Bioinformatics - protocols  D

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Basic sequence manipulation

>cDNA_C_At
CAAGCAACCTTTACACATATAGAAGAAGAAAAACACTTCTTTGTTTCTGTCATTAATTCCCTCCCTCTAT
ATATATATATTTAAATCTATTATGACAAAACAATCCAATACTGGATACTTTTTACAACAACATGCACAGA
CAAAGCTGAGATCTCACCTTAAGAAACTAATGAGATTGAGTTATGTTCGTCTTCATCAGACGAAGAGGTT
GAAGACGACGACGAAGAAGAAGAAGATTGTCTTCGTCCAACGAGTCCAGGAAGAGGTTGTGGCATCATTG
GATTCACTGGAACAGGAAACTTATGAGCAGAACTAACCATTGTTCTTTCGTTTATCATCCCTACTTCTTT
GCAAACTCTGTCTACTACTCCAAGGAAGTCTCTAACCACCAAGAATATTCTAAACGGATGCGCTTCTTCT
TTAGCCGAGTTTCCATGGAAATACTCTGTGATTTCTTTTACAAGTGATAACGCTACGCTCTCTTGAGCTT
GTACTCTGATGATCTCTTCCTCAGCTCTTTTCAGAAACNTTTTCATCGATTCCGNNAACCTCTGACTGTT
GCTTTCTTCTGTGATTGTTGATTGGACTTGGATTGCTTCGTTGATCTTGGCAATGNNTTGAGGAAAGCTT
GGAGANGTAGCTGCTTAGTACTTCTGAGTCCATCNNAGC

• Look at the coding capacity of your cDNA using either the BCM sequence utilities 6 Frame Translation function, or the ExPASy Translation Tool . Make sure that you are NOT using the default "Verbose" option on ExPASy (or just try it once so that you know why avoiding it :-). (You might want  to look also at other translation tools such as Nucleic Acid to Amino Acid Translation).
• Select the longest reading frame (it will be in antisense orientation, frame -2) and keep it as a FASTA file. (NOTE: you might not necessarily get a reasonable ORF - assume that this was a raw first run sequence, containing possible frameshifts). Try to convert the cDNA sequence to antisense using the BCM sequence utilities Reverse Complement function.

Sequence similarity searches and domain structure analysis

• Use the protein sequence from the previous excercise as a query for a standard protein FASTA search of the largest available non-redundant protein database on both available French servers. Keep default parameters where possible, take care of query format. If you were unable to perform the search for server-dependent reasons, go to the SOS page. Explore the results. You should be able to produce a working hypothesis about the possible function of your cDNA already at this stage! Why did both servers give different results?
• From the Infobiogen FASTA results page, retrieve the protein sequences corresponding to the first two and last two hits (by clicking the link to the left from the gene description). Copy the sequences in FASTA format from the bottom of each file opened (see Figure) and save them in a text file with the extension *.aa.


The file format should be FASTA, i.e.
>protein1
HERECOMESTHESEQUENCEOFPROTEINONE
>protein2
ANDHERETHESEQUENCEOFPROTEINTWO
etc.
(If the server is down, go to the SOS page).
• Take the first sequence from your file and run a ProfileScan on it, looking for both Prosite Profiles and Prosite Patterns, and examine the output. Then run a ProDom search using the BLAST facility of the server, and examine the results. Discuss the results with group C who used a different method to analyze this sequence. (If any of the servers was down, you can get the output from the SOS page).

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 you just created. 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.
• (Optional): repeat the last task using a different scoring matrix.

