iCLIPro usage¶
When calculating the start site overlap ratio a default flanking distance of 5 nt is used (parameter -s, see paper). A ratio well above 1 suggests to use the start sites of iCLIP fragments to detect binding sites (e.g., mean overlap start site ratio of 1.31 for U2AF65). Download latest version of Microsoft Word for Windows. Safe and Virus Free. IClip is the premier multiple clipboard & scrapbook tool that enables you to keep a history of almost anything you copy, instead of only the last thing. Would you like to see iClip in action? Click the Web Site link on the right to see a video. iClip was designed to be accessed as simply as the Mac’s Dock.
Usage: iCLIPro [options] in.bam
-o FOLDER | output folder (default is cwd - current working directory) |
-b INT | genomic bin size (100.1000, default: 300) |
-r INT | number of reads required in bin (20.500, default: 50) |
-s INT | flanking distances when calculating start site overlap ratio (3.15, default: 5) |
-q INT | use only reads with minimum mapping quality (mapq) (0.100, default: 10) |
-g LIST | read len groups (e.g.: “A:16-39,A1:16-25,A2:26-32,A3:33-39,L:20,B:42”) |
-p LIST | generate read overlap maps based on these comparisons (e.g.: “A1-A3,A2-A3,A1-B,A2-B,A3-B,L-B,A-B”) |
-f INT | flanking region for read overlap maps (default: 50) |
-h | longer help |
For given input BAM file [in.bam] the script will generatea number of output files that can be used to check for and diagnosesystematic misassignments in iCLIP data.
Main result is stored in file [in_report.txt], for given [in.bam] BAM file.
Read (query template) names in BAM files should include a recordof form expressed with this regular expression: :rbc[ATCGN]+:.The ending colon can be omitted if random barcode record is placedat the end of the name. Some valid examples:
Random barcodes can be specified at the end of the name butmust be preceded by colon, for example:
If no random barcode information is available, then iCLIPro willmost likely be able to work with the original read names. In suchcase, please check that the read names do not include any textthat conforms to the rules for specifying random barcode as itmay mislead iCLIPro.
The generated report file includes a list of random barcodesidentified by iCLIPro. You should check it first and make surethat proper random barcode information is being used.
- Parameters -b and -r:
- Specifies the bin size to use when segmenting the genome.Only bins with enough reads (parameter -r) are then consideredin the read overlap testing.
- Parameter -s:
- Flanking region when calculating the mean and the median startsite overlap ratios.
- Parameter -q:
- Consider only reads that pass the minimum mapping quality,ignore the rest.
- Parameter -g:
- iCLIPro needs to group reads based on their length.Any number of (overlapping) groups can be specified. For eachgroup, an interval (INT1-INT2) or single value (INT) of theread lengths in the group can be specified.
- Parameter -p (read overlap maps):
- Specifies which groups of reads to compare. When performing acomparison (G1-G2), cross-linked sites identified based ongroup G2 are used as reference (zero position). The relativepositioning of sites identified in G1 is the computed andshown in read overlap maps.
- Parameter -f:
- Width of the flanking region relative to reference pointshown in read overlap maps.
Method¶
A typical (i)CLIP experiment may result in the detection of RNAfragments of different lengths. Under the assumptions ofconventional iCLIP, the start sites of iCLIP fragments shouldcoincide at the cross-linking position in a fragmentlength-independent fashion.
This interpretation may not hold for some iCLIP libraries (e.g.,substantial read-through, binding to long RNA stretches etc). Fordetails, see associated paper by Hauer and coauthors. In summary,we identified a previously unrecognized effect of iCLIP fragmentlength on the position of fragment start sites and thus assignedbinding sites for some RBPs.
iCLIPro is a robust analysis approach that examines this effectand thus can improve the assignment of binding sites from iCLIPdata.
iCLIPro’s main function is to visualize coinciding andnon-coinciding fragment start sites in order to examine the bestway how to analyze iCLIP data.
With iCLIPro you can test test and compare the overlap ofdifferent reference points in the iCLIP fragments:
- one nucleotide before first mapped nucleotide (conventional assumption)
- center of the read
- end of the read
![Free Free](https://www.researchgate.net/profile/Zhen_Wang88/publication/312483407/figure/fig1/AS:452201727434755@1484824701854/An-overview-of-methods-and-experiments-a-A-simplified-schematic-of-the-iCLIP-protocol.png)
iCLIPro identifies regions (bins in genome, parameter -b) with asufficient number of reads (parameter -r) for an read overlaptest. Reads from each selected bin are processed separately.Reads get grouped based on their length (parameter -g) and sitesfrom different groups are compared.
The main output of iCLIPro are read overlap heatmaps thatidentify the best mode of analysis.
Read overlap heatmaps¶
Read overlap maps are generated by comparing fragment start,center and end sites in the test and reference groups.
The data underlying the high-resolution overlap heatmaps isused to calculate a ratio of overlapping and non-overlappingstart sites thus enabling the decision to be made as to whetherthe start or the center of the fragments should be used as areference point for most accurately defining the binding site.This overlap start site ratio is reported at the end of thegenerated report file. When calculating the start site overlapratio a default flanking distance of 5 nt is used(parameter -s, see paper).
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A ratio well above 1 suggests to use the start sites of iCLIPfragments to detect binding sites (e.g., mean overlap startsite ratio of 1.31 for U2AF65). A ratio below 1 favors theuse of the center position for binding site assignment(e.g., mean overlap start site ratio of 0.88 for eIF4A3,see paper for details).
Sites identified based on a reference group are used to definethe reference (zero) position in the map. The regions (-50 to +50,x-axis on plots, parameter -f) relative to the reference positionsare then scanned and number of co-occuring sites in test group is recorded.
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The x-axis shows the offset of the sites of the test group(shorter reads) relative to the sites of the reference group(usually longer reads). The y-axis shows the fragment length.The color in the heatmap represents the number of fragmentsthat co-occur at a given offset relative to the longer referencefragments.
Resample 1 1 5. In case of the fragment start sites, a peak at the start referenceposition 0 corresponds to coinciding start sites, whereas adistribution downstream of the reference position 0 arises fromstart sites of smaller fragments that occur at length-dependentoffsets from the reference start sites.