Macie Duran Week 4

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Macie M. Duran

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Purpose

The purpose of this assignment is to gain an understanding of how a biological database such as GenBank works, and to use it to access nucleotide and amino acid sequences for further analysis.

Method & Results

Part 1: GenBank

  • I used GenBank to find the complete nucleotide sequence for SARS-like coronavirus WIV16 from the Data & Resources section of the Week 4 assignment page.
  • I viewed the full record and FASTA file for the sequence.
    • The accession number is KT444582.
    • The GenBank record provided:
      • Host: Rhinolophus sinicus (Chinese rufous horseshoe bat)
      • Isolation source: feces
      • Country: China
      • Collection date: 21-Jul-2013
    • I downloaded the nucleotide sequence file in FASTA format, and viewed it in Microsoft Word to confirm that the sequence was correct.
  • Next, I found the spike protein sequence for the SARS-like coronavirus WIV16. I downloaded it in FASTA format and opened it in Microsoft Word.
    • The accession number is ALK02457.
    • The sequence is:
>ALK02457.1 spike protein [SARS-like coronavirus WIV16]
MFIFLFFLTLTSGSDLESCTTFDDVQAPNYPQHSSSRRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFH
TINHRFDNPVIPFKDGVYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAV
SKPTGTQTHTMIFDNAFNCTFEYISDSFSLDVAEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLP
SGFNILKPIFKLPLGINITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPATFMLKYDENGTITDAVDCSQ
NPLAELKCSVKSFEIDKGIYQTSNFRVAPSKEVVRFPNITNLCPFGEVFNATTFPSVYAWERKRISNCVA
DYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFTGCV
LAWNTRNIDATQTGNYNYKYRSLRHGKLRPFERDISNVPFSPDGKPCTPPAFNCYWPLNDYGFYITNGIG
YQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVLDFTD
SVRDPKTSEILDISPCSFGGVSVITPGTNTSSEVAVLYQDVNCTDVPVAIHADQLTPSWRVYSTGNNVFQ
TQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSSLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNF
SISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAVEQDRNTREVFAQVKQM
YKTPTLKDFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGL
TVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFN
KAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLIT
GRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYV
PSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGSCDVVIGIINNTVY
DPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ
YIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGVKLHYT
  • I added the spike protein sequence to the Talk page, where the rest of the class also compiled their sequences.

Part 2: Creating a phylogenetic tree with Phylogeny.fr

  • I navigated to Phylogeny.fr. I scrolled to the section titled 'Phylogeny Analysis' and selected 'One Click'.
  • I copied the list of sequences on the talk page and pasted them into the text field labeled 'Upload your set of sequences in FASTA, EMBL, or NEXUS format’. Then, I clicked 'Submit'.
  • I found the numbered tabs located beneath the text One Click Mode, and selected tab 3. Alignment.
    • The alignment was color coded to indicate conservation among the sequences. Blue indicated high conservation, grey indicated lower conservation, and white indicated very little, if any, conservation.
  • Under the section labeled Outputs, I selected Alignment in Clustal Format. The alignments displayed in a text format with symbols indicating conservation, rather than a color code. "*" indicates invariant conservation, ":" indicates high conservation, "." indicates weak conservation, and a space indicates no conservation.
  • I copied the sequence alignment text and have included it here:
QDF43825.1      ---------MKLLVLV-----FATLVSSYTIEKCTDFD------DRTPPSNTQFLSSHRG
AGZ48818.1      ---------MKLLVLV-----FATLVSSYTIEKCLDFD------DRTPPANTQFLSSHRG
ALK02457.1      ----------MFIFLF-----FLTLTSGSDLESCTTFD------DVQAPNYPQHSSSRRG
AAS10463.1      ----------MFIFLL-----FLTLTSGSDLDRCTTFD------DVQAPNYTQHTSSMRG
AAP13441.1      ----------MFIFLL-----FLTLTSGSDLDRCTTFD------DVQAPNYTQHTSSMRG
AAP13567.1      ----------MFIFLL-----FLTLTSGSDLDRCTTFD------DVQAPNYTQHTSSMRG
QHD43416.1      ----------MFVFLV-----LLPLVSSQ----CVNLT------TRTQLPPAYTNSFTRG
AVP78031.1      -----------MLFFL-----FLQFALVN--SQCVNLT------GRTPLNPNYTNSSQRG
ABD75323.1      --------MKILIFAF-----LVTLVKAQ--EGCGVIN------LRTQPKLTQVSSSRRG
QDF43835.1      --------MKVLIVLL-----CLGLVTAQ--DGCGHIS------TKPQPLLDKFSSSRRG
ABD75332.1      --------MKVLIFAL-----LFSLAKAQ--EGCGIIS------RKPQPKMEKVSSSRRG
QDF43820.1      --------MKILIFAF-----LVTLVEAQ--EGCGIIS------RKPQPKMAQVSSSRRG
AAZ67052.1      --------MKILILAF-----LASLAKAQ--EGCGIIS------RKPQPKMAQVSSSRRG
AFS88936.1      ----MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKA-DG
YP_0010399      MTLLMCLLMSLLIFVRGCDSQFVDMSPASNTSECLESQVDAAAFSKLMWPYPIDPSKVDG
                           ::.          .        *                     .   *

QDF43825.1      VYYPDDIFRSNVLHLVQDHFLPFDSNVTRFITFGLN-------------FDN---PIIPF
AGZ48818.1      VYYPDDIFRSNVLHLVQDHFLPFDSNVTRFITFGLN-------------FDN---PIIPF
ALK02457.1      VYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHR-------------FDN---PVIPF
AAS10463.1      VYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHT-------------FDD---PVIPF
AAP13441.1      VYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHT-------------FGN---PVIPF
AAP13567.1      VYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHT-------------FDN---PVIPF
QHD43416.1      VYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVS------GTNGTKRFDN---PVLPF
AVP78031.1      VYYPDTIYRSDTLVLSQGYFLPFYSNVSWYYSLTTN-------NAATKRTDN---PILDF
ABD75323.1      VYYNDDIFRSDVLHLTQDYFLPFHSNLTQYFSLNIE-------SDKIVYFDN---PILKF
QDF43835.1      VYYNDDIFRSDVLHLTQDYFLPFDTNLTRYLSFNMD-------SATKVYFDN---PTLPF
ABD75332.1      VYYNDDIFRSDVLHLTQDYFLPFDSNLTQYFSLNID-------SNKYTYFDN---PILDF
QDF43820.1      VYYNDDIFRSDVLHLTQDYFLPFDSNLTQYFSLNVD-------SDRYTYFDN---PILDF
AAZ67052.1      VYYNDDIFRSNVLHLTQDYFLPFDSNLTQYFSLNVD-------SDRFTYFDN---PILDF
AFS88936.1      IIYPQGRTYSNITITYQGLF-PYQGDHGDMYVYSAG--HATGTTPQKLFVANYSQDVKQF
YP_0010399      IIYPLGRTYSNITLAYTGLF-PLQGDLGSQYLYSVSHAVGHDGDPTKAYISNYSLLVNDF
                : *      *.      . * *   :                         :       * 

