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ID X03006; SV 1; linear; mRNA; STD; MAM; 620 BP. XX AC X03006; XX SV X03006.1 XX DT 28-JAN-1986 (Rel. 08, Create

ID X03006; SV 1; linear; mRNA; STD; MAM; 620 BP. XX AC X03006; XX SV X03006.1 XX DT 28-JAN-1986 (Rel. 08, Created) DT 12-SEP-1993 (Rel. 36, Last updated, Version 2) XX DE Bovine mRNA for lens beta-s-crystallin XX KW beta-crystallin; beta-gamma-crystallin; crystallin. XX

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ID X03006; SV 1; linear; mRNA; STD; MAM; 620 BP. XX AC X03006; XX SV X03006.1 XX DT 28-JAN-1986 (Rel. 08, Create

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  1. ID X03006; SV 1; linear; mRNA; STD; MAM; 620 BP. XX AC X03006; XX SV X03006.1 XX DT 28-JAN-1986 (Rel. 08, Created) DT 12-SEP-1993 (Rel. 36, Last updated, Version 2) XX DE Bovine mRNA for lens beta-s-crystallin XX KW beta-crystallin; beta-gamma-crystallin; crystallin. XX OS Bos taurus (cow) OC Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; OC Eutheria; Laurasiatheria; Cetartiodactyla; Ruminantia; Pecora; Bovidae; OC Bovinae; Bos. XX RN [1] RP 1-620 RX PUBMED; 4054100. RA Quax-Jeuken Y.E.F.M., Driessen H., Leunissen J., Quax W.J., de Jong W., RA Bloemendal H.; RT "Beta-s-crystallin: structure and evolution of a distinct member of the RT beta-gamma-superfamily"; RL EMBO J. 4(10):2597-2602(1985). XX CC Data kindly reviewed (06-MAR-1986) by Y. Quax-Jeuken XX ... EMBL

  2. Index index parser flatfile

  3. Retrieve index display parser entries

  4. SRS Sequence Retrieval System an indexing and retrieval system for flat file databases

  5. http://srs.bioinformatics.nl

  6. http://srs.ebi.ac.uk

  7. Q: Which sequences in EMBL [do not] encode for a protein for which the 3D structure is known?

  8. Command line SRS Using getz

  9. Retrieve the UniProt entry for the protein with accession number P19558: getz "[uniprot-acc:P19558]" -e Count the human proteins in the UniProt database: getz "[uniprot-org:human]" –c Print sequence of the rice proteins in the UniProt database that have a length between 10 and 50 aa: getz "[uniprot-org:rice]&[uniprot-sl#10:50]" -f sl

  10. Give the id and description for all A.thal proteins that have at least 8 transmembrane domains: getz '[swissprot-org:arabidopsis thaliana]< ([swissprot-CountedItem:transmem] &[swissprot-CountedN#8:]))' -f "id des"

  11. Count the human protein sequences in the NCBI RefSeq database: getz "[refseqp-org:human]" –c Count the human mRNA sequences in the NCBI RefSeq database: getz "[refseq-org:human]&[refseq-mol:mrna]" –c Retrieve the mRNA sequences for all human proteins in the NCBI RefSeq database in fasta format : getz "[refseqp-org:human]>[refseq-mol:mrna]" –d –sffasta

  12. MRS: A fast and compact retrieval system for biological data. Hekkelman M.L., Vriend G. http://mrs.cmbi.ru.nl/

  13. European Molecular Biology Open Software Suite

  14. EMBOSS "European Molecular Biology Open Software Suite" http://emboss.sourceforge.net/ Toolbox with bioinformatics applications

  15. http://emboss.bioinformatics.nl/

  16. http://main.g2.bx.psu.edu/

  17. command line / shell

  18. Useful EMBOSS commands

  19. Get help from EMBOSS itself # showdb Shows the currently available databases # tfmwossname How to use a EMBOSS command? Just (r)tfm it # wossname alignment Which commands can handle alignments? # seealsoseqret Are there any other commands able to do the similar thing?

