Hi All,

I am trying to generate a codon-aware nucleotide alignment using the protein alignment (output from Orthofinder) and their respective nucleotide sequence.

For that I am using pal2nal with the following command:

perl pal2nal.pl pep_algn.fa nucl_seq.fa -nogap -nomismatch -output palm > nucl_algn.fa

That works for 4 out of the 36 alignments that I am testing. For all the other sequences I get this error message:

#---  ERROR: inconsistency between the following pep and nuc seqs  ---#

I get the same error message if I run it on the pal2nal website.

Here is one example of sequences resulting in this error message:

>Edil_01564-mRNA
MEQLGEGQAVVVGSAGGTVQVVQMGQGGQAMMLPQAIQVAAPNGQIQVVPVSSLTSTGQQ
IVIQQPQTPQIIQTPDGQTYIYQPVQLEGQVQQAQPTVININGNLMQIAGTTSQTTTTAA
TTTPVQPLASPTATASQAGNVVMMVPGNSGQTQFQRVALPNAEVFEEEPLVNAKQYRRIL
KRRQARAKLEAEGKIPKERPKYLHESRHRHAMNRIRGEGGRFHSGQVKKRNRINENAMIT
QHITTSTSTNTVRTIAIAAANVGVQYHDTDNMASTIVIEKQGIPLQDMISEDDIVTSNNC
LM

>Edil_01564-mRNA
ATGGAACAACTGGGAGAAGGGCAGGCGGTTGTGGTTGGTAGTGCCGGAGGAACCGTCCAAGTTGTTCAGATGGGCCAAGGAGGCCAAGCAATGATGTTGCCACAAGCTATACAAGTTGCAGCACCAAATGGACAAATAC
AAGTTGTTCCTGTATCTAGCTTAACCAGTACAGGTCAACAAATTGTAATTCAACAACCGCAAACACCTCAAATAATCCAAACTCCAGATGGACAAACTTATATTTATCAACCAGTTCAGTTGGAAGGTCAAGTTCAACA
AGCACAACCAACAGTAATTAATATCAATGGGAATCTTATGCAAATTGCTGGAACTACGTCACAAACCACAACCACAGCAGCAACCACAACACCAGTACAACCATTAGCGAGTCCTACAGCTACAGCGTCTCAGGCAGGA
AATGTTGTCATGATGGTGCCAGGAAATAGCGGGCAGACACAATTTCAAAGGGTAGCATTGCCAAATGCAGAAGTTTTTGAAGAAGAACCTTTATAAGTAAATGCTAAACAATATAGACGTATATTAAAACGCCGTCAAG
CTCGGGCAAAATTAGAAGCTGAAGGAAAAATACCTAAGGAAAGACCTAAATACCTTCATGAATCGCGTCATCGGCATGCCATGAACAGAATTCGGGGTGAAGGTGGTCGTTTTCATTCTGGTCAAGTAAAAAAGCGAAA
TAGAATAAACGAAAACGCCATGATTACCCAGCACATCACAACTTCGACCAGCACCAATACCGTTCGTACTATAGCAATAGCAGCAGCAAATGTAGGTGTTCAGTATCATGACACAGATAATATGGCCTCTACAATTGTC
ATCGAAAAACAAGGTATTCCTCTTCAAGATATGATCTCTGAAGACGATATCGTTACCTCAAACAATTGTTTAATG

I run blastx to see the inconsistency and it seems to be one stop codon that was removed in the protein alignment but was kept in the nucleotide sequence. I understand that pal2nal is able to deal with gaps and mismatches between the protein and nucleotide sequences. But even though I include these options in the command line, it doesn't apply.

Does anyone have had similar issues? I really appreciate any thoughts on that.

Thank you,
Priscila