QDF43825.1      RDGVYF----AATEKSNVIRG-------------WVFGSTMNNKSQ---------SVIIM
AGZ48818.1      KDGIYF----AATEKSNVIRG-------------WVFGSTMNNKSQ---------SVIIM
ALK02457.1      KDGVYF----AATEKSNVVRG-------------WVFGSTMNNKSQ---------SVIII
AAS10463.1      KDGIYF----AATEKSNVVRG-------------WVFGSTMNNKSQ---------SVIII
AAP13441.1      KDGIYF----AATEKSNVVRG-------------WVFGSTMNNKSQ---------SVIII
AAP13567.1      KDGIYF----AATEKSNVVRG-------------WVFGSTMNNKSQ---------SVIII
QHD43416.1      NDGVYF----ASTEKSNIIRG-------------WIFGTTLDSKTQ---------SLLIV
AVP78031.1      KDGIYF----AATEHSNIIRG-------------WIFGTTLDNTSQ---------SLLIV
ABD75323.1      GDGVYF----AATEKSNVIRG-------------WVFGSTFDNTTQ---------SAIIV
QDF43835.1      GDGIYF----AATEKSNVVRG-------------WIFGSTMDNTTQ---------SAIIV
ABD75332.1      GDGVYF----AATEKSNVIRG-------------WIFGSSFDNTTQ---------SAIIV
QDF43820.1      GDGVYF----AATEKSNVIRG-------------WIFGSTFDNTTQ---------SAVIV
AAZ67052.1      GDGVYF----AATEKSNVIRG-------------WIFGSTFDNTTQ---------SAVIV
AFS88936.1      ANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLL
YP_0010399      DNGFVVRIGAAANSTGTIVISPSVNTKIKKAYPAFILGSSLTNTSAGQ-PLYANYSLTII
                 :*. .    *:.. ..:: .             :::*::. . :          :  ::

QDF43825.1      NNSTNLVIRACNFELCDNPFFVVLRSNNTQIPSY------IFNNAFN-CTFEYVSKDFNL
AGZ48818.1      NNSTNLVIRACNFELCDNPFFVVLKSNNTQIPSY------IFNNAFN-CTFEYVSKDFNL
ALK02457.1      NNSTNVVIRACNFELCDNPFFAVSKPTGTQTHTM------IFDNAFN-CTFEYISDSFSL
AAS10463.1      NNSTNVVIRACNFELCDNPFFVVSKPMGTRTHTM------IFDNAFN-CTFEYISDAFSL
AAP13441.1      NNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTM------IFDNAFN-CTFEYISDAFSL
AAP13567.1      NNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTM------IFDNAFN-CTFEYISDAFSL
QHD43416.1      NNATNVVIKVCEFQFCNDPFLGVYY--HKNNKSWMESEFRVYSSANN-CTFEYVSQPFLM
AVP78031.1      NNATNVIIKVCNFDFCYDP-YLSGY--YHNNKTWSIREFAVYSSYAN-CTFEYVSKSFML
ABD75323.1      NNSTHIIIRVCYFNLCKDPMYTVSA--GTQKSSW------VYQSAFN-CTYDRVEKSFQL
QDF43835.1      NNSTHIIIRVCYFNLCKEPMYAISN--EQHYKSW------VYQNAYN-CTYDRVEQSFQL
ABD75332.1      NNSTHIIIRVCNFNLCKEPMYTVSK--GTQQSSW------VYQSAFN-CTYDRVEKSFQL
QDF43820.1      NNSTHIIIRVCNFNLCKEPMYTVSR--GTQQSSW------VYQSAFN-CTYDRVERSFQL
AAZ67052.1      NNSTHIIIRVCNFNLCKEPMYTVSR--GAQQSSW------VYQSAFN-CTYDRVEKSFQL
AFS88936.1      PDGCGTLLRAFYCIL--EPRSGNHCPAGNSYTSF-----ATYHTPATDCSDGNYNRNASL
YP_0010399      PDGCGTVLHAFYCIL--KPRTVNRCPSGTGYVSY-----FIYETVHNDCQ-STINRNASL
                 :.   ::..    :  .*             :        : .  . *     .    :

QDF43825.1      DIGEKPGNFKDLREFVFRNKDG--------FLHVYSGYQPISAASGLPTGF--NALKPIF
AGZ48818.1      DLGEKPGNFKDLREFVFRNKDG--------FLHVYSGYQPISAASGLPTGF--NALKPIF
ALK02457.1      DVAEKSGNFKHLREFVFKNKDG--------FLYVYKGYQPIDVVRDLPSGF--NILKPIF
AAS10463.1      DVSEKSGNFKHLREFVFKNKDG--------FLYVYKGYQPIDVVRDLPSGF--NTLKPIF
AAP13441.1      DVSEKSGNFKHLREFVFKNKDG--------FLYVYKGYQPIDVVRDLPSGF--NTLKPIF
AAP13567.1      DVSEKSGNFKHLREFVFKNKDG--------FLYVYKGYQPIDVVRDLPSGF--NTLKPIF
QHD43416.1      DLEGKQGNFKNLREFVFKNIDG--------YFKIYSKHTPINLVRDLPQGF--SALEPLV
AVP78031.1      NISGNGGLFNTLREFVFRNVDG--------HFKIYSKFTPVNLNRGLPTGL--SVLQPLV
ABD75323.1      DTSPKTGNFTDLREFVFKNRDG--------FFTAYQTYTPVNLLRGLPSGL--SVLKPIL
QDF43835.1      DTAPQTGNFKDLREYVFKNKDG--------FLSVYNAYSPIDIPRGLPVGF--SVLKPIL
ABD75332.1      DTAPKTGNFKDLREYVFKNKGG--------FLRVYQTYTAVNLPRGFPAGF--SVLRPIL
QDF43820.1      DTAPKTGNFKDLREYVFKNRDG--------FLSVYQTYTAVNLPRGLPIGF--SVLRPIL
AAZ67052.1      DTAPKTGNFKDLREYVFKNRDG--------FLSVYQTYTAVNLPRGLPIGF--SVLRPIL
AFS88936.1      NSFKE---YFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFA
YP_0010399      NSFK---SFFDLVNCTFFNSWDITADETKEWFGITQDTQGVHLYSSRKGDLYGGNMFRFA
                :       :  * : .*    .         :   .    :    .   .:  . :  : 

