1. Computational Biology
Dr. Jens Allmer
Lecture Slides Week 3

2. MBG404 Overview
Processing
Pipelining
Generation
Data
Storage
Mining

3. Initialization Files There are many types of initialization files
Often a plain text format is used such as
Name=value || Name:value
Usually separated by line breaks
Nearly always commenting parts of the information is supported (here ;)
XML and JSON are becomming more and more popular in this area
Many programs adopt JSON or XML formatted ini files
Name=value

4. File Formats FASTA Consists of William R. Pearson Program Suite
Definition line (starts with ‘>’)
Ends with line break or carriage return or both
Following the definition line are the sequences
ONLY IUPAC characters
Nucleotides
Amino Acids
Ends with
Next >
End of file
GenBank gi|gi-number|gb|accession|locus
EMBL Data Library gi|gi-number|emb|accession|locus
DDBJ, DNA Database of Japan gi|gi-number|dbj|accession|locus
NBRF PIR pir||entry
Protein Research Foundation prf||name
SWISS-PROT sp|accession|name
Brookhaven Protein Data Bank pdb|entry|chain
Brookhaven Protein Data Bank entry:chain|PDBID|CHAIN|SEQUENCE
Patents pat|country|number
GenInfo Backbone Id bbs|number
General database identifier gnl|database|identifier
NCBI Reference Sequence ref|accession|locus
Local Sequence identifier lcl|identifier
>gi|532319|pir|TVFV2E|TVFV2E envelope protein
ELRLRYCAPAGFALLKCNDADYDGFKTNCSNVSVVHCTNLMNTTVTTGLLLNGSYSENRT
QIWQKHRTSNDSALILLNKHYNLTVTCKRPGNKTVLPVTIMAGLVFHSQKYNLRLRQAWC
HFPSNWKGAWKEVKEEIVNLPKERYRGTNDPKRIFFQRQWGDPETANLWFNCHGEFFYCK
MDWFLNYLNNLTVDADHNECKNTSGTKSGNKRAPGPCVQRTYVACHIRSVIIWLETISKK
TYAPPREGHLECTSTVTGMTVELNYIPKNRTNVTLSPQIESIWAAELDRYKLVEITPIGF
APTEVRRYTGGHERQKRVPFVXXXXXXXXXXXXXXXXXXXXXXVQSQHLLAGILQQQKNL
LAAVEAQQQMLKLTIWGVK

5. FASTA Soft Rules Depend on the impementation of a specific program
No space directly after ‘>’
Should not contain more than 80 characters
May not contain whitespace
Space
Tabulator
Other control characters
May only contain IUPAC nucleotides and amino acids
Note: often all foreign characters are simply ignored

6. eXtensible Markup Language
Aims to describe data
Each item in an XML file can be clearly defined and understood
Can be validated
Is humanly readable
Could be used to invent HTML
Can be used instead of HTML
Contains no formatting instructions (HTML, CSS, XSLT)

7. XML A tag An element consist of an opening and a closing tag
Is a word enclosed in angle brackets ‘<>’
<tag>
An element consist of an opening and a closing tag
<tag> opening tag
</tag> closing tag (note slash ‘/’)
An element can be abbreviated if it contains no further elements
<tag /> open and closed in one statement
An element may contain any number of elements
<tag1><tag2></tag2></tag1>
An element may contain text information
<tag>Some important information about tags</tag>

8. XML
An element can contain attributes which describe the element further
<tag attribute=“a green tag” />
The name of an element should describe its content
<Sequences></Sequences>
Should contain .. Of course .. Sequences
Children of an element should be related to the parent
<Sequences>
<Sequence></Sequence><Sequence></Sequence>
</Sequences>
Attributes should extent the information logically
<Sequences content=“1”>
<Sequence id=“gi|532319|pir|TVFV2E|TVFV2E envelope protein” length=“35”>
THEACTUALSEQUENCETHATISDESCRIBEDINTHISELEMENT
</Sequence>

9. XML Elements and Attributes can be used interchangebly
<Sequence>
<identifier>gi|532319|pir|TVFV2E|TVFV2E envelope protein</identifier>
<length>35</length>
<seq type=“english”>
THEACTUALSEQUENCETHATISDESCRIBEDINTHISELEMENT
</seq>
</Sequence>
Attributes and Elements can be mixed
If the elements are properly nested and the tags are appropriately opened, closed and matched and the attributes are properly quoted ...
Then the XML is well formed
It is not neccessarily valid

10. XML Validation Data can be precisely described
Only elements defined may be used in an XML file if it is to be valid
Validation styles
Document Type Definitions DTD
XML Schema
Validation ensures that only expected data is in the document
Program can then ensure that they understand the document

11. An XML File Declaration for the consumer of the XML file
<?xml version=“1.0” encoding=“UTF-8” ?>
A root element which will contain the complete data needs to be defined
<Sequences> <!-- Rest of thefile --> </Sequences>
An XML file is like a tree consiting of
Roots? No .. 1 root
Trunk elements immediately following the root
Branches (Elements containing further elements [nested])
Leaves (Elements with no contained elements)
Where are the attributes?

