Bioinformatics - protocols  A

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Basic sequence manipulation

CCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAAT
TCGGTGACCCCGGCAAAGCTTGCTTAATCCGAAGACGTTTCTGTTTCATC
TTCTTAAATCCGGGCCAACNGCGTTTACGAGACTAAACGCGTTTCTCTTT
AGGGCTTAATTATTATCCAGAGATGGCTCATCATAGCAAATGTTTACAAA
CGTTGGATTTAGCTTGTAAAGAGCTGAGATCTCGTGGCTTGTTTGTGAAG
CTTTTGGAGGCAATACTTAAAGCTGGAAACAGAATGAACGCGGGTACCGC
GAGAGGAAACGCTCAAGCGTTTAATCTAACCGCGCTTTTGAAGCTTTCGG
ATGTTAAAAGCGTTGATGGGAAGACTTCTTTGCTTAACTTTGTAGTGGAG
GAAGTTGTTAGATCGGAAGGAAAACGTTGTGTTATGAATAGAAGAAGCCA
TAGCTTAACACGAAGCGGTAGTAGTAACTACAATGGTGGTAATAGTAGTC
TTCAGGTTATGTCGAAAGAAGAGCAAGAGAAAGAGTACTTGAAGCTTGGT
TTACCAGTTGTTGGTGGATTGAGCTCTGAGTTTTCAAACGTGAAGAAAGC
TGCTTGTGTGGACTATGAAACGGTTGTTGCAACTTGTTCTGCTCTTGCGG
TTAGAGCGAAAGATGCGAAAACGGTGATTGGAGAATGTGAAGATGGAGAA
GGAGGGAGGTTTGTGAAAACGATGATGACGTTTCNTGATTCGGTAGAGGA
AGAGGTGAAAATAGCGAAAGGTGAAGAGAGGAAAGTGATCCCTGA

• Copy the sequence into a text file and convert it to the FASTA format. You can either do this manually by adding a header line (>mysequence) or using the BCM sequence utilities Readseq function.
• Perform restriction analysis to identify sites from the vector polylinker and remove vector sequences so that you have only clean cDNA left. Use either the BCM sequence utilitiesWebCutter interface or one of the other WebCutter or Cutter sites. Check the vector-insert boundary using the map provided below and remove vector sequences. Save the clean cDNA sequence (in FASTA format) as a text file.

Sequence similarity searches and domain structure analysis

• Use the clean cDNA sequence from the previous excercise as a query for a standard nucleotide BLAST search of the non-redundant NCBI database. How many different significant matches did you find in the A. thaliana genome?
• From the BLAST results page, retrieve the GenBank entries corresponding to all hits with E-values better (i.e. lower) than 0.05 (by clicking the links with entry numbers to the left from the gene description). IMPORTANT: open the links in a new window, otherwise your BLAST results may expire. If this happens, or if the BLAST server is down, go to the SOS page.
• Examine the GenBank entries and locate the sites of the BLAST hits using sequence co-ordinates from the bottom (alignment) half of the Blast output (Location of the match can be found on the Sbjct line of the alignments.) Make sure that you retrieve the correct CDS corresponding to a gene that spans the aligned BLAST match.
• Retrieve the predicted protein sequences.and save them into a FASTA format file with extension *.aa (named e.g. A1.aa). Fasta format file looks like this:

>protein1
HERECOMESTHESEQUENCEOFPROTEINONE
>protein2
ANDHERETHESEQUENCEOFPROTEINTWO
etc.
If you were unable to retrieve the sequences for server-dependent reasons, go to the SOS page.
• Perform a domain structure analysis of the protein corresponding to the best match to your cDNA using the SMART Sequence Analysis utility. Paste only clean single-letter code sequence into the sequence box (no FASTA header) and include also Outlier homologues, PFAM domains, signal peptides and internal repeats (for explanation what this means see the SMART page).

If you found any domains within the sequence, look up the domain description (available by clicking the domain in the SMART output). (If server was down, you can get the SMART output from the SOS page).

