The Argonne Soil Metagenomics Meeting is in its 4th year and this year’s meeting will be held October 3rd to 5th at the Indian Lakes Resort Conference Center, right outside of Chicago. The meeting, like past years, will focus on all aspects of soil metagenomics. There’s a whole lot of great speakers lined up, in fact a whole lot who are addressing fungi in soils. Meeting registration is open.
I’m starting a new series of short tutorials. In a selfish way, these posts are for me – a vehicle for me to clarify my own comprehension of a given topic. I might also tell you about a solution to a problem I have been troubleshooting.
The first post in this series is about fosmids. There seems to be some public confusion as to what they are or what they can be used for – basically, I have been confused. Apparently I had forgotten my microbiology coursework along the way. Many genome sequencing projects – such as the human genome project – have utilized fosmids to create libraries prior to sequencing, but it wasn’t until hearing about JGI’s fungal and metagenomic sequencing initiatives did I hear the term fosmid mentioned frequently.
Fosmids are used when preparing genomic libraries for genome sequencing. Fosmids are circular DNA of bacterial origin – technically plasmids – but where typical plasmids exist in high copy number (up to 100 copies per cell) and possess small (3 to 6 kb) inserts, fosmids are present as a single copy in a cell and may possess inserts upwards of 40 kb. Fosmids are advantageous because they produce stable libraries for genome sequencing. They have a tendency to provide fairly uniform coverage, so they are optimal for closing gaps in whole genome alignments. In addition to genome sequencing, they have also been used for metagenomics and expression studies.
Fosmids are derived from the fertility plasmid (or F-plasmid) and are responsible for the formation of the sex pilus during bacterial conjugation. This plasmid contains both origin and partitioning genes derived from the F’-episome and as a result, the plasmid is kept as a single copy clone, which comes in handy during genomic DNA library construction. Fosmid vectors are derived from random shearing – which yields more uniform coverage when comparing against other library cloning methods.
Cosmids may also be useful for genome sequencing projects, but unlike fosmids, they are multi-copy vectors that are generally present at anywhere from 20-70 copies per cell and this high copy number leads to instability and lost segments of genomic DNA. This can be an issue for closing gaps in genome alignment, but if you’ve got high sequencing depth and a small genome to sequence, it may not be much of an issue. Most importantly, with high copy number plasmids, such as cosmids, the chance of recombination increases which can disrupt and rearrange genomic DNA inserts prior to sequencing.
Lastly, fosmids can be useful for chromosome specific sequencing and as cytological markers for chromosome identification. The image above — which comes from this paper — shows the identification of chloroplast genome isolation and sequencing from fosmids; a similar technique can be used to isolate and sequence specific chromosomes. Also, fosmids may be used as cytological markers with in situ hybridization on metaphase karyotypes and sorted using flow cytometric methods.
There’s another series of workshops for both microbial genomics and metagenomics presented by the U.S. Department of Energy’s Joint Genome Institute in 2012. These workshops include two days of seminars and three days of hands-on tutorials for both microbial genomics and metagenomics. These workshops are centered on the use of the following bioinformatic tools: IMG, IMG/M, IMG-ER, IMG-EDU, VISTA, GREENGENES and ARB. Registration for this workshop can be found here.
A very interesting paper recently appeared in the PLOS ONE journal, authored by Flores et al. entitled “Microbial Biogeography of Public Restroom Surfaces”. This study, conducted by the Noah Fierer and Rob Knight labs at University of Colorado – Boulder, addressed the diversity of bacteria found at various places in public restrooms. The novel aspect of this research is the use of culture-independent next-generation sequencing to determine bacterial species found in discriminating locations in public restrooms.
The restroom has been one of the greatest inventions in human history – especially from a public health perspective. Without toilets and sinks – not failing to mention the plumbing infrastructure to get waste away from living spaces – disease causing bacteria (and let’s not forget other infectious organisms of the human gut, such as intestinal worms) associated with human waste easily spread from human to human, especially in close living quarters. A fascinating brief overview of the microbial history of toilets (including some great anecdotes featuring toilet visionary Sir Thomas Crapper) and a commentary of this scientific paper, written by Rob Dunn, can be found on the Scientific American Blogs site.
Using barcoded pyrosequencing of the 16S rRNA gene marker, Flores et al. observed bacterial species on ten different surface types (door handles & stall handles – both in and out, faucet handles, soap dispenser, toilet seat, toilet flush handle, floor around toilet and floor around sink) in twelve different (six male and six female) restrooms on the UC-Boulder campus on a single day.
