ordinating the writing and submission of a manuscript, carefully steering our paper through the reviewing and editorial process. And so, finally, we ended up with every academic’s dream-come-true—a paper in Science magazine!
Of course, my account of things here is heavily biased towards the role of sequencing and bioinformatics in this project. It is also important to recognise the key role played by our archaeological collaborators in framing the right questions, gathering the right samples, performing the palaeo-environmental analyses and providing the relevant contextual interpretation of the findings.
And this success brings a new challenge: what on earth is Warwick Medical School going to do with this high-impact paper in Science for REF2020, as I cannot see it flying with the clinical medicine Unit of Assessment! But we have five years to work on that problem!
Let me close by raising a figurative glass to toast the role of Birmingham’s Staff House Bar in all this! A note to all PVCs for Estates: shouldn’t all universities be investing in similar drinking establishments to catalyse new projects and facilitate collegiality? And a note to the relevant promotion panel in Warwick: shouldn’t it soon be Professor Robin Allaby. I’ll drink to both points!
Robin and I celebrating success at the top of the Shard.
Commentary on the Paper in Science: http://www.sciencemag.org/content/347/6225/945.summary
September 23, 2014
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
July 16, 2014
Writing about web page http://mbio.asm.org/content/5/4/e01337-14
Diagnosing a 700-Year-Old Infection
Last summer, Warwick Professor of microbial genomics Mark Pallenand colleagues described recovering tuberculosis genomes from the lung tissue of a 215-year-old mummy from Hungary in the New England Journal of Medicine.Soon afterwards, news of his interest in metagenomic analyses on historical samples spread, and materials started to flow in.
Italian anthropologist Raffaella Bianucci asked Pallen if he would look for pathogens in archaeological samples from Belgium and Sardinia, an island off the coast of Italy, and he agreed. The relationship led to recovering a genome of the bacterium Brucella melitensis from a 700-year-old skeleton found in the ruins of a Medieval Italian village.
Reporting this week in mBio®, the authors describe using a technique called shotgun metagenomics to sequence DNA from a calcified nodule in the pelvic region of a middle-aged male skeleton excavated from the Sardinian settlement of Geridu, thought to have been abandoned in the late 14th century. Shotgun metagenomics allows scientists to sequence DNA without looking for a specific target.
From this sample, the researchers recovered the genome of Brucella melitensis, which causes an infection called brucellosis in livestock and humans. In humans, brucellosis is usually acquired by ingesting unpasteurized dairy products or from direct contact with infected animals. Symptoms include fevers, arthritis and swelling of the heart and liver. The disease is still found in the Mediterranean region.
“Normally when you think of calcified material in human or animal remains you think about tuberculosis, because that’s the most common infection that leads to calcification,” Pallen says. “We were a bit surprised to get Brucella instead.”
The skeleton contained 32 hardened nodules the size of a penny in the pelvic area, though Pallen says it’s unclear if they originated in the pelvis, or higher up in the chest or other body part. The team took care to sample the interior of a nodule, to eliminate the risk of contamination from soil.
In additional experiments, the research team showed that the DNA fragments extracted had the appearance of aged DNA – they were shorter than contemporary strands, with only 100 base pairs, and had characteristic G-A or C-T mutations at the ends. They also found that the medieval Brucella strain, which they called Geridu-1, was closely related to a recent Brucellastrain called Ether, identified in Italy in 1961, and two other Italian strains identified in 2006 and 2007. They confirmed their findings by comparing the distribution of genetic insertions and deletions located in Geridu-1 with those found in other Brucella strains.
The study “confirms that whole-genome sequences from bacterial pathogens can be recovered from human remains by metagenomics hundreds or even thousands of years postmortem,” Pallen says.
Pallen’s team is now testing shotgun metagenomics on a range of additional samples, including historical material from Hungarian mummies; Egyptian mummies; a Korean mummy from the 16th or 17th century; and lung tissue from a French queen from the Merovingian dynasty, which ruled France from the 5th to 8th centuries; as well as contemporary sputum samples from the Gambia in Africa.
“Metagenomics stands ready to document past and present infections, shedding light on the emergence, evolution and spread of microbial pathogens,” Pallen says. “We’re cranking through all of these samples and we’re hopeful that we’re going to find new things.”