Gene building: searching for coding sequences in chromosomal DNA

>locusA
cccctataaaaagtattaaaaaggactgatacaataatgtatataaatat
cctaaaagatcttaattttgtaaatttattgttgtatattctaaacccgc
aatattagaatgatgatttagtaaacaagaaagacaaaataaataattaa
ttttagctagaaaagatgaaataaacactcatgatttaagccatacaaat
cgaagccccttgggttcagcatttctcaccaagtaaataccatcacctct
ggaaacccatttacgtacttgaccacatcttttattagcggctcctctgt
atgctctccatatgttatacacactatgatgccttaagatttattcacga
cgatttaatcagatacgcttatggattgccaaagatgatgccatctactt
agagaaaaacaatggaaagcgagaacgcatgtataattggaataaaaatt
aatatggttttcatatatctaaaaaattggacatttgaagccttaataaa
ttatactatgtaaaaatacttgtttatgaatgtaaattataataaattac
gatttaattagggaaatattgactatatatttcacccaaatattgaatgt
aaattttattttccaatacttttgcacatttaagaaattttcggatgtat
ttcctaaagaatattaccttttttgttttttaaaccatgcctttttgttt
tacacgttcataaatgcatgttccatacgcattaccataatttaatttga
acttaattttctctaggaatggtgatgatccactaccactatcattgatt
tcattccatattcctttgaccgactgaaattacgttggaaatagtatatt
ttgatgaataatttatttactcggaaaaaagaggtcaagttattaatagt
aagtacatatacattatcaattaagaattcaattgagttttaaggaaaat
cctattaatttgtttggtattcggtatttgttagttctaaggaattgaat
ttcccgattatacatcattataacgttctcaagttccaaacttgcaaccc
acattttgtcgatattctcaaatgtgaattcattcaatttcccatagaaa
acataaatttgcacttaaagttaacaattgaaatcgtatctaaatgggaa
tgtttttggcttttagtgttagacttccaaagcgtcaaaaatatttctag
aaagagcacaaaaaataagcaacgccactacttttggacaaagtcaacga
taacacacatcaaccgcaccagctccataaaagtccatctcacgaaaacg
attctagtcaaactacctaaaacacccttatatttacatacaacccaatc
ccactaacaagggtattttcgtcaatcacaaaatttatcaccgacccggg
aagaagaagaagaacagatcaactaatttctgctttcaactccacattaa
accaaaacctccaaaaagaatcatttatttaaattatcttcccgttttaa
gttcctgagatttttgggaattgtaaatttgaagaaaattaaacaaagac
gtgttttcatttttttttttgtttcctttattgatctctctctatctctc
taaatgagctaaatcgttaatggctgccatgtttaatcatccatggccta
atttaaccctaatttacttcttcttcatcgtcgttttaccattccaatca
ctttctcaatttgattctcctcaaaatatcgaaactttcttccccatctc
ttcactctcccctgttccaccaccgcttcttccaccttcgtcaaacccat
ctccgccgtcgaataattcatcatcttcggataaaaaaacaatcaccaaa
gctgtccttataacagcagcaagtactttacttgtagctggagttttctt
cttctgcctccaaagatgtatcatcgcacggagacggagagacagagttg
gaccagtcagagtcgaaaacactttacctccgtatcctcctcctccgatg
acgtcggcggcggtgactacgactactttggctagagaaggattcacgag
gtttggtggtgtgaaaggtttgattcttgatgagaatggtcttgatgtgt
tgtattggagaaagctacagagtcagagagaaagaagtgggagtttcagg
aaacagatcgtcaccggagaagaagaagacgagaaagaagttatttatta
caagaacaagaagaaaacagagcccgttacagagattcctcttcttagag
gaagatcatctacttctcacagtgttatccataacgaagatcatcagccg
ccaccgcaggtgaaacagagtgaaccaacaccaccaccgccaccaccgtc
aattgcggtgaaacagagtgcaccaacgccatcgccacctcctccgatta
agaagggttcttcaccatcgccaccgccacctccaccggtgaaaaaggtt
ggagctttatcatcatcagcttcgaaaccaccacctgcgccggttagagg
