I have obtained sequencing data for Forward and Reverse Primers, intending to use them in the construction of a Phylogenetic tree. Initially, I have aligned the Forward sequence with the reverse complement of the Reverse sequence. When I opt to generate a consensus sequence after alignment, I observe nucleotide symbols such as K, Y, R, H, D, M, N, etc.
Consensus Sequence:
ATCTCCGTTGCACTCTTTGCAAGCATTACCGCTTGTGGWYCGTTCGGTGGTCTGCCAAGCCTTAAAAGWWTCCTGAGCGAGAGCACAGTTCCAGGAAMAACGAAACTGTAAAAACWTTTCTTCCTTACGGAACAGTAATCAACWWWWATGGTTACATCAAACCAGWWCAAGCGCCGGACGGTTTGRCGATGGAAGCAAAAAAGCATACTACCTCTACGTTTGGGTACCTGCCGTTATCGCTGAAATGGGAGTCCGTATGATTTCCCCAACAGGCGAAATCGGTGAACCCGGCGACGGAGATTTAGTAAGCGATGCTRTCAAAGCGGCAACCCCAGAGGAAAAATCAATGCCTAACTGGTTTGATACCTGGATTCGTGTTGAAAGAATGTCAGCGATTATGCCTGACCAAATTGCAAAAGCTGCAAAGGCAAAACCAGTTCAAAAGCTGGACGATGATGATGATGGT
Should these W's, Y's, R's, M's, be removed or replaced ?
these codes are IUPAC codes for mixed bases so W is A or T, R is A or G and y is C or T. Polymorphic bases are common and the individual bases will form recognisable haplotypes . You cannot remove these bases as the comparison software will see base deletions which is not true but all phylogenic software will recognise these codes and deal with them appropriately
Dear Manohar. I have checked your consensus and I see now that it is for Leptospira mayottensis. If you get your seq data from chromato file (Sanger results) I highly recommend doubling-checking these ambiguous bases and seeing if there are any sequencing errors (by checking the highest wave). Other sequencing platforms (NGS) may have tools that polish reads if you can access the Fastq files.
These ambiguous bases are mostly due to sequencing errors. Deleting them may introduce deletion mutations that can negatively affect Phylogenetic algorithms. keeping them may introduce uncertainty but will keep the base position with a chance that one of the uncertain bases is the right one (R is A or G). For Phylogeny, it is the best action to keep them if you cannot find the right bases by genome editing or polishing.
Ibrahim H M Abualghusein Hello, i just would like to ask, instead of deleting these ambiguous sequences, is it okay to manually replace them with their probable bases? (like replacing W into A)
You can check the quality value of these bases in both forward and reverse sequences and chose the base with high Q value to replace the ambiguous codes
These letters other than GCAT indicates low quality or noisy peaks in sequences. As you have two sequences, you have to check at which nucleotide position, R,T,W etc is present. Then check peak with good quality. And replace R,T W with G, C,A,T. Your tree topology will also be improved.
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