  20. Command line options • All EMBOSS programs react to a number of command line options. The most important ones are –help Get help –help –verbose Get elaborate help –auto “no questions asked” –stdout Write to standard output –filter Read stdin, write stdout

  21. SEQRET parameters zonnebloem> seqret -help Standard (Mandatory) qualifiers: [-sequence] seqall (Gapped) sequence(s) filename and optional format, or reference (input USA) [-outseq] seqoutall [<sequence>.<format>] Sequence set(s) filename and optional format (output USA) Additional (Optional) qualifiers: (none) Advanced (Unprompted) qualifiers: -feature boolean Use feature information -firstonlyboolean Read one sequence and stop General qualifiers: -help boolean Report command line options. More information on associated and general qualifiers can be found with -help -verbose

  22. SEQRET parameters zonnebloem> seqret -help -verbose Standard (Mandatory) qualifiers: [-sequence] seqall (Gapped) sequence(s) filename and optional format, or reference (input USA) [-outseq] seqoutall [<sequence>.<format>] Sequence set(s) filename and optional format (output USA) Additional (Optional) qualifiers: (none) Advanced (Unprompted) qualifiers: -feature boolean Use feature information -firstonlyboolean Read one sequence and stop Associated qualifiers: "-sequence" associated qualifiers -sbegin1 integer Start of each sequence to be used ///

  23. SEQRET parameters /// "-sequence" associated qualifiers -sbegin1 integer Start of each sequence to be used -send1 integer End of each sequence to be used -sreverse1 boolean Reverse (if DNA) -sask1 boolean Ask for begin/end/reverse -snucleotide1 boolean Sequence is nucleotide -sprotein1 boolean Sequence is protein -slower1 boolean Make lower case -supper1 boolean Make upper case -sformat1 string Input sequence format -sdbname1 string Database name -sid1 string Entryname -ufo1 string UFO features -fformat1 string Features format -fopenfile1 string Features file name ///

  24. SEQRET parameters /// "-outseq" associated qualifiers -osformat2 string Output seq format -osextension2 string File name extension -osname2 string Base file name -osdirectory2 string Output directory -osdbname2 string Database name to add -ossingle2 boolean Separate file for each entry -oufo2 string UFO features -offormat2 string Features format -ofname2 string Features file name -ofdirectory2 string Output directory ///

  25. SEQRET parameters /// General qualifiers: -auto boolean Turn off prompts -stdoutboolean Write standard output -filter boolean Read standard input, write standard output -options boolean Prompt for standard and additional values -debug boolean Write debug output to program.dbg -verbose boolean Report some/full command line options -help boolean Report command line options. More information on associated and general qualifiers can be found with -help -verbose -warning boolean Report warnings -error boolean Report errors -fatal boolean Report fatal errors -die boolean Report dying program messages

  26. Universal Sequence Address

  27. Universal Sequence Address Each of the above can have '[start : end]' or '[start : end : r]' appended to them. The 'file' and 'dbname' forms of USA can have 'format::' in front of them (although a database knows which format it is and so this is redundant and error-prone)

  28. Walk through exercise For a protein with UniProt Accession number: Q5ZKN6 find the nucleotide sequence that encodes this (repeated) amino acid fragment: VAEEVAEE

  29. Getting the sequence seqret -auto uniprot:Q5ZKN6 -stdout >Q5ZKN6_CHICK Q5ZKN6 SubName: Full=Putative uncharacterized protein; MADNLPSEFDVVVIGTGLPESIIAAACARSGQRVLHVDSRNYYGGNWASFSFSGLLSWIK ENQQNTDIKDECEDWRKLILENEEVISLNKKDKTIQHVEAFCFDDQDAAEDVEEAGALAR LPAYGASVAEEVAEEPEKECSPLESAVPGAENLESEKATSVDPASAAEGNVTEINAESES SHDSASGESTLESGKTEAALSEISAQEPKKITYSQIVREGRRFNIDLVSKLLYSRGLLIE LLIKSNVSRYAEFKNATRILAFREGKVEQVPCSRADVFNSRQLAMVEKRMLMKFLTFCLE YEQHPDEYQDYKNSTFAQFLKTRKLTPSLQHFILHSIAMVSEKDCNTLEGLQATRKFLQC LGRYGNTPFLFPLYGQGEIPQCFCRMCAVFGGIYCLRHSVQCLVVDKESGRCKAVVDHFG QRISANYFIVEDSYLSESVCENVCYRQLSRAVLITDQSVLKTDSEQQVSILMVPPVDLGQ PAVCVIELCSSTMTCMKDTYLVHLTCPSTKTAREDLEPVVQKLFSLNAETEKETEDEVLE KPRVLWALYFNMRDSSGIDRNSYSGLPSNVYVCSGPDSALGNDCAVKQAETIFQEMFPTE EFCPAPPNPEDIIYDEDEIASEETGFNNSPETKPESSLQESSSRGSSTAVKEHIEE