agcaagtggaggagagacttcgaaacaagtaaagttgaagcctttacatt
gggataaagtaaaccctgattccgatcattcaatggtttgggacaaaatc
gatcgtggatcattcaggtatatatttatttcgaaagttagggcttttgc
ttcaatcaattgaaaaaaccctaatttgtttttgtttcttctcagtttcg
atggcgatttaatggaagctctgtttggatacgttgccgtggggaagaaa
tcaccagaacaaggcgatgagaaaaaccctaaatcaacgcaaatattcat
acttgatccgagaaagtctcaaaacacagcgattgtgctcaaatcattag
gtatgacacgtgaagagcttgttgaatcactcatagaaggaaacgatttc
gtgccagacactcttgagaggttagctagaatagctccaacgaaagaaga
acaatcagccattcttgaattcgacggtgacacggcaaagcttgctgatg
cggagacgtttctgtttcatcttcttaaatccgtgccaaccgcgtttacg
agactaaacgcgtttctctttagggctaattattatccagagatggctca
tcatagcaaatgtttacaaacgttggatttagcttgtaaagagctgagat
ctcgtggcttgtttgtgaagcttttggaggcaatacttaaagctggaaac
agaatgaacgcgggtaccgcgagaggaaacgctcaagcgtttaatctaac
cgcgcttttgaagctttcggatgttaaaagcgttgatgggaagacttctt
tgcttaactttgtagtggaggaagttgttagatcggaaggaaaacgttgt
gttatgaatagaagaagccatagcttaacacgaagcggtagtagtaacta
caatggtggtaatagtagtcttcaggttatgtcgaaagaagagcaagaga
aagagtacttgaagcttggtttaccagttgttggtggattgagctctgag
ttttcaaacgtgaagaaagctgcttgtgtggactatgaaacggttgttgc
aacttgttctgctcttgcggttagagcgaaagatgcgaaaacggtgattg
gagaatgtgaagatggagaaggagggaggtttgtgaaaacgatgatgacg
tttcttgattcggtagaggaagaggtgaaaatagcgaaaggtgaagagag
gaaagtgatggagcttgtgaaacgtacaacggattattatcaagcaggag
ctgttacaaaggggaagaatccacttcatttgtttgttatcgttagagat
tttcttgccatggttgataaagtttgcttagatattatgagaaatatgca
gaggaggaaggttggtagtccgatatcgccttcttcgcagcggaatgcgg
tgaaattcccggttttgcctccgaatttcatgtcggacagagcttggagt
gattctggtgggtcggattctgatatgtgagagtcaagatttgttatatg
taaatactaaatagtagaagcattttgggtattgattagcattgaaagat
gttgaattgtttatagatttatcagtccaaagcattggacttgagtataa
tttgttccttgtataaataaacaattttgctttaagacctttccatgttt
atgaacatgtcttctttaacttcacatagaccttttgtttacgtaagaac
taataatactaaattgtttgataattctaaatgtgaaagtgaaccactat
atagtgtgaacttggctttattgaattctttttaaaaaaatttctccaga
gctttagatgtaggagttaatattttcacctaacatagcctcttttttat
gtttctctatcaactaacactaaatttgtggatgaagactaaattaacat
aagtttatctattaactaacaacctaccagtttgatgcttgtaaatatga
aacttcaacgttataaagactatatggtgtgaactttttatccatcttta
ttgacttttaaaattttcttaatttgagtaaacaaaagcagaagcttttt
aaaggatgcaggagttgatttttgtatatgaacaaaacatatacttctcc
cttagacgaatttggagctatcattcttggtttcaaactttttaataatt
tgagctttaaagcaaaatggcaactttatattgatcactagtccacaaca
ctttctctgccttttcctcaatagcaacgcgtagtcaagaagaagaacgt
gtttaacatggaccaatcttgattaagataatagtatgatcaaatgctta
tataaacacactaaaaaggaatcaaatttaaccattccacaaatcaccaa
caaaatttaatgaatcatgtctctgcttctaaagatgttattattttcct
tattcttcttctatatggcttcaatttctcaatgctcagacccaaccggt
ggacagtttagcttcaacggttacttgtacaccgatggagttgcggatct
aaacccggacggtttgttcaaactcataacttcaaagaca

