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check points while preparing for sequencing

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10 replies to this topic

#1 nightingale

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Posted 09 September 2011 - 09:10 AM

Dear All,

i would like to ask ur honours about sequencing ...

when u r to do sequencing,, here are the steps i follow :-

1) PCR.
2) gel detection.
3') if my sample gave a single clear band, i go for PCR purification.
3'') if it gave me multiple bands, i go for gel slice purification.
4) cycle sequencing.
5) Dye-X purification.
6) denaturation & chilling.
7) sequencing.

now, in order to have a good sequencing i do :-

1) nested PCR.
2) gel detection.
3) here, i add isopropanol after adding the buffer & i go 2 times washing, & in the final step i only add 15 ul elution buffer.
4) cycle sequencing ( in which all the mix including my sample is 10 ul ).after i take my sample from the thermal cycler, i add another 10 ul nuclease free water.
5) i go for Dye-X purification to the whole 20 uls.
6) i prepare a 30 ul mix : 25ul nuclease free water + 5u from the Dey-X pure product, i do denaturation for 5 minutes at 95C then chilling for 10 minutes.
7) sequencing, adding the whole 30uls in the sequencer tube. before i run my sample i go for capillary test.

in times i get verrrryyyy nice results doing so, when i BLAST the sequence i.e :
2% gaps ...
in others .... unfortunately, NO Posted Image
8% gaps ...



& what drives me crazy, is that i do NOTHING different than what am used to ...

my question :-
how to check, if am in the right direction, before i reach the sequencing step ???
would be glad if u kindly share ur experiences.

forgive my bothering please.

grateful to the time spent reading the post.

thanks in advance,
nightingale.

Edited by nightingale, 09 September 2011 - 12:05 PM.

" The more you learn, the more you realize how little you know ... "

#2 Adrian K

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Posted 09 September 2011 - 10:00 AM

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?
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#3 pito

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Posted 09 September 2011 - 10:50 AM

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.

Edited by pito, 09 September 2011 - 10:51 AM.

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#4 nightingale

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Posted 09 September 2011 - 12:01 PM

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?


forgive me!
i mean : when u BLAST ur sequence, it will give u the gap% ..
so, sometimes it gave me 2% others 8% ...

i don't clone anything ...

i do sequencing in order to diffrentiate between virus strains ...
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#5 nightingale

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Posted 09 September 2011 - 12:16 PM


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)

forgive me please for not clarifying my point ...
i mean when u BLAST the sequence, it will give u the gaps% ...
so, we approve : 2% , 3% ... maximum : 4%
But, more than that ... we don't approve ...

the reason why i said : 2% & 8% is that:-
because 2% is acceptable to most of the ppl i think &
8% is rejected ...

so, just gave examples of acceptable vs. not acceptable gap% results ...




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.

this is the point ! that i sometimes get nice & very clean sequences & others no ...
following the same protocol ....

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...

many times ...



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.

thanks alot for trying to clarify my point to Adrian ...


i have modified the post, by adding that i BLAST the sequence ... so, it is now more clear i think ...
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#6 hobglobin

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Posted 10 September 2011 - 03:02 AM

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?
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#7 nightingale

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Posted 11 September 2011 - 12:54 AM

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?


thanks Hobglobin for ur passing ...
u kindly go for comparing sequences, after having clean sequences in the first place ... right ??
we do the very same thing ...
but, how much gap% do u approve ur sequence will have ( before comparing it with others ) ??

in my case :-
i receive field samples, in which they suspect a virus infection.
following PCR i can tell what is the virus ... but can't tell it's specific strain ...
so i go for sequencing ...
here, we try to reach a sequence with the least gap% so it will give us what is the strain when BLASTed.

after having this clean sequence, i go for making comparisons ... & differentiating strains.

i had a case yesterday ; in which it gave me 9% gaps, repeating it ... gave me 3% gaps ...
...i have done nothing different ...Posted Image
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#8 Trof

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Posted 13 September 2011 - 12:23 AM

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"?

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#9 nightingale

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Posted 14 September 2011 - 01:15 PM

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"?


thanks Trof for ur passing ...

am dealing with a virus that turns to mutate alot ...

i do of course check the electropherogram ...

to explain something :-
when u look at the sequence, you will find "Ns" ...
& when u BLAST it, you will find "gaps" ...
so, u won't be seeing real gaps in the sequence output ...

i will try to post you one ...
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#10 Trof

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Posted 15 September 2011 - 11:53 PM

thanks Trof for ur passing ...

am dealing with a virus that turns to mutate alot ...

i do of course check the electropherogram ...

to explain something :-
when u look at the sequence, you will find "Ns" ...
& when u BLAST it, you will find "gaps" ...
so, u won't be seeing real gaps in the sequence output ...

i will try to post you one ...

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.

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#11 nightingale

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Posted 16 September 2011 - 02:01 AM

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.


yes, mismatches ... on ABI 310 genetic analyzer.
i will try ... but, not sure if i can.. but will try ...
thanks again Posted Image
" The more you learn, the more you realize how little you know ... "




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