I have sequenced DNA data, now i would like to remove the primers from the sequence which is in fasta format.
I have tried with BioEdit s/w, had aligned using clustalw and generated contig.
In this i couldn't find my primer, i have checked with both Forward and complement.
Can someone shed light on this protocol.
If this is Sanger sequencing then there is no forward sequence of the forward primer or for the next 30 bases because the sequencing process starts from the 3'end of the sequencing primer and is very poor quality for the next 30 bases.This is normal but for sequences less than 800 bases you should see the reverse complement of the reverse primer in the forward sequence if the quality of your sequencing is good, The same applies to the reverse primer sequence when you cannot see the reverse primer
by running the PCR product in the Gel, and purification by specific kite (PCR clean-up Gel extraction - Macherey Nagel) you can ride of the primers.
If this is Sanger sequencing then there is no forward sequence of the forward primer or for the next 30 bases because the sequencing process starts from the 3'end of the sequencing primer and is very poor quality for the next 30 bases.This is normal but for sequences less than 800 bases you should see the reverse complement of the reverse primer in the forward sequence if the quality of your sequencing is good, The same applies to the reverse primer sequence when you cannot see the reverse primer
Agree with Paul Rutland Sir's answer, i need to know how would i go with the DNA trimming, where i could remove primers.
When I was doing sequencing, I used the "Applied Biosystems DNA Sequencing Analysis" software to track the outcome of each sequencing run.
To compare it with my "expected" sequence, i was importing the data from the BioEdit you mentioned, to a program called "SeqScape". In that program you are able to extract whichever region you are interested, thus "trimming" the primers as you suggested.
To achieve better quality of results, I suggest always doing a forward sequencing AND a reverse sequencing. That way you eliminating the poor quality at the beginning of the sequencing that Paul mentioned above.
I hope this helped.
Cheers,
Michalis
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