QDF43825.1      KLPLGINITNFRTLLTAF------PPNPGYWGTSAAAYFVGYLKPTTFMLKYDENGTITD
AGZ48818.1      KLPLGINITNFRTLLTAF------PPRPDYWGTSAAAYFVGYLKPTTFMLKYDENGTITD
ALK02457.1      KLPLGINITNFRAILTAF------LPAQDTWGTSAAAYFVGYLKPATFMLKYDENGTITD
AAS10463.1      KLPLGINITNFRAILTAF------SPAQDTWGTSAAAYFVGYLKPTTFMLKYDENGTITD
AAP13441.1      KLPLGINITNFRAILTAF------SPAQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITD
AAP13567.1      KLPLGINITNFRAILTAF------SPAQDTWGTSAAAYFVGYLKPTTFMLKYDENGTITD
QHD43416.1      DLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITD
AVP78031.1      ELPVSINITKFRTLLTIHRGD---PMPNNGWTAFSAAYFVGYLKPRTFMLKYNENGTITD
ABD75323.1      KLPFGINITSFRVVMAMF------SKTTSNYVPESAAYYVGNLKQSTFMLSFNQNGTIVD
QDF43835.1      KLPIGINITSFKVVMSMF------SRTTSNFLPEVAAYFVGNLKYSTFMLNFNENGTITD
ABD75332.1      KLPFGINITSYRVVMTMF------SQFNSNFLPESAAYYVGNLKYTTFMLSFNENGTITD
QDF43820.1      KLPFGINITSYRVVMAMF------SQTTSNFLPESAAYYVGNLKYTTFMLRFNENGTITD
AAZ67052.1      KLPFGINITSYRVVMAMF------SQTTSNFLPESAAYYVGNLKYTTFMLSFNENGTITN
AFS88936.1      TLPVYDTIKYYSIIPHSIRSI---QSDRKAW----AAFYVYKLQPLTFLLDFSVDGYIRR
YP_0010399      TLPVYEGIKYYTVIPRSFRSK---ANKREAW----AAFYVYKLHQLTYLLDFSVDGYIRR
                 **.   *. :  :                :    **::*  *:  *::* :. :* *  
QDF43825.1      AVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVAPSKEVVRFPNITNLCPFGEVFNATTF
AGZ48818.1      AVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVAPSKEVVRFPNITNLCPFGEVFNATTF
ALK02457.1      AVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVAPSKEVVRFPNITNLCPFGEVFNATTF
AAS10463.1      AVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKF
AAP13441.1      AVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKF
AAP13567.1      AVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKF
QHD43416.1      AVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRF
AVP78031.1      AVDCALDPLSETKCTLKSLTVQKGIYQTSNFRVQPTQSVVRFPNITNVCPFHKVFNATRF
ABD75323.1      AVDCSQDPLAELKCTTKSFNVSKGIYQTSNFRVSPVTEVVRFPNITNLCPFDKVFNATRF
QDF43835.1      AIDCAQNPLSELKCTIKNFNVSKGIYQTSNFRVSPTHEVIRFPNITNRCPFDKVFNASRF
ABD75332.1      AVDCSQNPLAELKCTIKNFNVSKGIYQTSNFRVTPTQEVVRFPNITNRCPFDKVFNASRF
QDF43820.1      AIDCAQNPLAELKCTIKNFNVSKGIYQTSNFRVSPTQEVVRFPNITNRCPFDKVFNASRF
AAZ67052.1      AIDCAQNPLAELKCTIKNFNVSKGIYQTSNFRVSPTQEVIRFPNITNRCPFDKVFNATRF
AFS88936.1      AIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVE-CDFSPLLSGTP-
YP_0010399      AIDCGHDDLSQLHCSYTSFEVDTGVYSVSSYEASATGTFIEQPNATE-CDFSPMLTGVA-
                *:**. : *:: :*:  .: :..*:*..*.: . .   .:  .: .: * *  ::..   
QDF43825.1      PSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDV
AGZ48818.1      PSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDV
ALK02457.1      PSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDV
AAS10463.1      PSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDV
AAP13441.1      PSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDV
AAP13567.1      PSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDV
QHD43416.1      ASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEV
AVP78031.1      PSVYAWERTKISDCIADYTVFYNSTSFSTFKCYGVSPSKLIDLCFTSVYADTFLIRFSEV
ABD75323.1      PSVYAWERTKISDCVADYTVFYNSTSFSTFNCYGVSPSKLIDLCFTSVYADTFLIRFSEV
QDF43835.1      PNVYAWERTKISDCVADYTVLYNSTSFSTFKCYGVSPSKLIDLCFTSVYADTFLIRSSEV
ABD75332.1      PNVYAWERTKISDCVADYTVLYNSTSFSTFKCYGVSPSKLIDLCFTSVYADTFLIRSSEV
QDF43820.1      PNVYAWERTKISDCVADYTVLYNSTSFSTFKCYGVSPSKLIDLCFTSVYADTFLIRSSEV
AAZ67052.1      PNVYAWERTKISDCVADYTVLYNSTSFSTFKCYGVSPSKLIDLCFTSVYADTFLIRSSEV
AFS88936.1      PQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMK
YP_0010399      PQVYNFKRLVFSNCNYNLTKLLSLFAVDEFSCNGISPDSIARGCYSTLTVDYFAYPLSMK
                ..** ::*  :::*  : : : .   .. *.*  :*.  :   *::.:  * *    .  
QDF43825.1      RQIAPGQTGVIADYNYKLPDDFMGC-VLAWNTRNIDATSTGNYNYKYRSLRHGKLRPFER
AGZ48818.1      RQIAPGQTGVIADYNYKLPDDFTGC-VLAWNTRNIDATQTGNYNYKYRSLRHGKLRPFER
ALK02457.1      RQIAPGQTGVIADYNYKLPDDFTGC-VLAWNTRNIDATQTGNYNYKYRSLRHGKLRPFER
AAS10463.1      RQIAPGQTGVIADYNYKLPDDFMGC-VLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFER
AAP13441.1      RQIAPGQTGVIADYNYKLPDDFMGC-VLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFER
AAP13567.1      RQIAPGQTGVIADYNYKLPDDFMGC-VLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFER
QHD43416.1      RQIAPGQTGKIADYNYKLPDDFTGC-VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER
AVP78031.1      RQVAPGQTGVIADYNYKLPDDFTGC-VIAWNTAKQD---VGNYF--YRSHRSTKLKPFER
ABD75323.1      RQVAPGQTGVIADYNYKLPDDFTGC-VIAWNTAKQD---VGSYF--YRSHRSSKLKPFER
QDF43835.1      RQVAPGETGVIADYNYKLPDDFTGC-VIAWNTAKQD---QGQYY--YRSSRKTKLKPFER
ABD75332.1      RQVAPGETGVIADYNYKLPDDFTGC-VIAWNTAQQD---QGQYY--YRSYRKEKLKPFER
QDF43820.1      RQVAPGETGVIADYNYKLPDDFTGC-VIAWNTAKQD---TGHYY--YRSHRKTKLKPFER
AAZ67052.1      RQVAPGETGVIADYNYKLPDDFTGC-VIAWNTAKQD---QGQYY--YRSHRKTKLKPFER
AFS88936.1      SDLSVSSAGPISQFNYKQSFSNPTC-LILATVPHNLTTITKPLKYSYINKCSRLLSDDRT
