From a sample of 10 marine bacteria I was able to succesfully identify these bacteria down to genus level using only one primer (forward). II then sent for the reverse primer to be sequenced. When I aligned the two sequences and carried out all neccessary bioinformatic procedures, I unfortunately, for all samples could not identify down to species level, instead the futhest confirmed identification I could make was down to genus level, again. Using both primer sequences did not further my results, as opposed to only using just one primer. Is there a reason as to why this would be?
Dear Catherine,
We have recently cloned the 16S region of several putative pseudomonads by PCR into a plasmid, and then determined the sequence using plasmid-specific primers to produce a single contig. As there is very little sequence variation within this 16S region amongst the pseudomonads, it made little difference to our BLAST analyses if we used the first 300 n, the last 300 n, or the whole sequence. You may have a similar problem - with so little sequence variation in the 16S region you may simply not be able to identify your strains below genus level. You may be able to improve your phylogenetic resolution by sequencing beyond the common region of 16S. Regards, Andrew.
Dear Catherine,
We have recently cloned the 16S region of several putative pseudomonads by PCR into a plasmid, and then determined the sequence using plasmid-specific primers to produce a single contig. As there is very little sequence variation within this 16S region amongst the pseudomonads, it made little difference to our BLAST analyses if we used the first 300 n, the last 300 n, or the whole sequence. You may have a similar problem - with so little sequence variation in the 16S region you may simply not be able to identify your strains below genus level. You may be able to improve your phylogenetic resolution by sequencing beyond the common region of 16S. Regards, Andrew.
Increase the length of the PCR product (of 16S) to more than 2000 bp and sequence the entire length using both Forward and Reverse primers. If necessary, make one more sequence primer in the middle of the region to get proper and complete sequence. This could help to identify the bacteria in species and subspecies level.
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