Gene building: searching for coding sequences in chromosomal DNA

>D_gene
TTTAATAAAATAAAAATCCACTCGCATTTTTATTTTCAACATTGTGCGTA
CGGTGCAATTCAATGAACAGTGTTTACTTTCAGTGTGTACACTTCTGCGG
ACTATTACAAAGTCCACGTCTTATCCTACGTGTTATAATCTCATATGTTA
CTGTCTGAAATGGACCCCACTACGTAAAAATAAAATTAAGAATCAACCAC
TCTTCTTCCATCACCTCTTTTGGCTTTCTCTCTACTCTCTCTACTACTCT
CTCACCATCACTGAGTTAAGAGAACAAACCAAAAACAAAATTATCAAACC
ATCACCAGCAGAATCTTAGCTGGATTCATCACTCTATTCAAAAAGTTTCT
CTCTTCTCTTTTCTCAGATCTTGAACTCTTGAAGAAGAAAGAAGAAGATA
ACACAATGCTCTTCTTCTTATTCTTCTTCTACTTACTCTTATCTTCATCC
TCCGATCTAGTCTTCGCCGACCGTCGTGTACTCCACGAACCATTCTTCCC
TATAGATTCACCACCACCGTCACCACCATCACCACCACCACTTCCTAAAC
TACCATTCTCTTCAACCACTCCTCCATCTTCATCAGACCCAAATGCTTCT
CCTTTCTTCCCTTTATACCCTTCATCTCCACCACCACCTTCTCCAGCCTC
CTTCGCTTCTTTTCCGGCGAATATCTCATCTCTAATCGTCCCTCACGCCA
CTAAATCCCCACCTAACTCCAAAAAACTCCTTATCGTCGCTATCTCCGCC
GTTTCCTCCGCTGCTTTAGTCGCTCTACTTATCGCTTTACTCTATTGGCG
AAGAAGCAAACGTAACCAAGATCTTAACTTCTCCGATGATAGCAAAACAT
ACACCACCGACAGTAGCCGCCGTGTCTACCCTCCTCCTCCGGCAACGGCG
CCTCCAACACGACGCAATGCGGAGGCTAGAAGTAAACAGAGGACCACCAC
GAGCTCCACCAATAACAACAGCTCTGAGTTTCTTTACTTAGGAACAATGG
TGAATCAAAGAGGAATCGATGAACAATCTCTTAGTAATAATGGATCAAGC
TCAAGAAAACTTGAATCTCCAGATCTTCAACCACTTCCTCCATTGATGAA
ACGAAGTTTCCGTTTAAATCCAGATGTTGGTTCAATCGGAGAAGAAGATG
AAGAAGATGAGTTTTACTCTCCACGTGGCTCACAAAGCGGGCGAGAACCG
TTAAACCGGGTCGGACTTCCGGGTCAAAATCCTAGATCTGTTAACAATGA
CACTATCTCTTGCTCATCTTCAAGCTCTGGTTCACCAGGAAGATCAACAT
TTATCAGTATCTCTCCTTCAATGAGTCCTAAGAGATCTGAACCAAAACCG
CCGGTTATCTCCACACCAGAACCGGCGGAGTTAACCGATTATAGATTTGT
TCGGTCTCCGTCACTGTCGTTAGCTTCTTTATCGTCGGGATTGAAAAACT
CCGATGAAGTAGGATTGAATCAAATCTTTAGATCTCCGACGGTTACATCT
CTAACAACTTCACCGGAGAATAACAAAAAAGAGAACTCTCCATTATCATC
TACTTCAACTTCACCGGAACGACGACCAAATGATACACCAGAAGCTTACT
TGAGATCTCCGTCGCATTCTTCTGCTTCTACATCACCGTATAGATGTTTT
CAGAAATCTCCGGAGGTCTTACCGGCGTTTATGAGTAATCTCCGGCAAGG
TTTGCAATCTCAGTTACTATCTTCTCCTTCTAACTCTCATGGAGGACAAG
GTTTCCTTAAGCAGTTAGATGCATTACGTTCTCGTTCACCGTCGTCGTCT
TCTTCTTCTGTTTGTTCTTCACCGGAGAAAGCTTCTCATAAGTCACCAGT
TACATCTCCTAAGTTATCTTCCCGGAATTCGCAGTCTCTATCATCTTCTC
CGGATAGAGATTTTAGTCATAGCTTAGATGTATCACCACGGATATCGAAC
ATTTCACCTCAAATTTTACAGTCTCGTGTGCCTCCGCCTCCTCCTCCTCC
CCCACCGTTGCCGTTGTGGGGACGACGGAGTCAGGTGACTACTAAAGCGG
ACACAATCTCGAGACCGCCTTCTCTTACACCGCCTTCACATCCTTTTGTG
ATCCCATCTGAAAACTTACCAGTGACTTCGTCTCCTATGGAGACTCCAGA
GACGGTTTGTGCGAGTGAGGCGGCGGAGGAAACTCCGAAACCGAAGCTAA