YP_0010399      SYIRPGSAGNIPLYNYKQSFANPTCRVMASVLANVTITKPHAYG--YIS-KCSRLTGANQ
                  :  ..:* *. :*** .     * ::     :            *       *     
QDF43825.1      DISNVPFSPDGKPCTPP-AF-NCYW-----------PLNDYGFFTTNGIGYQPYRVVVLS
AGZ48818.1      DISNVPFSPDGKPCTPP-AF-NCYW-----------PLNDYGFYITNGIGYQPYRVVVLS
ALK02457.1      DISNVPFSPDGKPCTPP-AF-NCYW-----------PLNDYGFYITNGIGYQPYRVVVLS
AAS10463.1      DISNVPFSPDGKPCTPP-AP-NCYW-----------PLNGYGFYTTSGIGYQPYRVVVLS
AAP13441.1      DISNVPFSPDGKPCTPP-AL-NCYW-----------PLNDYGFYTTTGIGYQPYRVVVLS
AAP13567.1      DISNVPFSPDGKPCTPP-AL-NCYW-----------PLNDYGFYTTTGIGYQPYRVVVLS
QHD43416.1      DISTEIYQAGSTPCNGVEGF-NCYF-----------PLQSYGFQPTNGVGYQPYRVVVLS
AVP78031.1      DLSSDE---------------NGVR-----------TLSTYDFNPNVPLEYQATRVVVLS
ABD75323.1      DLSSEE---------------NGVR-----------TLSTYDFNQNVPLEYQATRVVVLS
QDF43835.1      DLTSDE---------------NGVR-----------TLSTYDFYPNVPIEYQATRVVVLS
ABD75332.1      DLSSDE---------------NGVY-----------TLSTYDFYPSIPVEYQATRVVVLS
QDF43820.1      DLSSDDG--------------NGVY-----------TLSTYDFNPNVPVAYQATRVVVLS
AAZ67052.1      DLSSDE---------------NGVR-----------TLSTYDFYPSVPVAYQATRVVVLS
AFS88936.1      EVPQLVNANQYSPCVSI-VP-STVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMG
YP_0010399      DVETPLYINPGEYSICRDFSPGGFSEDGQVFKRTLTQFEGGGLLIGVGTRVPMTDNLQMS
                ::                   .               :.  .              : :.
QDF43825.1      FELL----NAPATVC-----GPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQF
AGZ48818.1      FELL----NAPATVC-----GPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQF
ALK02457.1      FELL----NAPATVC-----GPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQF
AAS10463.1      FELL----NAPATVC-----GPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQF
AAP13441.1      FELL----NAPATVC-----GPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQF
AAP13567.1      FELL----NAPATVC-----GPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQF
QHD43416.1      FELL----HAPATVC-----GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQF
AVP78031.1      FELL----NAPATVC-----GPKLSTQLVKNQCVNFNFNGLKGTGVLTDSSKRFQSFQQF
ABD75323.1      FELL----NAPATVC-----GPKLSTSLVKNQCVNFNFNGFKGTGVLTDSSKTFQSFQQF
QDF43835.1      FELL----NAPATVC-----GPKLSTGLVKNQCVNFNFNGLRGTGVLTDSSKRFQSFQQF
ABD75332.1      FELL----NAPATVC-----GPKLSTQLVKNQCVNFNFNGLRGTGVLTTSSKRFQSFQQF
QDF43820.1      FELL----NAPATVC-----GPKLSTQLVKNQCVNFNFNGLKGTGVLTDSSKRFQSFQQF
AAZ67052.1      FELL----NAPATVC-----GPKLSTQLVKNQCVNFNFNGLKGTGVLTESSKRFQSFQQF
AFS88936.1      FGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRF
YP_0010399      FIISVQYGTGTDSVCPMLDLGDSLTITNRLGKCVDYSLYGVTGRGVFQNCTAVGVKQQRF
                * :       . :**     . . .     .:**::.: *. * **:  ..      *.*
QDF43825.1      GRDVSD-FTDSVRDPKTSEILDISPCSFGGVSVITPGTNTSSEVAVLYQDVNCTDVPVAI
AGZ48818.1      GRDVSD-FTDSVRDPKTSEILDISPCSFGGVSVITPGTNTSSEVAVLYQDVNCTDVPVAI
ALK02457.1      GRDVLD-FTDSVRDPKTSEILDISPCSFGGVSVITPGTNTSSEVAVLYQDVNCTDVPVAI
AAS10463.1      GRDVSD-FTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTLI
AAP13441.1      GRDVSD-FTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAI
AAP13567.1      GRDVSD-FTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAI
QHD43416.1      GRDIAD-TTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAI
AVP78031.1      GKDASD-FIDSVRDPQTLEILDITPCSFGGVSVITPGTNTSLEVAVLYQDVNCTDVPTTI
ABD75323.1      GRDASD-FTDSVRDPQTLRILDISPCSFGGVSVITPGTNTSSAVAVLYQDVNCTDVPRTI
QDF43835.1      GRDTSD-FTDSVRDPQTLEILDITPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVPTAI
ABD75332.1      GRDTSD-FTDSVRDPQTLEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVPTSI
QDF43820.1      GRDTSD-FTDSVRDPQTLEILDITPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVPTAI
AAZ67052.1      GRDTSD-FTDSVRDPQTLEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVPAAI
AFS88936.1      VYDAYQNLVGYYSDDGNYYCLR--ACVSVPVSVIY--DKETKTHATLFGSVACEHISSTM
YP_0010399      VYDSFDNLVGYYSDDGNYYCVR--PCVSVPVSVIY--DKSTNLHATLFGSVACEHVTTMM
                  *  :   .   *  .   :   .*    ****    : :   *.*: .* *  :.  :
QDF43825.1      --HADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSY---ECDIPIGAGICASYHTVSS
AGZ48818.1      --HADQLTPSWRVYSTGNNVFQTQAGCLIGAEHVDTSY---ECDIPIGAGICASYHTVSS
ALK02457.1      --HADQLTPSWRVYSTGNNVFQTQAGCLIGAEHVDTSY---ECDIPIGAGICASYHTVSS
AAS10463.1      --HAEQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSY---ECDIPIGAGICASYHTVSS
AAP13441.1      --HADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSY---ECDIPIGAGICASYHTVSL
AAP13567.1      --HADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSY---ECDIPIGAGICASYHTVSL
QHD43416.1      --HADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSY---ECDIPIGAGICASYQTQTN
AVP78031.1      --HADQLTPAWRIYATGTNVFQTQAGCLIGAEHVNASY---ECDIPIGAGICASYHTASI
ABD75323.1      --QADQLAPSWRVYTTGPYVFQTQAGCLIGAEHVNASY---QCDIPIGAGICASYHTASH
