Run CSB-Aligner
What is a CSB-alignment?Many of the multi-species conserved sequences (MCSs) within enhancers are thought to consist of overlapping binding sites for transcriptional regulators. MCSs consist of conserved sequences blocks (CSBs), continuous blocs of conserved sequences averaging about 13 bp in length that may contain overlapping binding sites for transcriptional regulators. Comparison of CSBs from different enhancers reveal that they contain elements common to enhancers of different function as well as elements that bear the hallmarks of being specific for enhancers of similarly expressed genes. CSB-alignment compares CSBs of similarly expressed genes and of divergently expressed genes to discover the presence of sequence elements shared by enhancers. The output of CSB-alignment consists of cis-Decoder tags (cDTs) that represents the shared sequence elements common to different enhancers.
Generating a CSB-alignment
The first step in cis-DECODER analysis is the generation of EvoPrints using the EvoPrinter software. Full documentation for the EvoPrinter program is provided at the EvoPrinter home page.
The second step is the parsing of EvoPrints. EvoPrint-parser takes the output of EvoPrinter and converts it to a form that can be used by cis-DECODER. EvoPrint Parser extracts and annotates conserved Sequence Blocks (CSBs) in forward and reverse-complement directions. The default minimum length is set at six bases. Type the name of the enhancer into the appropriate window.
CSB-alignment requires two inputs: (1) the upper window accepts CSBs generated by a parser program (forward and reverse-complement sequences) (2) the lower window accepts either the EvoPrint or forward direction CSBs. The lower window should contain forward direction sequences only (i.e. not the reverse-complement).
After inputting the name of the parsed segments and the name of the sequence to be analyzed and selecting the minimum length of sequence element to be discovered (usually 6 or 7 bases), an alignment can be achieved by selecting the 'Align' button.
Alignments can be saved by selecting the 'File' button of the alignment window and saving. The saved alignment can then be copied into Microsoft Word with the format features preserved. To make the readout appear as it does in the HTML file, do the following: 1) change the page orientation to horizontal (in the 'Page setup' option of the file menu of Word), 2) convert the font to Courier and 3) reduce the font size to 8.
Generation cis-Decoder tag libraries from CSB alignments
A cDT-library represents a collection of cDTs that are shared by various enhancers examined. A tissue-specific library contains cDTs that are shared by a group of enhancers of co-expressed genes but are absent from a second set of enhancers from divergently regulated genes. A common cDTs library, contains cDTs that are shared between sets of enhancers of divergently regulated genes.
All libraries are generated from readouts of CSB-aligner. Making enhancer-type specific libraries requires two CSB-libraries, a library from the tissue of interest (e.g. neural), and a second library that serves as an outgroup (e.g. mesodermal). For the generation of a neural cDT-library, neural CSBs in both forward and reverse directions were copy/pasted into both upper and lower windows of CSB-aligner. The resulting cDTs from this alignment were listed in the 'Result of CSB alignment table' of the CSB-aligner output, in the column titled 'Motif.' Duplicated sequences were removed using the Java applet Puzzamatic 1.0 (http://www.mcn.org/c/rsurratt/Puzzamatic/WordSearchJar.html), a freeware created by Ron Surratt. Alphabetization and sorting of cDTs by size was also accomplished using Puzzamatic 1.0. The cDTs, constituting a raw neural cDT-library, were then copy/pasted into a Microsoft Word document. A second CSB-alignment was performed with the neural CSBs in the top window of CSB-aligner, and mesodermal CSBs in the lower window. The cDTs from this alignment were freed of duplicates. These cDTs constituted an unedited shared cDT-library. The shared cDT-library was copy/pasted into the same Microsoft Word document that contained the raw neural cDT-library. Sequences common to the two cDT libraries (present in the first and second alignments) are removed using the JavaScript program cDT-cleaner, leaving the neural-specific sequences. Neural enriched and common cDTs were curated from the unedited shared cDT-library.
A example of a CSB alignment is given below. The nerfin-1 #33 CSB (from a neural enhancer region) was aligned with a library of neural and mesodermal CSBs (designated as 'neuro' and 'meso' in the cDT name). The resultant alignment was saved and copy-pasted in Microsoft Word. Indicated in blue text (as modified in Microsoft Word) are the common cDTs that align with the nerfin-1 CSB. Indicated in red (as modified in Microsoft Word) are the neural specific cDTs.
| EvoPrinter |
|
Please feel free to contact Dr. Thomas Brody at brodyt@ninds.nih.gov or Dr. Ward Odenwald at odenwaldw@ninds.nih.gov if you have any questions about EvoPrinter or its applications.
Discover multispecies conserved sequences using EvoPrinter.
Return to cis-Decoder home .