Human Microbiome journal clubMarch 17th 2011
Jennifer StearnsPostdoctoral fellow
Farncombe Family Digestive Health Research InstituteMcMaster University
A second primer talk
• Current work• Favourite talks• Things to consider
Currently…
• A large emphasis on culture-independent studies – especially metagenomics
• A focus on optimizing protocols and ferreting-out both trends and sources of error
• Large-scale sample collection
• Sequencing• 454, Illumina,
IonTorrent• Phylochips• Culturing• Microfluidic• Bioreactor
Many body sites have been targeted
• Obesity• IBD• CF• Diabetes• Age• Diet • Newborns• Pregnancy • Vaccines• Nationality• Probiotics• Longitudinal studies (stable or
variable over time?)• Viriome
Skin
Airways
Oral cavity
VaginaForeskin
Heart plaqueGI tract
Distal colon/stool
Models in mice
• Germ-free• Humanized – fecal
transplant, transgenic, etc…• Can do more complete
longitudinal studies• Long-term effects of
antibiotics• Colonizaiton resistance• Normal variation in the
system• Variables are far easier to
control
Types of data
• 16S rRNA – for community studies• Whole sample shotgun sequence – for metagenomic studies• WGS of host – for correlation of host genetic factors to
differences in the microbiome• Reference genomes – of cultured organisms and single cells• RNA sequencing and PCR – for microbial gene expression
within a sample• Metabolic studies – to measure function of the bacterial
community
Reference genomes
• Sequencing complete genomes for both environmental and human associated strains
• Can be used as a framework for assembly of metagenomic sequences
• Used to study LGTThe Human Microbiome Jumpstart Reference Strains Consortium
Science 2010;328:994-999
What to do with all the data
http://hmjournalclub.wordpress.com/tips-tricks-tools-and-resources
Things to consider
• In terms of 16S rRNA sequencing, there is considerable variability between people• This is not surprising considering the variables involved
(i.e. genetics, lifestyle, diet, health…)• A the functional level the microbiome seems to have
core functions but even here there are few studies that see clear trends or “smoking guns”
Often no obvious trends – why?
• Analysis of the HMP data• 16 body sites on 103 individuals… no core microbiome
at the “species” level yet there is a functional core, but there is enzyme diversity at each functional pathway
• MetaHit – similar results• Still sifting through the data• Still developing tools and standards• No quantitative DNA isolation• Inability to remove environmental DNA
Things to consider
It is estimated that there are millions of microbial species on the planet, relatively few of which have been isolated in culture. Despite the recognized importance of microorganisms, we still know little about the magnitude and variability of microbial biodiversity in natural environments relative to what is known about plants and animals. This is a major knowledge gap, given that microbes are critical components of our planet, responsible for key ecosystems services including the production of agriculturally critical small molecules, the degradation of environmental contaminants, and the regulation of human host phenotypes.
Sharpton et al., 2001 PLOS Comp. Biol. 7(1): e1001061
Things to consider
• With large differences it’s easy to see trends• The question becomes: why use high throughput
sequencing when another method might be just as informative?
• Variation is often subtle, this creates a few situations:• Are the differences due to real biological effects/causes?• How much variation is introduced by your sampling,
sequencing and analysis methods?
Lessons?
• We should have clear questions that we want to answer with our microbiome studies
• Good experimental design to increase chances of getting answers
• Much of the data analysis relies on databases populated with reliable reference material from studies of the organisms and enzymes themselves
Thank you for your interestPlease sign up to present a paper
Visit the blog at hmjournalclub.wordpress.com
Dennis Kunkel