QDF43835.1      --RADQLTPAWRVYSTGINVFQTQAGCLIGAEHVNASY---ECDIPIGAGICASYHTAST
ABD75332.1      --HADQLTPAWRVYSTGVNVFQTQAGCLIGAEHVNASY---ECDIPIGAGICASYHTASV
QDF43820.1      --RADQLTPAWRVYSTGVNVFQTQAGCLIGAEHVNASY---ECDIPIGAGICASYHTAST
AAZ67052.1      --HADQLTPAWRVYSTGTNVFQTQAGCLIGAEHVNASY---ECDIPIGAGICASYHTAST
AFS88936.1      SQYSRSTRSMLKRRDSTYGPLQTPVGCVLGL--VNSSLFVEDCKLPLGQSLCALPDTPST
YP_0010399      S-QFSRLTQSNLRRRDSNIPLQTAVGCVIGLS--NNSLVVSDCKLPLGQSLCAV-PPVST
                                    :** .**::*    : *    :*.:*:* .:**   . : 
QDF43825.1      ----LRSTS----QKSI--------VAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVM
AGZ48818.1      ----LRSTS----QKSI--------VAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVM
ALK02457.1      ----LRSTS----QKSI--------VAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVM
AAS10463.1      ----LRSTS----QKSI--------VAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVM
AAP13441.1      ----LRSTS----QKSI--------VAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVM
AAP13567.1      ----LRSTS----QKSI--------VAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVM
QHD43416.1      SPRRARSVA----SQSI--------IAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEIL
AVP78031.1      ----LRSTS----QKAI--------VAYTMSLGAENSIAYANNSIAIPTNFSISVTTEVM
ABD75323.1      ----LRSTG----QKSI--------VAYTMSLGAENSVAYANNSIAIPTNFSISVTTEVM
QDF43835.1      ----LRSVG----QKSI--------VAYTMSLGAENSIAYANNSIAIPTNFSISVTTEVM
ABD75332.1      ----LRSTG----QKSI--------VAYTMSLGAENSIAYANNSIAIPTNFSISVTTEVM
QDF43820.1      ----LRSVG----QKSI--------VAYTMSLGAENSIAYANNSIAIPTNFSISVTTEVM
AAZ67052.1      ----LRSVG----QKSI--------VAYTMSLGAENSIAYANNSIAIPTNFSISVTTEVM
AFS88936.1      ----LTPRS----VRSVPGEMRLASIAFNHPIQVDQ-LNSSYFKLSIPTNFSFGVTQEYI
YP_0010399      ----FRSYSASQFQLAV--------LNYTSPIVV-TPINSSGFTAAIPTNFSFSVTQEYI
                      . .      ::        : :. .: .   :  :  . :*****::.:* * :
QDF43825.1      PVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAVEQDRNTREVFAQVKQ
AGZ48818.1      PVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAVEQDRNTREVFAQVKQ
ALK02457.1      PVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAVEQDRNTREVFAQVKQ
AAS10463.1      PVSMAKTSVDCNMYICGDSTECANLLLQYGSFCRQLNRALSGIAAEQDRNTREVFVQVKQ
AAP13441.1      PVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQ
AAP13567.1      PVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQ
QHD43416.1      PVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQ
AVP78031.1      PVSMAKTSVDCTMYICGDSIECSNLLLQYGSFCTQLNRALSGIAIEQDKNTQEVFAQVKQ
ABD75323.1      PVSMAKTSVDCTMYICGDSLECSNLLLQYGSFCTQLNRALSGIAVEQDKNTQEVFAQVKQ
QDF43835.1      PVSMSKTSVDCTMYICGDSQECSNLLLQYGSFCTQLNRALTGIAIEQDKNTQEVFAQVKQ
ABD75332.1      PVSIAKTSVDCTMYICGDSLECSNLLLQYGSFCTQLNRALTGIAIEQDKNTQEVFAQVKQ
QDF43820.1      PVSMAKTSVDCTMYICGDSQECSNLLLQYGSFCTQLNRALTGVALEQDKNTQEVFAQVKQ
AAZ67052.1      PVSMAKTSVDCTMYICGDSLECSNLLLQYGSFCTQLNRALSGIAIEQDKNTQEVFAQVKQ
AFS88936.1      QTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKS
YP_0010399      ETSIQKVTVDCKQYVCNGFTRCEKLLVEYGQFCSKINQALHGANLRQDESVYSLYSNIKT
                 .:: *.:***. *:*..   * :** :**.** ::*.** *    ** .. .:: .:* 
QDF43825.1      MYKTPTLKD-FGG-FNFSQILPDPLKPTKRSF---IEDLLFNKVTLADAGFMKQYGECL-
AGZ48818.1      MYKTPTLKD-FGG-FNFSQILPDPLKPTKRSF---IEDLLFNKVTLADAGFMKQYGECL-
ALK02457.1      MYKTPTLKD-FGG-FNFSQILPDPLKPTKRSF---IEDLLFNKVTLADAGFMKQYGECL-
AAS10463.1      MYKTPTLKD-FGG-FNFSQILPDPLKPTKRSF---IEDLLFNKVTLADAGFMKQYGECL-
AAP13441.1      MYKTPTLKY-FGG-FNFSQILPDPLKPTKRSF---IEDLLFNKVTLADAGFMKQYGECL-
AAP13567.1      MYKTPTLKY-FGG-FNFSQILPDPLKPTKRSF---IEDLLFNKVTLADAGFMKQYGECL-
QHD43416.1      IYKTPPIKD-FGG-FNFSQILPDPSKPSKRSF---IEDLLFNKVTLADAGFIKQYGDCL-
AVP78031.1      IYKTPPIKD-FGG-FNFSQILPDPSKPSKRSF---IEDLLFNKVTLADAGFIKQYGDCL-
ABD75323.1      MYKTPTIRD-FGG-FNFSQILPDPLKPTKRSF---IEDLLYNKVTLADAGFMKQYADCL-
QDF43835.1      MYKTPAIKD-FGG-FNFSQILPDPSKPTKRSF---IEDLLFNKVTLADAGFMKQYGECL-
ABD75332.1      MYKTPAIKD-FGG-FNFSQILPDPSKPTKRSF---IEDLLFNKVTLADAGFMKQYGECL-
QDF43820.1      MYKTPAIKD-FGG-FNFSQILPDPSKPTKRSF---IEDLLFNKVTLADAGFMKQYGECL-
AAZ67052.1      MYKTPAIKD-FGG-FNFSQILPDPSKPTKRSF---IEDLLFNKVTLADAGFMKQYGECL-
AFS88936.1      SQSSPIIPG-FGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQ
YP_0010399      T-STQTLEYGLNGDFNLTLLQVPQIGGSSSSYRSAIEDLLFDKVTIADPGYMQGYDDCMK
                  .:  :   :.* **:: :        . *    *****::***:**.*::: * :*: 
QDF43825.1      -GDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAM
AGZ48818.1      -GDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAM
ALK02457.1      -GDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAM
AAS10463.1      -GDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAM
AAP13441.1      -GDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAM
AAP13567.1      -GDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAM
QHD43416.1      -GDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM
AVP78031.1      -GGISARDLICAQKFNGLTVLPPLLTDEMIAAYTAALISGTATAGWTFGAGAALQIPFAM
ABD75323.1      -GGINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALISGTATAGWTFGAGAALQIPFAM
QDF43835.1      -GDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAM
ABD75332.1      -GDISARDLICAQKFNGLTVLPPLLTDEMIAAYTAALVSGTATAGWTFGAGSALQIPFAM
QDF43820.1      -GDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAM
AAZ67052.1      -GDISARDLICAQKFNGLTVLPPLLTDEMIAAYTAALVSGTATAGWTFGAGSALQIPFAM
AFS88936.1      QGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQ
YP_0010399      QGPQSARDLICAQYVSGYKVLPPLYDPNMEAAYTSSLLGSIAGAGWTAGLSSFAAIPFAQ
                 *   ******** . * .*****   :* * **::*:..    *** * .:   **** 
QDF43825.1      QMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALN
AGZ48818.1      QMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALN
ALK02457.1      QMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALN
AAS10463.1      QMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALN
AAP13441.1      QMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALN
AAP13567.1      QMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALN
QHD43416.1      QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN
AVP78031.1      QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQESLTSTASALGKLQDVVNQNAQALN
ABD75323.1      QMAYRFNGIGVTQNVLYENQKQIANQFNKAITQIQESLTTTSTALGKLQDVVNQNAQALN
QDF43835.1      QMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALN
ABD75332.1      QMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALN
QDF43820.1      QMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALN
AAZ67052.1      QMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALN
AFS88936.1      SIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFQKVQDAVNNNAQALS
YP_0010399      SMFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTSNLAFSKVQDAVNANAQALS
                .: **:**:*:**:** **** ***:**.*:  :* .::::  *: *:**.** *****.
QDF43825.1      TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
AGZ48818.1      TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
ALK02457.1      TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
AAS10463.1      TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
AAP13441.1      TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
AAP13567.1      TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
QHD43416.1      TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
AVP78031.1      TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
ABD75323.1      TLVKQLSSNFGAISSALNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
QDF43835.1      TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
ABD75332.1      TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
QDF43820.1      TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
AAZ67052.1      TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA
AFS88936.1      KLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAAL
YP_0010399      KLASELSNTFGAISSSISDILARLDTVEQDAQIDRLINGRLISLNAFVSQQLVRSETAAR
                .*..:**..*****: :.**: *** :* :.******.*** :*:::*:***:*:     
QDF43825.1      SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPA
AGZ48818.1      SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPA
ALK02457.1      SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPA
AAS10463.1      SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPA
AAP13441.1      SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPA
AAP13567.1      SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPA
QHD43416.1      SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPA
AVP78031.1      SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYIPSQEKNFTTAPA
ABD75323.1      SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPSQEKNFTTAPA
QDF43835.1      SANLAATKMSECVLGQSKRVDFCGRGYHLMSFPQAAPHGVVFLHVTYVPSQEKNFTTAPA
ABD75332.1      SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPA
QDF43820.1      SANLAATKMSECVLGQSKRVDFCGRGYHLMSFPQAAPHGVVFLHVTYVPSQEKNFTTAPA
AAZ67052.1      SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPA
AFS88936.1      SAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYG
YP_0010399      SAQLASDKVNECVKSQSKRNGFCGSGTHIVSFVVNAPNGFYFFHVGYVPTNYTNVTAAYG
                **:**  *:.*** .**** .*** * *::**   **:*. *:** * *::  :..:* .
QDF43825.1      ICHEGK---AYFPREGVFVFNGTS-------WFITQRNFFSPQIITTDNT-FVSGSCDVV
AGZ48818.1      ICHEGK---AYFPREGVFVFNGTS-------WFITQRNFFSPQIITTDNT-FVSGSCDVV
ALK02457.1      ICHEGK---AYFPREGVFVFNGTS-------WFITQRNFFSPQIITTDNT-FVSGSCDVV
AAS10463.1      ICHEGK---AYFPREGVFVFNGTS-------WFITQRNFFSPQIITTDNT-FVSGNCDVV
AAP13441.1      ICHEGK---AYFPREGVFVFNGTS-------WFITQRNFFSPQIITTDNT-FVSGNCDVV
AAP13567.1      ICHEGK---AYFPREGVFVFNGTS-------WFITQRNFFSPQIITTDNT-FVSGNCDVV
QHD43416.1      ICHDGK---AHFPREGVFVSNGTH-------WFVTQRNFYEPQIITTDNT-FVSGNCDVV