AGGCGTTACATTGGGATAAAGTTAGAGCAAGTTCGGATCGTGAGATGGTT
TGGGATCATCTTCGATCAAGCTCTTTCAAGTGAGTTAATGTGACATACTC
GTTTATATGATACTATATGCTTTTAGTGAGAATGTGGTTGTTGAGATTAT
GAATGTGGTTTGCAGATTAGATGAGGAGATGATTGAGACGTTGTTTGTGG
CGAAGTCGTTAAACAACAAACCAAATCAGAGTCAGACAACTCCAAGATGT
GTTCTCCCGAGCCCGAACCAAGAGAACAGAGTCCTGGACCCGAAGAAGGC
TCAGAATATTGCCATCTTGCTTCGTGCACTTAATGTCACTATAGAAGAAG
TTTGTGAGGCTCTTCTTGAAGGTAAACTATGCTGTCACATACATAGTTTC
TCATTTTCTTCTCCTTTGATCTCCAGAATTAGAGTTCTTATGCATTTGTT
AATGGTTTTTCGATGATATGGTTGAGTTATTCTGAAAGCTTTGCTTCTTT
GATGGTGTGGAGATTCTTGGTTACATTGATGTTCTTAGTTATGCTTTTTC
AGGCAATGCTGATACACTGGGGACTGAACTTCTTGAGAGCTTACTGAAGA
TGGCACCGACAAAAGAAGAAGAGCGCAAGTTGAAAGCGTACAATGATGAT
TCGCCTGTTAAGCTTGGACATGCTGAGAAATTCCTTAAGGCAATGTTGGA
CATCCCTTTCGCCTTTAAAAGAGTTGATGCAATGCTCTATGTAGCCAACT
TTGAGTCCGAGGTTGAATACTTGAAGAAATCTTTTGAGACTCTTGAGGTA
TATATTACAAGCTATTCTCTCTCTTTTTACCATATGGTTGTATTGTAACA
GATTATGACTTCATTTCTATTGTTTGTGTAGGCTGCTTGTGAAGAACTGA
GGAACAGTAGGATGTTCTTAAAGCTTCTTGAAGCGGTTCTAAAGACAGGA
AACCGTATGAACGTTGGAACAAACCGAGGAGATGCACATGCGTTCAAGCT
TGATACACTTCTCAAGCTAGTCGATGTCAAAGGCGCTGATGGGAAAACAA
CTCTCTTGCATTTCGTTGTACAAGAGATAATCCGAGCAGAAGGCACACGT
CTCTCAGGTAACAATACACAAACAGATGACATTAAATGCCGGAAACTAGG
TCTCCAAGTTGTATCAAGTCTCTGTTCTGAGCTTAGTAACGTCAAGAAAG
CTGCTGCGATGGACTCAGAAGTACTAAGCAGCTACGTCTCCAAGCTTTCT
CAAGGCATTGCCAAGATCAACGAAGCAATCCAAGTCCAATCAACAATCAC
AGAAGAAAGCAACAGTCAGAGGTTTTCGGAATCGATGAAAACGTTTCTGA
AAAGAGCTGAGGAAGAGATCATCAGAGTACAAGCTCAAGAGAGCGTAGCG
TTATCACTTGTAAAAGAAATCACAGAGTATTTCCATGGAAACTCGGCTAA
AGAAGAAGCGCATCCGTTTAGAATATTCTTGGTGGTTAGAGACTTCCTTG
GAGTAGTAGACAGAGTTTGCAAAGAAGTAGGGATGATAAACGAAAGAACA
ATGGTTAGTTCTGCTCATAAGTTTCCTGTTCCAGTGAATCCAATGATGCC
ACAACCTCTTCCTGGACTCGTTGGACGAAGACAATCTTCTTCTTCTTCGT
CGTCGTCTTCAACCTCTTCGTCTGATGAAGACGAACATAACTCAATCTCA
TTAGTTTCTTAAGGTGAGATCTCAGCTTTGTCTGTGCATGTTGTTGTAAA
AAGTATCCAGTATTGGATTGTTTTGTCATAATAGATTTAAATATATATAT
ATAGAGGGAGGGAATTAATGACAGAAACAAAGAAGTGTTTTTCTTTTCTG
CATTTGTGTAAAAAAAATAATATAGGTTTACCTTAAAATTTGTTCATCTT
AAATTAATAATTTAAGAATCAAATAAATTTGTTTATCTGAACCGTGTGTA
CCACGAAAGAATGTGAGAGCAAACATATTACTTACTTACCCTTCGTTGCT
GAATATAATGATCATTATAAATCACTACCTCCAGTACCTTCTACCTTCTT
CAAAGAACCTTGTTGGATTTGAACCAAAGTTGGAACATAATTGACGAGAG
GTGAGCATCTAGATTCTGCATCGTGATGATGATCCACTTTTATCTATTTA

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 E or use links to results, if possible).