Try to predict the exon-intron structure using the MIT GenScan server (don`t forget to select organism = Arabidopsis!) and Gene Finder. (If any of the servers down, the results can be found on the  SOS page.). Compare the localisation of introns and exons found by both programs, as well as NetGene2/WebGene (contact group B or use links to results, if possible).
 

Construction and interpretation of a protein sequence alignment

• Open MACAW on your computer (should be OK over the browser). Go File... New Project..., select Sequence type= protein and Significance=number. Chose the PAM120 scoring matrix and import sequence (Sequence... Import...) from the file A1.aa you created previously. Save the Macaw file on your computer before proceeding further in order to decrease the likelihood of crash.
• Examine the performance of Gibbs sampler vs. Segment pair overlap methods and construct an alignment of the protein sequences.

>p140mDia
YKPEVQLRRPNWSKFVAEDLSQDCFWTKVKEDRFENNELFAKLTLAFSAQTKTSKAKKDQEGGEEKKSVQ
KKKVKELKVLDSKTAQNLSIFLGSFRMPYQEIKNVILEVNEAVLTESMIQNLIKQMPEPEQLKMLSELKE
EYDDLAESEQFGVVMGTVPRLRPRLNAILFKLQFSEQVENIKPEIVSVTAACEELRKSENFSSLLELTLL
VGNYMNAGSRNAGAFGFNISFLCKLRDTKSADQKMTLLHFLAELCENDHPEVLKFPDELAHVEKASRVSA
ENLQKSLDQMKKQIADVERDVQNFPAATDEKDKFVEKMTSFVKDAQEQYNKLRMMHSNMETLYKELGDYF
VFDPKKLSVEEFFMDLHNFRNMFLQAVKENQKRRETEEKMRRAKLAKEKAEKERLEKQQKREQLIDMNAE
GDETGVMDSLLEALQSGAAFRRKR
>Diaphanous Drosophila
WDVKNPMKRANWKAIVPAKMSDKAFWVKCQEDKLAQDDFLAELAVKFSSKPVKKEQKDAVDKPTTLTKKN
VDLRVLDSKTAQNLAIMLGGSLKHLSYEQIKICLLRCDTDILSSNILQQLIQYLPPPEQLKRLQEIKAKG
EPLPPIEQFAATIGEIKRLSPRLHNLNFKLTYADMVQDIKPDIVAGTAACEEIRNSKKFSKILELILLLG
NYMNSGSKNEAAFGFEISYLTKLSNTKDADNKQTLLHYLADLVEKKFPDALNFYDDLSHVNKASRVNMDA
IQKAMRQMNSAVKNLETDLQNNKVPQCDDDKFSEVMGKFAEECRQQVDVLGKMQLQMEKLYKDLSEYYAF
DPSKYTMEEFFADIKTFKDAFQAAHNDNVRVREELEKKRRLQEAREQSAREQQERQQRKKAVVDMDAPQT
QEGVMDSLLEALQTGSAFGQRNRQARRQRPAGAERRAQLSRSRSRTRVTNGQLMTREMILNEVLGSA
>Fugu formin
IKTKFRLPVFNWTALKPNQINGTVFNEIDDERELELERFEELFKTRAQGPIMDLSCTKSKVAQKAVNKVT
ILDANRSKNLAITLRKANKTFDLKTLPVDFVECLMRFLPTEMEVKALRQYERERRPLDQLAEEDRFMLLF
SKIERLTQRMNIITFIGNFSDNVAMLTPQLNAIIAASASVKSSPKLKRMLEIILALGNYMNSSKRGCVYG
FKLQSLDLLLDTKSTDRKMTLLHYIALIVKEKYPELANFYNELHFVDKAAAVSLENVLLDVRELGKGMDL
IRRECSLHDHSVLKGFLQASDTQLDKVQRDAKTAEEAFNNVVNYFGESAKTAPPSVFFPVFVRFLKAYKD
AVEENELRKKQEQAMREKLLAEEAKQQDPKVQAQKKRQQQHELIAELRKRQAKDHRPVYEGKDGTIEDII
TVLK
>Cappuccino Drosophila
PPTAPPATKEIWTEIEETPLDNIDEFTELFSRQAIAPVSKPKELKVKRAKSIKVLDPERSRNVGIIWRSL
HVPSSEIEHAIYHIDTSVVSLEALQHMSNIQATEDELQRIKEAAGGDIPLDHPEQFLLDISLISMASERI
SCIVFQAEFEESVTLLFRKLETVSQLSQQLIESEDLKLVFSIILTLGNYMNGGNRQRGQADGFNLDILGK
LKDVKSKESHTTLLHFIVRTYIAQRRKEGVHPLEIRLPIPEPADVERAAQMDFEEVQQQIFDLNKKFLGC
KRTTAKVLAASRPEIMEPFKSKMEEFVEGADKSMAKLHQSLDECRDLFLETMRFYHFSPKACTLTLAQCT
PDQFFEYWTNFTNDFKDIWKKEITSLLNELMKKSKQAQIESRRNVSTKVEKSGRISLKERMLMRRSKN
>Bni1 yeast
PRPHKKLKQLHWEKLDCTDNSIWGTGKAEKFADDLYEKGVLADLEKAFA
AREIKSLASKRKEDLQKITFLSRDISQQFGINLHMYSSLSVADLVKKILN
CDRDFLQTPSVVEFLSKSEIIEVSVNLARNYAPYSTDWEGVRNLEDAKPP
EKDPNDLQRADQIYLQLMVNLESYWGSRMRALTVVTSYEREYNELLAKLR
KVDKAVSALQESDNLRNVFNVILAVGNFMNDTSKQAQGFKLSTLQRLTFI
KDTTNSMTFLNYVEKIVRLNYPSFNDFLSELEPVLDVVKVSIEQLVNDCK
DFSQSIVNVERSVEIGNLSDSSKFHPLDKVLIKTLPVLPEARKKGDLLED
EVKLTIMEFESLMHTYGEDSGDKFAKISFFKKFADFINEYKKAQAQNLAA
EEEERLYIKHKKIVEEQQKRAQEKEKQKENSNSPSSEGNEEDEAEDRRAV
MDKLLEQLKNA

If you had difficulties with the alignment (which is likely), try a different scoring matrix: save the alignment, create a new project with the PAM250 or BLOSUM80 matrix, and open your file from within the new project (MACAW will now remember the matrix setting).

In the resulting alignment, try to select regions that would be suitable for phylogenetic analysis.