I'm looking for a method to collect preliminary information on changes in the gut microbiome in mice. 16s rRNA sequencing would be the ultimate goal, but I'm looking for a way to determine if sequencing will be worth my effort and money. Is there a faster/cheaper way to determine if there are differences in numbers or kinds of bacteria (between groups)?
You can try to check for changes in total enzymatic activities, like amylases, proteases, lipases and others. If you have differential changes comparing different conditions, you probably may have changes in microbiome and decide if you want to go for metagenomic sequencing.
It depends on your budget, bioinformatic availability and intention. The DNA busting methods of RFLP, DGGE and TGGE are the traditional approach, but give limited detail afterwards. In an established lab set up they can be quick and pretty cheap.
Alternatives would be phylum-level RT-PCR or heavily multiplexed next-generation sequencing of 16S amplicons. The latter needs lots of bioinformatics support but has the advantage of scaleability- if there's a message with a small number of sequence reads, it might be worth exploring with more.
Clinically, I use the following test panel to assess the GI microbiota:
http://www.gdx.net/product/gi-effects-comprehensive-stool-analysis-profile
If you are looking to characterize your gut microbiota you can sequence a sample (fecal). They will perform a 16s RNA amplification and sequencing and compare with known libraries. Then your population can be compared to others. Take a look at using ubiome for personal microbiome analysis.
For cultivable bacteria, you can use maldi-biotyper. The identification of colony is quite fast.
As Mitchel Jones says, firstly you need to analyze at least 2 or 3 samples of gDNA extracted from heces by pyrosequencing. Thus, you can quantify just 2 or 3 of the principal bacteria in all the feces samples by quantitative PCR using the same samples (high and low for each principal bacteria) run before, as standards to make quantitation.
Looks like it that you are looking for a method that gives you some indications of the changes in the major groups of gut microbiota following a dietary intervention for example. You can try FISH (with probes mainly at genus level) first and if the changes are significant or meaningful and further studies needed, then try what others suggested. See attached for more details.
I think you could use a similar approach as described in my paper: "The Gut Microbiotassay: a high-throughput qPCR approach combinable with next generation sequencing to study gut microbial diversity" - This paper describes how it's possible to quickly screen the gut microbiota, and afterwards judge which samples (if any) you wish to sequence. Try to read it, you might be inspired ;-)
If you know the principal components of the mice microbiota, just design primers for real time PCR and run them.
a panel of TaqMan assays might work, or targeted sequencing (which is more informative and data analysis is way easier- vs discovery seq)
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Genetic Analysis
- genetic techniques
- Sequencing