Hi, and why you don't want to sequence the complete product? Cheers, Nadine
Thanks for reply..
actually there is different charges for sequencing upto 1kb and 1.5kb and our service provider is saying that they will perform primer walking. so i am confused if info. provided by 1kb sequencing will be sufficient to identify bacteria after BLAST.
Hey! Just ask your service provider for run two sequencing reactions using forward and reverse primers. If you get sequence of approx. 800 bp in each reaction you can align them to make contig of both reactions. So, by this way you can bypass primer walking step and will get 1500 bp sequence, which is good for bacterial identification.
That's exactly what I would do too. On the other hand 1 kb is sufficient information.
I would sequence the whole fragment forward and reverse (if you have the money) like others have said but depends on the gene. What is it? Did you use primers to amplify 16S rDNA? If however, your amplified fragment is in the middle of a highly conserved housekeeping gene you may need another target.
I agree with Praveen that sequencing from both ends, and making a contig is the best solution. It will give you the longest sequence, and therefore the best phylogenetic information. On the other hand, in most cases, a single read (800 bps) will be enough to nail down the bacterial species. It is only when you are dealing with less characterized bacterial groups or a novel bacterial sequence, that you might need a longer sequence to be able to get an exact answer, or to get a reliable phylogenetic tree placement.
This is going to depend on what gene you have amplified (16S rRNA or something else) and what granularity of 'identification' you need. It's possible to get to strain/ecotype level using the V1-V2 region of 16S rRNA using 454 sequencing data (~250 bp, see http://www.nature.com/ismej/journal/vaop/ncurrent/full/ismej201332a.html), so unless you have highly similar bacterial species you are trying to separate, you might not need the full 1500 bp.
If you got BLASTn hit of 98-99%, 1Kb sequence will not help you to exactly identify your strain, although you may get a clue on the identity of the strain. If you want to describe a new taxon, the referee would for sure demand the complete sequence. ;-)
A 1 kb 16S rRNA gene amplicon sequence is plenty to identify bacteria. It all depends on the resolution that you want. Most 454 studies result in ~250 bp reads and faciliate genus level identification.
Thanks Colin for ur reply.. i am using universal primer for the amplification and i got 1.5Kb PCR product.
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