the best protocol for analyzing 16S rRNA gene to identify bacterial strain
Hi, you need to determine the primers that would be appropriate for amplifying the 16S rRNA. Although there are universal primers, sometimes it depends on the bacterial group that you are looking for.
Once determining the primers to use, you can also check the PCR protocol too. This would be dependent what kind of reagents or kits you are using. So please check.
Once you successfully amplified the 16S rRNA, you need to have it sequenced. Once you get the sequence you need to clean it and make sure it is of good quality. You can presumptively try to get the identification of your sample by doing BLAST. However, this is only for presumptive identification. What you need to do next is to get the type sequences of the bacterial group you are interested in and do a simple phylogenetic analysis to determine which bacterial species is close to your sample.
There are many pieces of literature out there that provide the detailed steps in using the 16S rRNA for determining bacterial identification. You can check out some of my publication that dealt with Methylobacteirum group.
Good luck!
Hi, I agree with Thaddeus but would add a few comments. The utility of the 16s rRNA gene for identification to the species level varies according to the phylogenetic taxon of the bacteria you are analyzing. Some families and genera have relatively little variation accross species in the 16s. A good example of this would be Mycobacterium spp. For these families and genera, it will be very useful to sequence portions of multiple genes. There are a number of reference (housekeeping) genes that are useful, but again their utility varies somewhat from genus to genus. Consider looking at ropB, 16s, and gyrB as a place to start. For strain-level (subspecies/biotype) identification you will almost always need to analyse multiple gene sequences. This method of bacterial strain identification is called multilocus sequence typing. Good luck!
There are many options to do this analysis, one way is use RDP database (https://rdp.cme.msu.edu/). If possible use multi locus sequence type to have a better identication .
I agree with the comments by Rodrigo. You have to consider the group you are looking at since some researchers have made progress in doing the molecular based identification. MLST is another approach too. Please do check the species or genera group.
However if you dont know a priori the kind of species you are dealing with. Meaning you need to know what could be the diversity. Then that's another story.
Best of luck
good primers are 341F-805R, they amplify the V3-V4 variable region as you can see in the following figure
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Microorganisms
- Bacteria