12. XML File Example <?xml version=“1.0” encoding=“UTF-8” ?>
<Sequences>
<Sequence>
<identifier>gi|532319|pir|TVFV2E|TVFV2E envelope protein</identifier>
<length>35</length>
<seq type=“nucleotides”>
THEACTUALSEQUENCETHATISDESCRIBEDINTHISELEMENT
</seq>
</Sequence>
<identifier>gi|732413|pir|PDP22E|PDP22E predicted protein</identifier>
ANOTHERSEQUENCETHATMAYBERECORDEDSOMEWHERE
</Sequences>

13. Styling of XML (4get HTML)
Cascading Stylesheets (CSS)
Define styles for the elements that shall be displayed
Sequence {
Font-famiy: Courier,Monospace;
Background-color:#DDDDDD;
display:block;
border-bottom: 1px outset green; }
Identifier {
Font-family: Arial,Helvetica
font-size: 1.2em;
font-weight: bold;
Length {
display:none;
Connecting XML and CSS
<?xml-stylesheet type="text/css" href="style.css" ?>
Add at the top of the file just after version information

14. Connecting to an XML Schema
The schema or definition is linked in the root element
<Sequences
xmlns="
xmlns:xsi="
xsi:schemaLocation=" >
First attribute namespace
Second attribute type of schema (here XML)
Third attribute schema location (namespace + URL)

15. JavaScript Object Notation
JSON models objects
An object is enclosed in curly braces ‘{ }’
An array is enclosed in braces ‘[ ]’
Pairs
“Name” : “Value”
Separated by commas
Example
{“Sequence“ : “ACGCTAGCCGCATCGTAGC”}
{“Sequence“ : “ACGCTAGCCGCATCGTAGC”,
“Identifier” : “gi|532319|pir|TVFV2E|TVFV2E envelope protein”,
“length” : 35 }
{“Sequences” : [ {“Sequence“ : “ACGCTAGCCGCATCGTAGC”,
“Identifier” : “gi|532319|pir|TVFV2E|TVFV2E envelope protein”,
“length” : 19
{“Sequence“ : “ACGCTAACGCTGATCAGCGGCATCGCCGCATCGTAGC”,
“Identifier” : “gi|764319|pir|AVBB2E|AVBB2E predicted protein”,
“length” : 38 ]

16. XML - JSON JSON is like XML because: JSON is UNlike XML because:
They are both 'self-describing' meaning that values are named, and thus 'human readable'
Both are hierarchical. (i.e. You can have values within values.)
Both can be parsed and used by lots of programming languages
Both can be passed around using AJAX (i.e. httpWebRequest)
JSON is UNlike XML because:
XML uses angle brackets, with a tag name at the start and end of an element: JSON uses squiggly brackets with the name only at the beginning of the element.
JSON is less verbose so it's definitely quicker for humans to write, and probably quicker for us to read.
JSON can be parsed trivially using the eval() procedure in JavaScript
JSON includes arrays {where each element doesn't have a name of its own}
In XML you can use any name you want for an element, in JSON you can't use reserved words from javascript

17. ASN.1 Format

18. End of Theory I
5 min Mindmapping
10 min break

19. Practice I

20. File Formats FASTA Create a FASTA formatted file
Goto mbg403.allmer.de/tools
Choose FASTAValidator and check if your FASTA file is correct.

21. File Formats
XML
Write an XML file which will model a sequence similar to FASTA
<?xml version=“1.0” ?>
<Sequences
xmlns="
xmlns:xsi="
xsi:schemaLocation="
<Sequence Identifier=“some id” Type=“nucleotides||amino acids”>
THE_SEQUENCE
</Sequence>
</Sequences>

22. Validation Validate the instance of the XML file that you wrote
Copy and paste your data in one of those forms and see whether your XML
is well formed
is valid

23. Style the File Use the provided CSS and XML file as a reference
Change the styling such that it is pleasant for you to view

24. U Design Write an XML file which models a person
No validation possible unless you can create the schema as well (not part of this course)
Add information about yourself in the file
Style the file using CSS
Example from mbg403.allmer.de
Present your creation

25. Theory II

26. Sequence Alignment Exact Approximate simple More difficult target
pattern
target
pattern

27. Sequence Alignment Exact pattern matching
Naive method aligns pattern with each location of the target
Boyer-Moore indexes the pattern to skip some alignments
Wu-Manber indexes many patterns and skips some alignments
Indexing
Suffix tree indexes target and then quickly finds each pattern
Many other methods

28. Sequence Alignment Approximate pattern matching Pairwise Multiple
Local
Smith Waterman
BLAST
FASTA
Global
Needlemann Wunsch
Multiple
T-Coffee
ClustalW
...