The researchers identified 19 different bacterial phyla on all of the surfaces sampled. The majority of sequences (approximately 92%) could be placed within four phyla, including the Actinobacteria, Bacteriodetes, Firmicutes, and Proteobacteria. Human-associated bacteria were found strongly associated with restroom surfaces, which is not surprising for indoor environments.
Bacterial communities could be categorized by the surfaces they inhabited. On toilets, gut-associated bacteria were the dominant group. Skin-associated bacteria were – not surprisingly – found on surfaces touched by hands, such as door handles. The restroom floor held the greatest diversity of bacteria – some of which were found in low abundance – as these surfaces contained soil associated, as well as human associated, bacteria. Quite interestingly, the researchers found that some of the toilet flush handles contained soil associated bacteria, implying that some restroom users flush toilets with their feet to avoid directly touching the handles.
There were no statistically significant differences between bacterial communities found in female and male restrooms, although the relative abundances of some bacterial groups were gender associated. The bacterial family, Lactobacillaceae, found associated with vaginas, were – not surprisingly – more abundant in and around female restroom toilets than male counterparts.
The authors used the newly developed software package, Source Tracker, to determine the similarity of bathroom surfaces to communities from expected and previously published sources, such as human skin, the human gut, urine, soil, and faucet water. It was predicted that human skin was the primary source of restroom surface bacteria. Human gut was a source of bacteria found on and around toilets. Despite the presence of many typical soil bacterial groups found on restroom floors, soil was not identified as a statistically significant source, probably because soil typically contains a highly diverse taxonomic array of species, many of which are rare. The authors state that custodial mops and ventilation systems may also have some influence on the floor surfaces but were not directly addressed in this study.
The authors show here that human-associated bacteria are the most common microbes found in public restroom surfaces. Human influenced source patterns can be determined from the bacterial community structure within the biogeography of restrooms. This study underscores the importance of hand washing, particularly when using public restrooms, and the techniques used in this paper could be used to track or determine likely pathogenic bacteria found on surfaces during incidents of infectious outbreaks.
It’s not too late to register for the EMBO (European Molecular Biology Organization) training course in Metagenomics. The course will be held at EMBL‘s German Laboratory which is located in one of my favorite cities: Heidelberg, Germany. Here is the link for more information about the course, and an overview of the discussion topics and instructors, and how to register for the course.
It’s been a busy summer, but I’m back to focusing on some recent research. In fact, there’s been a flurry of recent papers which I plan to highlight here. I’m exploring fungal and bacterial abundance in forest soils using pyrosequencing techniques with my own research, so I was interested to read this paper on bacterial activity in oceans off the Delaware coast.
In a study from the July 18th early online edition of the journal PNAS, researchers from the University of Delaware and University of Southern California sequenced the bacteria in seawater off the Delaware coast every month over the course of three years. The research, authored by Barbara Campbell and her colleagues, measured both 16s rDNA and rRNA using next generation pyrosequencing techniques. By measuring both the presence of DNA (a marker for species presence and overall abundance) and RNA (a marker for relative activity or, more accurately, ribosome activity) in this constantly shifting ecosystem, the authors hoped to explore and understand abundance of both rare and frequently found bacteria in a coastal ocean environment. I already told you about an article featuring the Rappemonad bacteria, some of which were studied in this paper.
It has been hypothesized in ocean ecosystems that abundant bacteria are found frequently because they have high growth rates and are better at competing against slower growing bacterial. Conversely, rare bacteria have long been considered to have slower growth rates, just be poor competitors to the more abundant bacteria, or have more streamlined genomes which are better suited to wait in dormancy until the right factor, most likely a specific nutrient, comes into play.
More than 600 OTUs (Operational Taxonomic Units – a term for individuals observed from the environment) were observed and these organisms formed a typical rank abundance curve that we have come to expect from environmental sampling, so there were no surprises in that finding.
What was more surprising, or should I say interesting, was what the authors found by comparing both DNA and RNA from their samples. After the quality control of their 454 pyrosequencing reads, the authors included more than 500,000 nucleotide samples in their analysis. More than half of the individual bacteria cycled between abundant and rare during the three years of sampling. Interestingly, almost half of the bacteria were always considered rare, and close to 12 percent remained rare and inactive, and less than 5 percent were considered to be always abundant throughout the sampling. The researchers used quantitative RT-PCR to validate specific DNA and RNA concentrations for five separate OTUs to verify the findings from the pyrosequencing portion of the study.
Also quite interesting was that the authors did not observe a pronounced seasonally affected microbial component or an environmental factor that could explain the abundance or scarcity in this ocean environment. It appears by all accounts that the microbial community observed in this study is constantly changing and may not be regulated by many other factors except the community itself. See here for a press release from the University of Delaware on this study.