My lab has done 16S and whole genome shotgun on mouse gut/fecal microbiome (wild-type vs knockout). Recently we found that the culturable part of these knockout mouse fecal samples are actually effective in our following assays. So we'd like to further analyze the abundance and composition difference of the culturable microbiome (wild-type vs knockout) at species level.
I could not find much references for this kind of "metagenome" NGS.
Some questions to discuss here:
Assuming there are fewer species of culturable bacteria (compared with fecal samples ), which approach are we supposed to use? 16S or whole genome shotgun sequencing? What kind of NGS depth? How many biological samples are needed to see the difference?
Your input would be appreciated.
If you have cultured isolates then 50X coverage for NGS will give a reasonable genome assembly for most bacterial isolates and allow identification
Sanger sequencing of 16S from each isolate would provide a guide to the diversity that you have, but would not provide differentiation of E. coli strains that could be gained from WGS of isolates.
Thanks for the reply. The sequencing I mentioned is not to do cultured isolate NGS for genome assembly. What we need to find is the differences between two sources of the fecal content microbiota in several days of liquid culture.
16S seq wouldn't provide you enough resolution to compare your results (with cultured strains). WGS would provide you information at species or even strain level (using marker genes by using softwares like Metaphlan https://bitbucket.org/biobakery/biobakery/wiki/Home). It's difficult to predict how many samples (replicates) are required to catch the differences. A good approach is to run 16S seq on a higher number of replicates. This helps you to see how many samples you need, what are the inter-individual differences, etc., and most importantly to use the 16S data to choose the most representative samples for each of your treatment groups and then perform WGS on those representative samples. There are tools for picking the most representative samples from 16S data such as MicroPita found in Galaxy http://huttenhower.sph.harvard.edu/galaxy/. As for the depth of sequencing, the rule of thumb is "the higher, the better coverage and resolution". But if you are not interested in profiling the metagenome (metabolic pathways), then you don't need to achieve very high depth which will let you save money. Hope it helps.
Thanks for all the replies. Those are helpful. For this particular project, I've decided to go with 16S first and then WGS. We have used Metaphlan but I was not aware of MicroPita. I am still interested in the metabolic pathways but I thought that should be done with metatransciptome?
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