AVP78031.1      ICHEGK---AHFPREGVFVSNGTH-------WFVTQRNFYEPKIITTDNT-FVSGNCDVV
ABD75323.1      ICHEGK---AYFPREGVFVSNGSS-------WFITQRNFYSPQIITTDNT-FVAGSCDVV
QDF43835.1      ICHEGK---AYFPREGVFVSNGTS-------WFITQRNFYSPQIITTDNT-FVAGSCDVV
ABD75332.1      ICHEGK---AYFPREGVFVSNGTS-------WFITQRNFYSPQIITTDNT-FVAGNCDVV
QDF43820.1      ICHEGK---AYFPREGVFVSNGTF-------WFITQRNFYSPQIITTDNT-FVAGNCDVV
AAZ67052.1      ICHEGK---AYFPREGVFVSNGTS-------WFITQRNFYSPQIITTDNT-FVAGSCDVV
AFS88936.1      LCDAANPTNCIAPVNGYFIKTNNT--RIVDEWSYTGSSFYAPEPITSLNTKYVA--PQVT
YP_0010399      LCNNNNPPLCIAPIDGYFITNQTTTYSVDTEWYYTGSSFYKPEPITQANSRYVS--SDVK
                :*   :   .  * :* *: . .        *  *  .*: *: **  *: :*:   :* 
QDF43825.1      IGIINNTVYDPL---QPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL
AGZ48818.1      IGIINNTVYDPL---QPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEINRL
ALK02457.1      IGIINNTVYDPL---QPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL
AAS10463.1      IGIINNTVYDPL---QPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQEEIDRL
AAP13441.1      IGIINNTVYDPL---QPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL
AAP13567.1      IGIINNTVYDPL---QPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL
QHD43416.1      IGIVNNTVYDPL---QPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL
AVP78031.1      IGIINNTVYDPL---QPELDSFKEELDKYFKNHTSPDIDLGDISGINASVVNIQKEIDRL
ABD75323.1      IGIINNTVYDPL---QPELDSFKQELDKYFKNHTSPDVDLGDISGINASVVDIQKEIDRL
QDF43835.1      IGIINNTVYDPL---QPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL
ABD75332.1      IGIINNTVYDPL---QPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL
QDF43820.1      IGIINNTVYDPL---QPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL
AAZ67052.1      IGIINNTVYDPL---QPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL
AFS88936.1      YQNISTNLPPPLLGNSTGID-FQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSL
YP_0010399      FDKLENNLPPPLLENSTDVD-FKDELEEFFKNVTSHGPNFAEISKINTTLLDLSDEMAML
                   :...:  **   .. :* *::**:::*** ::   ::..:: **::::::  *:  *
QDF43825.1      NEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKG
AGZ48818.1      NEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKG
ALK02457.1      NEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKG
AAS10463.1      NEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKG
AAP13441.1      NEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKG
AAP13567.1      NEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKG
QHD43416.1      NEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKG
AVP78031.1      NEVARNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKG
ABD75323.1      NEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLVGLFMAIILLCYFTSCCSCCKG
QDF43835.1      NEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMATILLCCMTSCCSCLKG
ABD75332.1      NEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKG
QDF43820.1      NEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMATILLCCMTSCCSCLKG
AAZ67052.1      NEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKG
AFS88936.1      QQVVKALNESYIDLKELGNYTYYNKWPWYIWLGFIAGLVALALCVFFILCCTGCGTNCMG
YP_0010399      QEVVKQLNDSYIDLKELGNYTYYNKWPWYVWLGFIAGLVALLLCVFFLLCCTGCGTSCLG
                ::*.. **:* ***:***:*  * *****:********:.: :  :::   *.* :   *
QDF43825.1      ACSCGSCC-KFDEDDSEPVLKGVKLHYT
AGZ48818.1      ACSCGSCC-KFDEDDSEPVLKGVKLHYT
ALK02457.1      ACSCGSCC-KFDEDDSEPVLKGVKLHYT
AAS10463.1      ACSCGSCC-KFDEDDSEPVLKGVKLHYT
AAP13441.1      ACSCGSCC-KFDEDDSEPVLKGVKLHYT
AAP13567.1      ACSCGSCC-KFDEDDSEPVLKGVKLHYT
QHD43416.1      CCSCGSCC-KFDEDDSEPVLKGVKLHYT
AVP78031.1      CCSCGSCC-KFDEDDSEPVLKGVKLHYT
ABD75323.1      MCSCGSCC-RFDEDDSEPVLKGVKLHYT
QDF43835.1      ACSCGSCC-KFDEDDSEPVLKGVKLHYT
ABD75332.1      ACSCGSCC-KFDEDDSEPVLKGVKLHYT
QDF43820.1      ACSCGSCC-KFDEDDSEPVLKGVKLHYT
AAZ67052.1      ACSCGSCC-KFDEDDSEPVLKGVKLHYT
AFS88936.1      KLKCNRCCDRYEEYDLEP----HKVHVH
YP_0010399      KMKCKNCCDSYEEYDVE------KIHVH
                  .*  **  ::* * *      *:*
  • I went back and selected 6. Tree Rendering, which created a phylogenetic tree of the sequences.
    • Horizontal lines represent individual evolutionary lineages.
    • Vertical lines (splits) represent mutation events, and the length of these lines has no biological meaning.
    • The left-most split is the root and represents a hypothesis of the most recent common ancestor of the sequences being analyzed.
    • The length of each horizontal line (branch) signifies percentage change in amino acid sequence occurring along that branch, relative to the scale bar located below the tree.
      • The scale bar shown is 0.5 (5%).
    • The red values shown on the branches represent the support values of each clade. A higher support value corresponds with a higher statistical confidence of that clade.
  • I saved the phylogenetic tree and have included it here:

DuranWk5Tree.png

  • The phylogenetic tree and the multiple sequence alignment show a lot of similarities. I first noticed that Human betacoronavirus (AFS88936) and Tyloncteris bat coronavirus (YP_001039953) are together in a clade separate from the rest of the sequences. When looking at the multiple sequence alignment, those two sequences are clearly very different from the rest of the sequences, and are fairly similar to one another. However, even though they make up their own clade, they do have a fair amount of differences in their sequences. This is also represented in the tree by the longer horizontal branch for Human betacoronavirus. The second major clade of the tree is broken into two subgroups. The sequence that I analyzed in Part 1 of the assignment (ALK02457) has three sister taxa shown in the tree. These three sequences are SARS coronavirus Urbani (AAP13441), SARS coronavirus CUHK-W1 (AAP13567), and SARS coronavirus (AAS10463). The alignment shows that the amino acid sequences for these four spike proteins are nearly identical.
  • Figure 3A of the Wan et al. paper shows a multiple sequence alignment for Human SARS 2002, Civet SARS 2002, Bat SARS 2013, and 2019-nCoV. The amino acid positions differ for each sequence, but it ranges from ~424 until ~494. I found the same section of amino acids in my sequence, SARS-like Coronavirus WIV16:
NTRNIDATQTGNYNYKYRSLRHGKLRPFERDISNVPFSPDGKPCTPPAFNCYWPLNDYGFYITNGIGYQPY
  • The first key residue highlighted in blue in Figure 3A is a tyrosine in human and civet SARS, a serine in bat SARS, and a leucine in 2019-nCoV. In my sequence, the residue in that position is a serine. This most closely resembles the bat SARS from 2002. The second highlighted residue is a leucine in both human and civet SARS and a phenylalanine in bat SARS and 2019-nCoV. In my sequence, it is also a phenylalanine. The third highlighted residue is asparagine in human and bat SARS, a lysine in civet, and a glutamine in 2019-nCoV. Similar to bat SARS, the amino acid in my sequence is an asparagine. The fourth key residue is aspartic acid in human, bat, and civet SARS, and a serine in 2019-nCoV. My sequence also has aspartic acid. The last key residue analyzed is a threonine in human SARS, a serine in civet SARS, and asparagine in bat SARS, 2019-nCov, and in my sequence. In all five key residue positions, my sequence (SARS-like coronavirus) has the same amino acids as the bat SARS sequence. This makes sense, as the sequence I analyzed was also obtained from a bat.
  • Figure 2 in the Wan et al. paper is a phylogenetic tree of the spike protein sequences used in the study. There are some noticeable differences between the study's tree and our class tree. First off, the study's tree shows an outgroup of BtSCoV PDF2386. This accession number cannot be found in GenBank, so the tree we generated does not have the same outgroup. Instead, Human betacoronavirus 2c (AFS88936) and Tyloncteris bat coronavirus HKU4 (YP_001039953) are outliers from the other sequences. We also only used about half of the sequences in the original study, so there are differences in that regard. However, overall, it seems to be fairly similar in its organization. The branches in the study's tree are much longer, but their scale is also much smaller at 0.01, so that would be expected.
  • For the most part, the study could be replicated by finding the sequences, aligning them, and creating a tree. However, because the outgroup accession number was incorrect, there is a slight issue in replicating the results. Another aspect that made comparison difficult was the fact that they included the accession numbers for the full sequences in their tree, and our tree included accession numbers for the spike protein sequences. I think it would have been helpful if they had used the spike protein accession numbers, as those were the sequences actually used in the study's analysis.

Scientific Conclusion

This assignment was useful for practicing how to use a database like GenBank, and create multiple sequence alignments and phylogenetic trees. I was able to use these skills to attempt to replicate the Wan et al. study, and gain a better understanding of their results.

Acknowledgements

  1. I contacted my homework partner, Nathan R. Beshai, over Zoom to discuss the protocol for generating a phylogenetic tree and multiple sequence alignment.
  2. I copied and modified the protocol outlined on the Week 4 Assignment page.
  3. I used the Wan et al. article to find spike protein sequences in GenBank, and in Part 2 of the assignment, where I compared Figures 2 and 3 to the phylogenetic tree and multiple sequence alignment I generated.
  4. I used sequences found on GenBank.
  5. I generated a multiple sequence alignment and phylogenetic tree using Phylogeny.fr.
  6. I obtained the class's spike protein sequences from the Week 4 Talk page.
  7. Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

(Macie Duran (talk) 19:54, 30 September 2020 (PDT))

References