I have obtained both the forward and reverse sequences of a PCR amplicon, which contain a few ambiguous bases and gaps.
I am considering manually editing the forward sequence using the reverse complementary sequence of the reverse sequence as a reference. Is this a recommended approach for resolving the ambiguous bases?
Furthermore, I intend to conduct a phylogenetic analysis,
But generating a consensus sequence from the forward sequence and reverse complement of the reverse sequence leads to a shortened sequence, with around 50 bases deleted from the beginning and 70 bases from the end.
In light of this, would it be more appropriate to merge the forward sequence and the reverse complement of the reverse sequence into a single CONTIG sequence, instead of creating a consensus sequence?
Can this contig sequence be submitted to the NCBI database, and is it acceptable to use for phylogenetic analysis?
You have many questions and looks like there is a need for you to get some basics and fundamentals of sequence analysis and some reading.
There are many things which can be directed to you but as this is not a tutorial, you need to figure out yourself. However, NO! you can never simply replace ambiguous base.
@Abhijeet Singh Thank you for the response. I am trying to find out the possible solution for ambiguous bases.
Could you please clear the confusion on whether can I use Contig sequence for NCBI submission and phylogeny analysis?
It is not contig sequence. It would be a consensus sequence which is generated by the forward and reverse overlap it is a standard procedure in Sanger sequence analysis.
Before any submission and analysis, you should think about if and whether the sequence is correct. Why ambiguous bases. I just think it is the sequencing and processing error which should be reclaimed before moving to next step.
@Abhijeet Singh No. I was asking about making Contig sequence by merging the Forward sequence and the reverse complement of Reverse sequence together in a single sequence.
I agree with Abhijeet Singh, you need to decide if your sequence is correct. Ask yourself some basic questions. Where are your ambiguous base reads? Are they close to the primer binding site where things are messy or in the middle of the consensus sequence containing defining characters? How long are the sequences in your alignment? A thousand bases? If you shorten your alignment by 120 bases, does that affect the results of your phylogenetic analysis? Almost all alignments will have gaps and a few ambiguous bases within them. I recommend Barry Hall's book on phylogentic trees to get some basic training on alignments.
John-Erich Haight Thank you.
My PCR amplicon length was 349 bp.
My forward sequence ambiguous bases within 59th position from starting. So, I trimmed the sequence from start to 59th base.
The reverse sequence has ambiguous bases within first 81 bases. So I trimmed it from the start to 81st base. After that I generated reverse complement and did alignment of Forward and reverse complement.
Now, after alignment, I have
F: ---(77)to(349)
R: (1)to(248)---
And generating consensus sequence by Bioedit, it gives me a consensus sequence of only 172 bp (only the overlapped region).
However, generating consensus by EMBOSS Cons gives the total 349 bp sequence.
Should I take the EMBOSS Cons sequence or should I take the Bioedit one ?
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