29. Basic Local Alignment Seach Tool
Input
Pattern
Target
Search parameters and settings
Output
Alignments in various formats
XML
Help

30. BLAST Target Target indexing Needs to be indexed Cannot be FASTA
Must fit to the pattern and BLAST variant
protein target and protein pattern can be searched using blastp
Target indexing
makeblastdb, in the BLAST package can index FASTA files
Needs sequence input (e.g. FASTA, asn.1)
Needs sequence type to be provided e.g.: protein

31. BLAST blastp Needs indexed database
Needs query sequence (can be unindexed FASTA)
Produces alignments

32. Poll class - How many of you have done a BLAST search before?
We’ll all get to do BLAST searches by the end of today
How do you do a BLAST search?
Determing factors about how effective you are a reaching your goals of identify homologs in the results
Scoring model – implict in BLAST program, distinguish true from false positives
Database – quantity and quality of information
Method – efficient and comprehensive

33. Blast flavors Query: DNA Protein DB: DNA Protein
BlastN - nt versus nt database
BlastP - protein versus protein database
BlastX - translated nt (6 frames) versus protein database
tBlastN - protein versus translated nt database (6 frames)
tBlastX - translated nt versus translated nt database (both 6 frames)

34. BLAST Output
XML
-outfmt 5
This switch leads to XML output

35. Download Blast
Get blastp and makeblastdb from mbg404 since you are not allowed to install anything
Download a Fasta file (protein, genome, collection of sequences in fasta format)
Database must consist of amino acids since we only have access to blastp today
Use makeblastdb from the Blast package to index the file
Several files will be created when you do it right

36. MakeDB Example More information?
makeblastdb -in seq.fasta -dbtype prot -out seqBl –title seqBlastDB
More information?
Go to the doc folder of BLAST
Documentation is there

37. BLAST Now that we have an indexed database try to run BLAST
Read documentation and try to solve the simplest case
You will need the indexed database and you will need a FASTA file as query
You could create queries from the database and slightly change them
Good luck

38. Theory II

39. Mass Spectra Recording (e.g. Triple Play)
4500
4505

40. Fragmentation Spectrum

41. MS/MS spectra MS/MS spectra can be assigned a peptide sequence (PSM)
Database search
De novo sequencing

42. PepNovo Performs de novo sequencing of MS/ MS spectra
Takes a single spectrum as input
Needs a mathematical model for its evaluation
Will display the results in the console
You will therefore need to redirect the output
Example
?>PepNovo.exe -dta MSMSSpectrum.dta -model tryp_model.txt

43. De Novo Sequencing 1016.4 901.6 772.4 901.6 129.2 114.8 ~ E (129.1)KA I A Q L E D Y E D LY D E L Q A I K

44. MS/MS spectra MS/MS spectra can be assigned a peptide sequence (PSM)
Database search
De novo sequencing

45. Correlation Database selection >1080ZR IAAYPGVSPGLMIHYNIGR
>1137RZ
AAYPGATQPGATELARRLGK
>1152RZ
GSGDAAYPGGPFFNLFNLGK
>1152ZR
>2360RZ
VDSGWGGVVVVALAPYNLGR
>240RZ
HPGVVCRPGRGGGCSRHIGK
HPGVVCCSRHRRSHTIGK
-0.10
-0.05
0.00
0.05
0.10 1 2 3 4 5 6

46. Initalization Files X!Tandem Running X!Tandem That was easy
Taxonomy.xml
Default_Input.xml
Input.xml
Running X!Tandem
?>tandem.exe input.xml
That was easy
But behold, what about the input?

47. Taxonomy XML <?xml version="1.0" ?>
<bioml label="x! taxon-to-file matching list">
<taxon label="chlamy">
  <file format="peptide" URL="test_chlre2.fasta.pro" />
  </taxon>
</bioml>

48. Input.xml Another input file Personally, I don’t approve of
<?xml version="1.0" ?>
<bioml>
  <note>Each one of the parameters for x! tandem is entered as a labeled note node. Any of the entries in the default_input.xml file can be over-ridden by adding a corresponding entry to this file. This file represents a minimum input file, with only entries for the default settings, the output file and the input spectra file name. See the taxonomy.xml file for a description of how FASTA sequence list files are linked to a taxon name.</note>
  <note type="input" label="list path, default parameters">default_input.xml</note>
  <note type="input" label="list path, taxonomy information">taxonomy.xml</note>
  <note type="input" label="protein, taxon">chlamy</note>
  <note type="input" label="spectrum, path">test_spectra.mgf</note>
  <note type="input" label="output, path">output.xml</note>
</bioml>
Another input file
Personally,
I don’t approve of
the XML used here

