check points while preparing for sequencing - (Sep/09/2011 )
I dont understand what you mean by 2% and 8% gap, mind further elaborate? Do you clone in vector and sequence it using primers on the vector?
Adrian K on Fri Sep 9 18:00:29 2011 said:
I dont understand what you mean by 2% and 8% gap, mind further elaborate? Do you clone in vector and sequence it using primers on the vector?
I think she means that sometimes she has "2% of gaps" when sequencing and sometimes 8% ..
Not sure if this makes it more clear? She just means she has a sequence result with 2% gaps.. or 8% gaps or sometimes even more and wonders why this is.
(why no uniform results, like 4% gaps each time , for example)
So what she is asking: is there any way to predict if your sample that you are going to sequence is ok to sequence..
I think the answer is : no.
(not concidering the real exceptions)
You cant predict it and you will always have weird results or results that differ.
Of course: its possible there is something wrong with your protocol or how you handle your samples, but if you sometimes have results that give a result as low as 2% then I doubt there is a problem there.
I just think that this is science: not always the same outcome..
I mean: how many times does it happen that you read a paper, read how they did it, and then when you do it, you get a totally different result...
About the protocol: cant tell anything special about that, not used to work with that a lot.
And if I am wrong about what the 2%, 8% mean etc.. then just ignore my post.
Adrian K on Fri Sep 9 18:00:29 2011 said:
I dont understand what you mean by 2% and 8% gap, mind further elaborate? Do you clone in vector and sequence it using primers on the vector?
pito on Fri Sep 9 18:50:56 2011 said:
Adrian K on Fri Sep 9 18:00:29 2011 said:
I dont understand what you mean by 2% and 8% gap, mind further elaborate? Do you clone in vector and sequence it using primers on the vector?
I think she means that sometimes she has "2% of gaps" when sequencing and sometimes 8% ..
Not sure if this makes it more clear? She just means she has a sequence result with 2% gaps.. or 8% gaps or sometimes even more and wonders why this is.
(why no uniform results, like 4% gaps each time , for example)
So what she is asking: is there any way to predict if your sample that you are going to sequence is ok to sequence..
I think the answer is : no.
(not concidering the real exceptions)
You cant predict it and you will always have weird results or results that differ.
Of course: its possible there is something wrong with your protocol or how you handle your samples, but if you sometimes have results that give a result as low as 2% then I doubt there is a problem there.
I just think that this is science: not always the same outcome..
I mean: how many times does it happen that you read a paper, read how they did it, and then when you do it, you get a totally different result...
About the protocol: cant tell anything special about that, not used to work with that a lot.
And if I am wrong about what the 2%, 8% mean etc.. then just ignore my post.
perhaps you should give an idea what you expect and what not and the aim of the experiments. If you want to differentiate virus strains, some difference should be good (I interpret the alignment gaps as sequence differences due to mutations, slippage, or indels etc), because otherwise you could not differentiate strains at all. Or did I get it completely wrong?
hobglobin on Sat Sep 10 11:02:15 2011 said:
perhaps you should give an idea what you expect and what not and the aim of the experiments. If you want to differentiate virus strains, some difference should be good (I interpret the alignment gaps as sequence differences due to mutations, slippage, or indels etc), because otherwise you could not differentiate strains at all. Or did I get it completely wrong?
The gaps in BLAST sequences comparison mean that there are small insertions/deletions in a sequence. If this is what you refer to, I can't imagine how you could get "gaps" in the sequence other way than in the PCR step (do you use proof-reading polymerase, especialy since you do nested?) or because there actually are insertions/deletions in the original virus sequence (viruses tend to mutate a lot).
Just for the record, do you check your sequence electrophoretogram before you BLAST it to be sure it's not just an error of basecalling? Can you post an image of your sequencing output with a "gap"?
Trof on Tue Sep 13 08:23:29 2011 said:
The gaps in BLAST sequences comparison mean that there are small insertions/deletions in a sequence. If this is what you refer to, I can't imagine how you could get "gaps" in the sequence other way than in the PCR step (do you use proof-reading polymerase, especialy since you do nested?) or because there actually are insertions/deletions in the original virus sequence (viruses tend to mutate a lot).
Just for the record, do you check your sequence electrophoretogram before you BLAST it to be sure it's not just an error of basecalling? Can you post an image of your sequencing output with a "gap"?
nightingale on Wed Sep 14 21:15:12 2011 said:
I see now. You mean mismatches, where there are peaks of different nucleotides in a single position.
Now this can be caused by the things I mentioned above, but also it may be too high sequencing noise.
Do you sequence on ABI machine? Can you share the sequencing file? That would be even better than an image, it may be possible to check the raw data of the sequence.