I was doing NCBI BLAST for a sequence and I found that in BLAST result it was matching as Reverse & Complimentary. Is it normal that DNA is matching this way?
When we sequence the DNA with 27F and 1492R, what is the binding pattern of primers e.g 27F bind to sense or non sense and same for 1492R?
From the chromatograms, should we generate complimentary sequence to know sense strand sequence? If yes, then for strand amplified by 27F or 1492R?
if any reference is provided for basics of sequencing and analysis, it will increase my understanding.
Thanks..
Arvind,
as said above, sense (F) and antisense (R) is just relative to something on the DNA. Just think about the two directions as parts of the two corresponding strands of the DNA. The DNA sequence in the database (and in your mind) is just one of the two strands, but do not forget about the other strand which is also there. This strand is not shown, because one can get its sequence easily by reverse complementing the shown sequence. Half of the genes/features are coded on one of the strands and the rest is coded on the other strand. There is absolutely no difference between the two strands and it is only accidental which particular strand's sequence was chosen for being put to the database as genomic sequence. On the other hand, if you take only a small part of the DNA with only one feature, like one gene, then for the convenience the coding strand is considered, and the "F" and "R" is relative to that strand (that feature), nevertheless in half of the cases this is the opposite orientation relative to the genomic sequence in the Genbank.
Now, the other problem that you mentioned is that you can not find your primers' sequences in the result of the sequencing company. This is also completely OK, as the sequencing technology works in a way that in each "read" the first dozens of bases can not be established, and this is exactly the same region where your primer exists. The primers you provided to the company are elongated in a special reaction according to the template and these elongated molecules are then observed by the equipment that gives you the series of of those four letters. Nevertheless the quality of this information is not the same throughout the observed span. The start is garbage (here is your primer and some more) then comes some high quality part that can be several hundred bases long, in lucky cases even more than a kb, and its quality decreases gradually. In your case what you needed was longer than 800 base long reads, so that the ends of these (one of them reverse complemented) overlap with each other, and hence they together give you a 1442 long sequence span, that was established by sequencing it inward from both ends.
Unless I am mistaken about the designation of your primers, then, since the distance of the two primers were 1465 bases apart from each other and the company has given you the 1442 bases in the middle, it seems to me that you only lost 23 bases, so the ends of your primers you may be able to found as the starting and ending bases of the resulted sequence.
The sequencing company does not know (and does not care) which strand you are interested in and their software choses one of the strands in a random manner. You can then get the sequence of the other strand simply by one click in most bioinformatics software, which is much easier than telling them which strand you care about.
Arvind,
When you do a bi-directional sequence analysis, you reverse primer will go you the sequence of the complementary strand.
Usually, mutation detection software, reverse complement the reverse strand data so analysis becomes simpler. But since you use BLAST to analyse your data, it gave you the sequence that was present in your query. Did you not BLAST the sequence obtained for the other primer then?
if you use only forward primer in a sequencing reaction then the result will be matching to the sense strand of the BLAST result.
and if you are using R primer for your sequencing reaction than the Query sequence (sequence result) will be Reverse and complementary.
I think it is normal.
Hi,
Sequencing with 27F should give you the sense sequence. Indeed, 27F binds on the antisense strand of DNA and so will allow the synthesis of the sense strand during the sequencing process.
1492R primer should give you the antisense sequence, because it binds on the sense strand of DNA and then will allowto synthesize the sense strand during the sequencing process.
Concerning BLAST analysis, if you submit a sequence obtained with 27F, it should match in sense orientation if the reference sequence (subject sequence) is orientated in the sense sequence (sequences can be submitted and puplished on GenBank in antisense orientation)
Moreover, do not forget that "sense" or "antisense" orientation of a sequence is a relative concept. Indeed, one can think that for a coding sequence (gene sequence), sense sequence correspond to the "reading frame". But the reading frame of a gene can be 3' -> 5' orientated on the genome sequence... Meaning that depending on the reference sequence (for example genome sequence or transcript sequence), your queried sequence can match in both orientation
thanks all for the help within same day. Now i understand the situation in better way.
I got a sequence, but the sequence does not indicate 27f, 1492r or even their complementary because sequence provided by company is internal to primers (total 1442 bases). may be company used different primer.
The sequence from 2 primers, assembled by company is matching as reverse and complimentary to sequences in BLAST search, means company did not provide it as sense strand. Bit strange for it's being reverse and complimentary.
Hope next i will download a software to check what DNA chromatograms show, till now I was using text file being provided.
whatever it is, sense or antisense it matches with same accession numbers in BLAST search. This may be beauty of BLAST to sense it better than my confused mind. Another lesson is that matching of Forward primers to sequence make it understand that it's a sense strand, I will remember this.
I tried other forwarding primers and those match within the sequences now after converting my strand to a Sense one.
thanks David Cohen for clearing my doubt in best way..
Arvind,
as said above, sense (F) and antisense (R) is just relative to something on the DNA. Just think about the two directions as parts of the two corresponding strands of the DNA. The DNA sequence in the database (and in your mind) is just one of the two strands, but do not forget about the other strand which is also there. This strand is not shown, because one can get its sequence easily by reverse complementing the shown sequence. Half of the genes/features are coded on one of the strands and the rest is coded on the other strand. There is absolutely no difference between the two strands and it is only accidental which particular strand's sequence was chosen for being put to the database as genomic sequence. On the other hand, if you take only a small part of the DNA with only one feature, like one gene, then for the convenience the coding strand is considered, and the "F" and "R" is relative to that strand (that feature), nevertheless in half of the cases this is the opposite orientation relative to the genomic sequence in the Genbank.
Now, the other problem that you mentioned is that you can not find your primers' sequences in the result of the sequencing company. This is also completely OK, as the sequencing technology works in a way that in each "read" the first dozens of bases can not be established, and this is exactly the same region where your primer exists. The primers you provided to the company are elongated in a special reaction according to the template and these elongated molecules are then observed by the equipment that gives you the series of of those four letters. Nevertheless the quality of this information is not the same throughout the observed span. The start is garbage (here is your primer and some more) then comes some high quality part that can be several hundred bases long, in lucky cases even more than a kb, and its quality decreases gradually. In your case what you needed was longer than 800 base long reads, so that the ends of these (one of them reverse complemented) overlap with each other, and hence they together give you a 1442 long sequence span, that was established by sequencing it inward from both ends.
Unless I am mistaken about the designation of your primers, then, since the distance of the two primers were 1465 bases apart from each other and the company has given you the 1442 bases in the middle, it seems to me that you only lost 23 bases, so the ends of your primers you may be able to found as the starting and ending bases of the resulted sequence.
The sequencing company does not know (and does not care) which strand you are interested in and their software choses one of the strands in a random manner. You can then get the sequence of the other strand simply by one click in most bioinformatics software, which is much easier than telling them which strand you care about.
Please accept my thanks for spending time to write explanations to my question.
When I have next-generation sequencing done, I never tell the company what the primers - that way they can't automatically cut them out (or part of them out) and instead only remove their added primers.
Please What is the difference between sequencing done with both forward and reverse primers and that done using only forward or reverse primer?
Forward and reverse primers are used in the PCR step prior to NGS. The sequencing typically uses other primers conjugated onto your DNA.
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