49. Default-input XML <?xml version="1.0"?>
<?xml-stylesheet type="text/xsl" href="tandem-input-style.xsl"?>
<bioml>
<note>list path parameters</note>
<note type="input" label="list path, default parameters">default_input.xml</note>
<note>This value is ignored when it is present in the default parameter
list path.</note>
<note type="input" label="list path, taxonomy information">taxonomy.xml</note>
<note>spectrum parameters</note>
<note type="input" label="spectrum, fragment monoisotopic mass error">0.4</note>
<note type="input" label="spectrum, parent monoisotopic mass error plus">100</note>
<note type="input" label="spectrum, parent monoisotopic mass error minus">100</note>
<note type="input" label="spectrum, parent monoisotopic mass isotope error">yes</note>
<note type="input" label="spectrum, fragment monoisotopic mass error units">Daltons</note>
<note>The value for this parameter may be 'Daltons' or 'ppm': all other values are ignored</note>
<note type="input" label="spectrum, parent monoisotopic mass error units">ppm</note>
<note type="input" label="spectrum, fragment mass type">monoisotopic</note>
<note>values are monoisotopic|average </note>
<note>spectrum conditioning parameters</note>
<note type="input" label="spectrum, dynamic range">100.0</note>
<note>The peaks read in are normalized so that the most intense peak
is set to the dynamic range value. All peaks with values of less that
1, using this normalization, are not used. This normalization has the
overall effect of setting a threshold value for peak intensities.</note>
<note type="input" label="spectrum, total peaks">50</note>
<note>If this value is 0, it is ignored. If it is greater than zero (lets say 50),
then the number of peaks in the spectrum with be limited to the 50 most intense
peaks in the spectrum. X! tandem does not do any peak finding: it only
limits the peaks used by this parameter, and the dynamic range parameter.</note>
<note type="input" label="spectrum, maximum parent charge">4</note>
<note type="input" label="spectrum, use noise suppression">yes</note>
<note type="input" label="spectrum, minimum parent m+h">500.0</note>
<note type="input" label="spectrum, minimum fragment mz">150.0</note>
<note type="input" label="spectrum, minimum peaks">15</note>
<note type="input" label="spectrum, threads">1</note>
<note type="input" label="spectrum, sequence batch size">1000</note>
<note>residue modification parameters</note>
</bioml>

50. Beautifying XML XML Formatting of XML Transformation (XSLT)
Only describes data
Formatting of XML
Additional files can be linked to beautify the display
Transformation (XSLT)
Translates XML into HTML
XML Styling (CSS)
Describes formatting to the elements and attributes used in the XML file
Both files need to be linked at the beginning of the XML file

51. XML What is an element? What is an attribute? Design a Person
What are attributes of a person?
Use elements for logical grouping
Use attributes for specific information

52. Styling Connect the example style Nothing will be styled ;)
Examine the CSS file and rename the styles such that your person XML will be somewhat styled

53. End Theory II
5 min mindmapping
10 min break

54. Practice II

55. View Spectra and Sequence
To view matching peaks of the PepNovo prediction and the spectrum at the same time
Use the DtaViewer from

56. Download Download PepNovo Download test file
Download test file

57. Try PepNovo Try to run PepNovo Aim Use the given input
Use the help information
Use the lecture slides
Use the lecture notes
Aim
Store the result in a text file

58. PepNovo Results are displayed in the console
We need to redirect the output into a file.
?>PepNovo.exe -dta MSMSSpectrum.dta
-model tryp_model.txt
> result.txt

59. X!Tandem Unzip folder and check Mgf formated spectra (file)
Database file (FASTA)
tandem-win folder
Used .xml configuration files (default_input.xml, input.xml and taxonomy.xml)
To get the same output given in zip folder;
Replace configuration files in «tandem-win\bin» folder with ones in «used» folder.
Also copy database file to «fasta» folder and .mgf file to «bin» in «tandem-win»

60. X!Tandem Console Application

61. X!Tandem Default Input
Parameters such as mass tolerances, enzyme type, number of charged for search can be reset in default_input.xml

62. X!Tandem Input.xml In input.xml file, you should specify path of:
taxonomy.xml
default_input.xml
Spectra filename
Output filename
NOTE: Here input.xml and all files above are in same folder(directory))

63. X!Tandem Taxonomy
In taxonomy file, you should specify «database file path». In this example, database file is in «fasta» folder in «Xtandem\tandem-win » folder.

64. X!Tandem Output