Adjusting the MAFFT command line algorithm to better account for gaps

I've been attempting to use the MAFFT command line tool as a means to identify coding regions within a genome. My general process is to align the amino acid consensus sequence of a gene to a translated reading frame of a target sequence. My method has been largely successful. However, I've noticed some peculiar alignments which will unfortunately impede my annotation method. The following is one such example (Note - I've also included a pairwise alignment from the Pairwise2 Biopython module to demonstrate my desired output. Unfortunately, the computation time for Pairwise2 is nearly 20 times slower than MAFFT command line):

from time import *
from Bio.SubsMat import MatrixInfo as matlist
from Bio import pairwise2
from Bio.pairwise2 import format_alignment
from Bio.Align.Applications import MafftCommandline

startTime = time()

sample_tList = [['>Frame 1', 'RIGVGSIPRHLYCQELPLAQPKTCCAETPFRDSPLQGRLGVCPHLASGVALLYGLSTPLTMSGILDRCTCTPNARVFMAEGQVYCTRCLSARSLLPLNLQVPELGVLGLFYRPEEPLRWTLPRAFPTVECSPAGACWLSAIFPIARMTSGNLNFQQRMVRVAAEIYRAGQLTPAVLKVLQVYERGCRWYPIVGPVPGVGVYANSLHVSDKPFPGATHVLTNLPLPQRPKPEDFCPFECAMADVYDIGHGAVMFVAGGKVSWAPRGGDEVRFETVPEELKLIANRLHISFPPHHLVDMSKFAFIVPGSGVSLRVEHQHGCLPADIVPKGNCWWCLFDLLPPGVQNREIRYANQFGYQTKHGVSGKYLQRRLQINGLRAVTDTHGPIVVQYFSVKESWIRHFRLAGEPSLPGFEDLLRIRVESNTSPLADKDEKIFRFGSHKWYGAGKRARKARSGATTTVAHRASSARETRQAKKHEGVDANNAAHLEHYSPPAEGNCGWHCISAIVNRMVNSNFETTLPERVRPSDDWATDEDFVNTIQILRLPAALDRNGACKSAKYVLKLEGEHWTVSVAPGMSPSLLPLECVQGCCEHKGGLGSPDAVEVSGFDPTCLDRLAEVMHLPSSVIPAALAEMSNNSDRPASLVNTAWTVSQFYARHTGGNHRDQVRLGKIISLCQVIEECCCHQNKTNRATPEEVAAKIDQYLRGATSLEECLIKLERVSPPSAADTSFDWNVVLPGVEAAGPTTEQPHANQCCAPVPVVTQEPLDKDSVPLTAFSLSNCYYPAQGDEVRHRERLNSVLSKLEEVVLEEYGLMPTGLGPRPVLPSGLDELKDQMEEDLLKLANAQATSEMMALAAEQVDLKAWVKSYPRWIPPPPPPKVQPRRMKPVKSLPENKPVPAPRRKVRSDPGKSILAVGGPLNFSTPSELVTPLGEPVLMPASQHVSRPVTPLSEPAPVPAPRRIVSRPMTPLSEPTFVFAPWRKSQQVEEANPAAATLTCQDEPLDLSASSQTEYEAYPLAPLENIGVLEAGGQEAEEVLSGISDILDNTNPAPVSSSSSLSSVKITRPKYSAQAIIDSGGPCSGHLQKEKEACLRIMREACDAARLGDPATQEWLSHMWDRVDVLTWRNTSVYQAFRTLDGRFGFLPKMILETPPPYPCGFVMLPHTPTPSVSAESDLTIGSVATEDVPRILGKTENTGNVLNQKPLALFEEEPVCDQPAKDSRTLSRESGDSTTAPPVGTGGAGLPTDLPPLDGVDADGGGLLRTAKGKAERFFDQLSRQVFNIVSHLPVFFSHLFKSDSGYSPGDWGFAAFTLFCLFLCYSYPFFGFAPLLGVFSGSSRRVRMGVFGCWLAFAVGLFKPVSDPVGAACEFDSPECRNILHSFELLKPWDPVRSLVVGPVGLGLAILGRLLGGARYIWHFLLRLGIVADCILAGAYVLSQGRCKKCWGSCIRTAPNEIAFNVFPFTRATRSSLIDLCDRFCAPKGMDPIFLATGWRGCWTGQSPIEQPSEKPIAFAQLDEKRITARTVVSQPYDPNQAVKCLRVLQAGGAMVAEAVPKVVKVSAIPFRAPFFPTGVKVDPECRIVVDPDTFTTALRSGYSTTNLVLGVGDFAQLNGLKIRQISKPSGGGPHLIAALHVACSMVLHMLAGVYVTAVGSCGTGTSDPWCANPFAVPGYGPGSLCTSRLCISQHGLTLPLTALVAGFGLQEIALVVLIFVSIGGMAHRLSCKADMLCILLAIASYVWVPLTWLLCVFPCWLRWFSLHPLTILWLVFFLISVNMPSGILAVVLLVSLWLLGRYTNIAGLVTPYDIHHYTSGPRGVAALATAPDGTYLAAVRRAALTGRTMLFTPSQLGSLLEGAFRTRKPSLNTVNVVGSSMGSGGVFTIDGRIKCVTAAHVLTGNSARVSGVGFNQMLDFDVKGDFAIADCPNWQGVAPKTQFCGDGWTGRAYWLTSSGVEPGVIGDGFAFCFTACGDSGSPVITEAGELVGVHTGSNKQGGGIVTRPSGQFCNVTPIKLSELSEFFAGPKVPLGDVKVGSHIIKDTSEVPSDLCALLAAKPELEGGLSTVQLLCVFFLLWRMMGHAWTPLVAVGFFILNEVLPAVLVRSVFSFGMFALSWLTPWSAQVLMIRLLTAALNRNRVSLIFYSLGAVTGFVADLATTQGHPLQAVMNLSTYAFLPRMMVVTSPVPAIACGVVHLLAIILYLFKYRCLHHVLVGDGAFSAAFFLRYFAEGKLREGVSQSCGMSHESLTGALAIKLSDEDLDFLTKWTDFKCFVSASNMRNAAGQFIEAAYAKALRIELAQLVQVDKVRGTLAKLEAFADTVAPQLSPGDIVVALGHTPVGSIFDLKVGSTKHTLQAIETRVLAGSKMTVARVVDPTPAPPPAPVPIPLPPKVLENGPNAWGGEDRLNKRKRRRMEAVGIFVMDGKKYQKFWDKNSGDVFYEEVHNSTDEWECLRAGDPADFDPETGIQCGHVTIEDKVYNVFTSPSGRRFLVPANPENRRIQWEAARLSVEQALGMMNVDGELTAKELEKLKRIIDKLQGLTKEQCLNCPPVAPAVVAAAWLLLRQRKNFTTGPSPDLTKWPVRLSRTRSSTTNIRLPNRLMVVLCSCAPLFLRLMSSPALMHLLSYLPATGRETLGLMARFGILRPRPPKRKSHLVRKYRLVTLGAVTHLKLVSLISCTLLGATLSGKEFYRIQGLETYLTEPPVTLEAQCMRLPASRPMLLRLMGVPSWPQPCPPVLSCMYRPFQRPSLIILILGLTALNSQSTVVRMLLGTSPNTICPPKALFCLEFFALCGSTCLPMWVSARPFIGLPLTLPRILWLEMGTDFQPRIFRASLKSTFCAHRLCEKTGKLLLLVPSRSSIVGRRRLGQYLALITLRWPTGQRVVLPRASKRHSTRPSPSEKTNLRNYILQFAGALKLILHPAIDPHLQLSAGSLPIFFMNSPVLKSIYRRTCLTAVTTYWLRSPARLREAACRLATRLPPCQTPFTAYMHSTWCSVTLKVVTLMAFCFCKTSSLRTCSRFNPSSIQTTSCCMPSLPPCQITTGGLNITLCVSKRTQRRQPQTRHHFVAGMGVSSLTVTGFLRPSPTIRQAMSLNTTPRRLQYLWTAVLVSMILSGLKSSWLVRSAPARTVTASQARRSSCPCGKNSGPIMKGRSPECAGTAEPRLRTPLPVASTSVLTTPISTSIVLSSGVATRRVLALVVSVNLPWEKAQVLWMRCNKSRISLRGLSCMWSRVSPLLTQVDTKLAADSPLGVASGETKLTCQTVIMPVPPCSPLVKRSTWSLSPPTCCAAGSSSVPPALGKHTGSSNRSRMVMSFTRQLTRPCLTLGLWGCAGSTSQRVRRCNSLPPLVPARGFASWPAVGVLVRIPFWTKQRIAITLMSGFLAKPPLPAEISNNSTRWVLTLIAMFLTSCLRPNRPSGDSDRISVMPSNQITGTNLCPWSTQPVPRWTNLSGMGKSSPPTTGTERTAPSLSTPVKVPHLMWLHCICPLKIHSTGNEPLLLSPGQDMQSSCMTHTGNCRACLIFLRKAHPSTSQCSVTSSSYIEITKNARLLRLAMEINSGLQTSALILSAPFVQIWKGRAPRSPKLHITWGSISHLIHSLLNSQQNSHPTGPWQPRTMKSGLIGWLPAFAPSINIAARALVQAIWWAPRCFAPQGLCHTTSQNLLGARLKCFLRQSSAPAELRIAGSTSMIGSEKLLSPSHMPSLATSKALPVGDVITSPPDTFRASFLRNQLRSGFLAPEKLQRQFAHQMCTSQILKRTSTQRPSPSAGKCWILEKSDWSGKTRRPIFNLKAAISPGINLQATPHTSEFLLILQCIWTPAWALPFATGGLLGPPIGELTSRSPLMITVPKSFCLVHTMVKCLQGTKFWRARSSRLTTQGTNTLGDLNRIQRICTSLLGMVRTGRIIMKRFGRARKGKFIRLLPPASFIFPRALSLNQLATEMKWGLCRASLTKLVNFLWMLSRNFWCPLLISSYFWPFCLASPSPAGWWSFASDWFAPRYSVRALPFTLSNYRRSYEAFLSQCQVDIPTWGVKHPLGILWHHKVSTLIDEMVSRRMYRIMEKAGQAAWKQVVSEATLSRISNLDVVAHFQHLAAIEAETYKYLASRLPMLHNLRMTGSNVTIVYNSTLNQVFAIFPTSGSRPRLHDSQQWLIAVHSSIFSSVVASCTLFVVLWLRIPMLRSVFGFRWLGAIFLLNSRITRCVRLASPGRPLLRSMNPVGLFGAGGMTDAVRTTMTNGSWFRLASAKATPVFTPGWRSCHSATRPSSIPRYLGGTVKFMLTSRTNSFAPSTTGRTPPCLAMTTFQPYFRPTTNIRSTAVIGFTNGCAPSFPLGWFMFRGFSGVRLQAMFQFKSFRHQDQHYRSIRLCCPPGHQLPVWRLAPSDGSQELSVPHGDRDTRVHHHHSQCHRELFTFFSPHAFLLPFLCFDEKGIQSGIWQCVRHRGCVCLYQLRPTCQGVHPTLLGSRSCATASFHDTDHEVGNRFSLSFCHPTGNLNVQVCWGNAPRAVTRNCFLCGVSCRSVLLCSSTPAATAALIFSFITRYVSMAQIGWQKDLTGQWRLLSFFLCLTLFPMEHSPPAIFLTRLVSLCPPPGSITGGMSVVSMRSVLWLRFASSLGLRRTACPGATLVLDTPTSFWTLRADSIVGGRPLLRKGVRLKSRVTSTSKELCLMVPWQPLPEFQRNNGVVSRRLLPHGSTKGAFGVFHYLYASDDICSKGKSRPTARASAPFDLPELCFYLRVHDIRALSEHKGRAHYGGSSCTSLGGVLSHRNLEIHHLQMPFVLARPQVHSGPCPPRRKCRGLSSDCGKPRICRPASRLHYGRHIGARVEKPRVGWQKSCTGSGKPCQICQITTASSKRERRGTASQSISCARCWVRSSPNKTSPEARDRGRKIIREARRSPIFLRLKKMSGTTSPLVSGNCVCRRSRLPLTRAPGHVPCQIQGGVTLWSLVCRRIILCASASQHHPQHDELAFFGHLGVMIGRMCGEWHLTLCLVTYSIRATVWGSLIGENHAAAIKKKKKKKK'], ['>ORF2_GP2', 'MKWGLCKASLTKLANFLWMLSRSFWCPLLISSYFWPFCLASQSPVGWWSFASDWFAPRYSVRALPFTLSNYRRSYEAFLSQCQVDIPTWGVKHPLGVLWHHKVSTLIDEMVSRRMYRIMEKAGQAAWKQVVSEATLSRISGLDVVAHFQHLAAIEAETCKYLASRLPMLHNLRLTGSNVTIVYNSTLDQVFAIFPTPGSRPKLHDFQQWLIAVHSSIFSSVAASCTLFVVLWLRIPMLRSVFGFRWLGATFLLNSW']]