-- Karen Blum, science journalist writing for Mbiosphere
Original blog posting here: http://mbioblog.asm.org/mbiosphere/2014/07/diagnosing-a-700-year-old-infection.html
April 14, 2014
Writing about web page http://www.biomedcentral.com/1756-0500/7/111
I came across this piece published in BMC Research Notes a few weeks ago, but have only just found time to comment on it: http://www.biomedcentral.com/1756-0500/7/111
- False positives complicate ancient pathogen identifications using high-throughput shotgun sequencing BMC Research Notes 2014, 7:111 doi:10.1186/1756-0500-7-111 Michael G Campana (email@example.com) Nelly Robles García (firstname.lastname@example.org) Frank J Rühli (email@example.com) Noreen Tuross (firstname.lastname@example.org)
I cannot say that I am too happy with the style of the comments therein on our recent publication of metagenomic recovery of a TB genome from mummified remains (http://www.nejm.org/doi/full/10.1056/NEJMc1302295):
Additionally, a recent study by Chan and colleagues  claiming the identification of multiple strains of pathogenic tuberculosis (Mycobacterium tuberculosis) through non- targeted metagenomic sequencing has demonstrated insufficient analytical rigor to support their conclusions. The authors aligned their sequences against a single strain of pathogenic tuberculosis, but did not account for misalignments or environmental contamination with ubiquitous soil mycobacteria. Chan and colleagues’ data merit reanalysis with appropriate environmental controls. We recommend that the authors of these three studies demonstrate the veracity of their findings using a targeted capture approach and further bioinformatic analysis.
I guess working in Harvard makes people prone to academic arrogance! Perhaps there is also a whiff of sour grapes: they couldn't find any pathogens in their samples by metagenomics so we can't have done too! But dealing with the substance of the comments is easy enough. And ironically, I agree entirely with their earlier comments that these two papers are highly suspect:
(NB they are misreferenced in this paper).
OK, so let's take their points on our study one by one...
- The authors aligned their sequences against a single strain of pathogenic tuberculosis, but did not account for misalignments or environmental contamination with ubiquitous soil mycobacteria.
- Mycobacterium tuberculosis is a genetically monomorphic species, so there is not much to be gained by aligning against multiple strain genomes. But we did also compare the SNP profiles of our genomes with the recent close relative 7199/99. In the standard filtering that we employed, SNPS with low/high coverage and low mapping scores were removed, thus avoiding problems with repetitive DNA. The fact the majority of the mixed SNPs matched those of 7199/99 and H37Rv confirms that they are real. Plus, we did discuss the presence of environmental Actinobacteria in the metagenome in the Supplementary Material, where we report the presence of a Nocardia sp at around 200X coverage and of a relative of Thermobifidia fusca at around 10X coverage. We binned contigs according to Z score and coverage to avoid mixing up reads from different species. And we obtained deep and even coverage of the M. tuberculosis genome, which cannot be accounted for by misinterpretation of matches to environmental species. We have seen such spurious matches in some analyses, but they appear only when a low-stringency approach is applied to mapping and are obvious because they show spikey coverage limited to conserved regions (e.g. rRNA genes) rather than across the whole genome.
- Chan and colleagues’ data merit reanalysis with appropriate environmental controls.
- And what might these controls be? We have analysed a piece of lung tissue from mummified remains from a casket rather than the soil. As detailed in previous papers (http://www.ncbi.nlm.nih.gov/pubmed/?term=12576588+12541332+18399990), rigorous efforts were taken to avoid contamination during sampling and storage. We have never grown M. tuberculosis or sequenced TB genomes in the lab. Where else could the M. tuberculosis DNA have come from other than the sampled individual?
- We recommend that the authors of these three studies demonstrate the veracity of their findings using a targeted capture approach and further bioinformatic analysis.
- There is some faulty logic here. We are indeed contemplating using a capture-based approach to increase the sensitivity of our analyses, but this will do nothing for the speciificity of the approach, since any contaminating sequences which map to the pathogenic reference strains in silico are likely to be captured in vitro anyway because of their similarity to the bait. The answer is instead to increase the stringency of mapping and look for a consilience of results from multiple sources of evidence (e.g. evenness of coverage, SNPs that allow an assignment within an established clade), which we have done.