  30. Getting the sequence seqret -auto uniprot:Q5ZKN6 -stdout >Q5ZKN6_CHICK Q5ZKN6 SubName: Full=Putative uncharacterized protein; MADNLPSEFDVVVIGTGLPESIIAAACARSGQRVLHVDSRNYYGGNWASFSFSGLLSWIK ENQQNTDIKDECEDWRKLILENEEVISLNKKDKTIQHVEAFCFDDQDAAEDVEEAGALAR LPAYGASVAEEVAEEPEKECSPLESAVPGAENLESEKATSVDPASAAEGNVTEINAESES SHDSASGESTLESGKTEAALSEISAQEPKKITYSQIVREGRRFNIDLVSKLLYSRGLLIE LLIKSNVSRYAEFKNATRILAFREGKVEQVPCSRADVFNSRQLAMVEKRMLMKFLTFCLE YEQHPDEYQDYKNSTFAQFLKTRKLTPSLQHFILHSIAMVSEKDCNTLEGLQATRKFLQC LGRYGNTPFLFPLYGQGEIPQCFCRMCAVFGGIYCLRHSVQCLVVDKESGRCKAVVDHFG QRISANYFIVEDSYLSESVCENVCYRQLSRAVLITDQSVLKTDSEQQVSILMVPPVDLGQ PAVCVIELCSSTMTCMKDTYLVHLTCPSTKTAREDLEPVVQKLFSLNAETEKETEDEVLE KPRVLWALYFNMRDSSGIDRNSYSGLPSNVYVCSGPDSALGNDCAVKQAETIFQEMFPTE EFCPAPPNPEDIIYDEDEIASEETGFNNSPETKPESSLQESSSRGSSTAVKEHIEE

  31. Run a program within Perl: 3 ways $seq = `seqret -auto uniprot:Q5ZKN6 stdout`; system("seqret -auto uniprot:Q5ZKN6 stdout"); open SEQRET,"seqret -auto uniprot:Q5ZKN6 stdout|"; while(my $line = <SEQRET>) { if($line !~ /^>/) { chomp($line); $seq .= $line; } } close SEQRET;

  32. my $lsOutput = `ls -l`; put shell commands or programs in backticks to run from Perl. The output can be stored in a variable.

  33. open LS,"ls -l|"; The open function can run a program and read its output. The pipe symbol "|" links the output to a filehandle.

  34. Find the fragment’s position my $seq = ""; open SEQRET,"seqret -auto uniprot:Q5ZKN6 stdout|"; while(my $line = <SEQRET>) { if($line !~ /^>/) { chomp($line); $seq .= $line; } } close SEQRET; # look for location of the repeat my $position = index($seq, "VAEEVAEE") + 1; # print the offset print "Position = ", $position, "\n";

  35. !~ opposite of "=~ "gives true if the search found no hits.

  36. Get a cross-reference to EMBL entret uniprot:Q5ZKN6 -auto stdout |grep "DR " Get the feature table of this protein entry

  37. Understand the cross-reference DR EMBL; AJ720048; CAG31707.1; -; mRNA. Read the detailed documentation of UniProt cross reference http://www.expasy.org/sprot/userman.html#DR_line Status identifier Link to EMBL Protein ID Molecule Type EMBL accession number Database cross reference The corresponding cross reference in EMBL

  38. Get a cross-reference to EMBL entret uniprot:Q5ZKN6 -auto stdout | grep "DR " |grep "EMBL;" In Perl, use a regular expression to locate the EMBL reference line, and extract the EMBL accession number and the protein-ID

  39. Link protein to coding DNA extractfeat embl:AJ720048 -value CAG31707.1 stdout Returns the DNA coding for protein CAG31707.1 (=Q5ZKN6)

  40. Figure out the offset in DNA Offset in amino acid sequence: 128 Offset in corresponding nucleotide sequence: ((128-1) x 3) + 1 OR (128 x 3)-2 = 382 Position is from 382 to (382 + 8x3)=406 Figure out the position of its corresponding coding DNA sequence (is there anything wrong here?)

  41. Extract the DNA sequence extractfeat embl:AJ720048 -value CAG31707.1 stdout| extractseq –filter -reg"382-406" Now we got the corresponding DNA sequence for the protein fragment It should be: “gttgctgaggaggttgctgaagaac” But is that correct? Let's translate it for verification…

  42. Verify the result extractfeat embl:AJ720048 -value CAG31707.1 stdout| extractseq –filter -reg "382-406" | transeq-filter Result is “VAEEVAEEX” but not “VAEEVAEE” What’s wrong here? Always try to verify your results: computers make very few errors, but that is not true for people...

  43. Exercise Build a pipeline in Perl to perform the previous steps of the walkthrough (from slide 34) Test it with the UniProt protein A0L7N9 Find the fragment at offset 305 that is 8 aa long Find out the coding DNA of this amino acid fragment and verify it

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