ex_file = open("newTempFile112233.fasta", "w")

for items in sample_tList:
    ex_file.write(items[0] + "\n")
    ex_file.write(items[1] + "\n")

ex_file.close()

in_file = '.../msa_example.fasta'
mafft_exe = '/usr/local/bin/mafft'

mafft_cline = MafftCommandline(mafft_exe, input=in_file) #have to change file path
#mafft_cline = MafftCommandline(mafft_exe, input=in_file, localpair=True, lexp=-1.5, lop=0.5)

stdout, stderr = mafft_cline()
print(stdout)
test_align = AlignIO.read(io.StringIO(stdout), "fasta")
#print(test_align)

os.remove("newTempFile112233.fasta")

print('Total time = ' + str(time() - startTime))
startTime = time()

matrix = matlist.blosum62 

pWise_align = pairwise2.align.localds(sample_tList[0][1], sample_tList[1][1], matrix, -6, -1)
print(format_alignment(*pWise_align[0]))

print('Total time = ' + str(time() - startTime))

I've attempted to change the MAFFT command line alignment algorithm by referencing the help document (http://mafft.cbrc.jp/alignment/software/manual/manual.html). I don't get any error messages, but the alignment output does not change. I'm unsure what adjustments need to be made. I believe that by increasing the gap extension penalty (which is zero by default), the alignment will be improved. I haven't been able to find many documentation examples where custom variables are used when using MAFFT command line on this forum or through Google search. Help is much appreciated. For reference, documentation on the Pairwise2 alignment parameters can be found here: http://biopython.org/DIST/docs/api/Bio.pairwise2-module.html

Managed to figure out a possible solution. The alignment of the example sequences provided results in a long terminal/end gap which should not be present. Changing the MAFFT alignment algorithm using localpair, lexp, and lop had no effect (causing me a good deal of confusion). However, I have noticed differences in the alignment output when each input sequence is reversed. Oddly, the only way I was able to remove the terminal/end gap was to set the lop (gap opening penalty) to a lesser amount relative to lexp (gap extension penalty). I suspect my solution is niche and may not be applicable to other similar occurrences of terminal gaps. Changing the alignment settings also likely reduces the optimal alignment.

Going forward, I plan to use an automated process to run alignments of consensus sequences to raw sequences. In the event I detect irregularities with the alignment output (specifically terminal gaps), I'll attempt to reverse the input sequences and apply custom alignment settings. I suppose if that isn't a consistent solution, I'll figure out a way to refine the alignment output directly.

For anyone curious, I used a lexp value of -1.5 and lop value of 0.5 (now included in a hashed out line in my example code).