We are continuing to perform metagenomics on mummified material from Vác and on other historical samples and will be publishing additional studies in due course. This is an exciting area of research and one does has to be careful in interpretation, but our findings stand firm. Anyone who wants to repeat the analyses we reported in Chan et al is welcome to do so. The reads are available here:http://www.ncbi.nlm.nih.gov/sra?LinkName=pubmed_sra&from_uid=23863071
But I am afraid I agree with Campana et al when they critiicise the other two papers, Thèves et al because, inter alia, you cannot tell Shigella from E. coli by 16S and Khairat et al, because no sequence data is available in the public domain. Caveat lector! But enjoy the excitement of progress in this field (see also http://www.ncbi.nlm.nih.gov/pubmed/?term=24708363+23765279)
April 09, 2013
Writing about web page http://jama.jamanetwork.com/article.aspx?articleid=1677374
A Culture-Independent Sequence-Based Metagenomics Approach to the Investigation of an Outbreak of Shiga-Toxigenic Escherichia coli O104:H4
Nicholas J. Loman, MBBS, PhD Chrystala Constantinidou, PhD Martin Christner, MD
Holger Rohde, MD Jacqueline Z.-M. Chan, PhD Joshua Quick, BSc Jacqueline C. Weir, MSci Christopher Quince, PhD Geoffrey P. Smith, PhD Jason R. Betley, PhD Martin Aepfelbacher, MD Mark J. Pallen, MA, MD, PhD
Ever since Hans Christian Gram and Robert Koch published their seminal papers in the early 1880s (1, 2), clinical diagnostic bacteriology has relied primarily on microscopy and culture in the laboratory to detect, identify and characterize human pathogens. Yet, there are many drawbacks to the use of these nineteenth-century approaches in the twenty-first century. Some important pathogenic bacteria can be isolated only after prolonged incubation (e.g. Mycobacterium tuberculosis) or under exacting culture conditions (e.g. anaerobic or microaerophilic organisms like Clostridium difficile or Campylobacter jejuni); in fact, several important pathogenic bacteria cannot be grown in vitro at all, including those that cause syphilis or leprosy. Variation in cultural requirements creates a need for many different culture media and many different procedures when processing clinical specimens and isolates—this results in complex and onerous workflows and a requirement for skilled staff (3).
So, what’s the solution? Well, 20 years ago, the polymerase chain reaction (PCR) brought the promise of a new model of diagnosis that might take us beyond the need for microscopy and culture.(4, 5) Gene-specific tests can be used to detect specific genetic determinants of importance in virulence and antimicrobial resistance. However, this approach suffers from the problem of finding only what you are looking for, so is not ideal for finding unknown virulence or resistance factors or even new combinations of known genetic determinants. Also, like culture, this approach requires a plethora of protocols each optimized for a particular pathogen or gene.
Today, we have published a paper in the Journal of the American Medical Association (JAMA) that presents proof of principle for a new approach that might augment or even replace the Koch-Gram paradigm that has dominated diagnostic bacteriology for so long. Metagenomics—the recovery of sequences directly from mixed populations of organisms without culture—has already proven a powerful approach to the analysis of complex microbial communities, particularly with the advent of high-throughput sequencing, which has delivered ultra-deep shotgun sequencing with unprecedented ease and cost-effectiveness (6, 7). This approach has seen widespread application in environmental microbiology and most recently in the cataloguing and investigation of the microbial communities associated with humans and other animals (8-10).
Diagnostic metagenomics—high-throughput sequencing of DNA extracted from clinical samples—provides an attractive alternative to the Gram-Koch paradigm in medical and public health bacteriology, avoiding the artifacts and inconvenience of culture and PCR, while potentially providing a simple but highly informative, one-size-fits-all approach to sample preparation and analysis. We have explored the potential of this approach by sequencing DNA extracted from a variety of human fecal samples, collected during and after the German STEC outbreak of 2011, in the hope of detecting and characterizing the outbreak strain and other bacterial pathogens without the need for microscopy, culture or PCR.
So how well did we do? Well, you can find out for yourself by reading the paper and associated commentary and blog post (coming soon from Bridget Kuehn) on the JAMA web site! Do not be put off by David Relman’s downbeat list of caveats: he is merely letting clinicians know that this is not a technique that is going to work out of the box in a routine diagnostic lab in the next week or two. But I would like to hope that there are parallels with the Wright brothers at Kitty Hawk: one day diagnostic metagenomics will be as routine as heavier-than-air human flight!
1. Koch R. Zur Untersuchung von pathogenen Organismen. Mitth. a. d. Kaiserl. Gesundheitsampte 1881; 1: 1-48. English translation Methods for the study of pathogenic organisms In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p101.
2. Gram HC. Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten. Fortschritte der Medizin 1884; 2: 185–189. English translation in Methods for the study of pathogenic organisms In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998. 1884.
3. Didelot X, Bowden R, Wilson DJ, Peto TE, Crook DW. Transforming clinical microbiology with bacterial genome sequencing. Nat Rev Genet. 2012;13:601-612.
4. Pallen MJ, Butcher PD. New strategies in microbiological diagnosis. J Hosp Infect. 1991;18 Suppl A:147-158.
5. Fredericks DN, Relman DA. Sequence-based identification of microbial pathogens: a reconsideration of Koch's postulates. Clin Microbiol Rev. 1996;9:18-33.
6. Gill SR, Pop M, Deboy RT et al. Metagenomic analysis of the human distal gut microbiome. Science. 2006;312:1355-1359.
7. Snyder LA, Loman N, Pallen MJ, Penn CW. Next-generation sequencing--the promise and perils of charting the great microbial unknown. Microb Ecol. 2009;57:1-3.
8. Caporaso JG, Lauber CL, Walters WA et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 2012;6:1621-1624.
9. Qin J, Li R, Raes J et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature. 2010;464:59-65.
10. A framework for human microbiome research. Nature. 2012;486:215-221.