Hi there!! I want to do quality check some Sanger sequence reads and realized that the reads contain some odd letters (N, K, Y, B etc) different from the normal 4 DNA base letters (ATGC). What can i do to edit away these strange nucleotides represented by the strange letters?
These letters represent multiple bases and can be polymorphism sor mutations and are important or just area of poor sequencing and are telling you to not trust sequence in this area if there are many Ns.Either way they are possibly important and should either be left alone or your sequencing improved if there are too many as the best way of dealing with them. Ns at the very beginning of the sequence and when the sequence is finished at the end of the sequence are not important
These are not strange nucleotides, but they represent ambiguous peaks at those positions, and polymorphism. Each of these letter specific combinations of signals and are defined in the IUPAC list of ambiguous nucleotides. You need to check these positions in your sequence electrpherograms and confirm. If you have peaks of comparable intensity, you may retain these letters while submitting your sequences to the GenBank. GenBank accept sequences with IUPAC codes. Fllow the link while revising your sequences:
https://www.bioinformatics.org/sms/iupac.html
SD
After going through the IUPAC nucleotide code list, everything now makes sense. It shows that the ambiguous positions could be mutations. Now the purpose of my analysis is to identify SNPs relative to reference sequences. How do I proceed with all the ambiguous nucleotide positions in almost every sequence. ?
Hi Nebangwa,
Probably, your secuence could be heterogeneous, this means that you could have different sequencing products, and as a result, the sequencing program can't discrimine them. If this is the case, you have to perform some improvements in your pcr program in order to obtain a single and homogeneous product. another option is that you could have a poor concentration of DNA, and all the peaks that you see could be noise.
Can you attached an image of the electropherogram of your sequence?
I do have a similar question and i did get lot of help from your question.
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