forkSense

DNAscent forkSense is a DNAscent subprogram that provides a probability estimate at each thymidine that a leftward- or rightward-moving fork moved through that position during the BrdU pulse.

Usage

To run DNAscent forkSense, do:
   DNAscent forkSense -d /path/to/BrdUCalls.detect -o /path/to/output.forkSense
Required arguments are:
  -d,--detect               path to output file from DNAscent detect,
  -o,--output               path to output file for forkSense.
Optional arguments are:
  -t,--threads              number of threads (default: 1 thread),
     --markOrigins             writes replication origin locations to a bed file (default: off),
     --markTerminations        writes replication termination locations to a bed file (default: off),
     --markForks               writes replication fork locations to a bed file (default: off).

The only required input of DNAscent forkSense is the output file produced by DNAscent detect. Note that the detect file must have been produced using the v2.0 ResNet algorithm; DNAscent forkSense is not compatible with legacy HMM-based detection.

If the --markOrigins flag is passed, DNAscent forkSense will use detected leftward- and rightward-moving forks to infer the locations of fired replication origins and write these to a bed file called origins_DNAscent_forkSense.bed in the working directory. Likewise, if the --markTerminations flag is passed, termination sites will be recorded in a bed file called terminations_DNAscent_forkSense.bed.

Output

If --markOrigins and/or --markTerminations were used, the resulting bed files has one called origin (for origins_DNAscent_forkSense.bed) or termination site (for terminations_DNAscent_forkSense.bed) per line and, in accordance with bed format, have the following space-separated columns:

  • chromosome name,
  • 5’ boundary of the origin (or terminiation site),
  • 3’ boundary of the origin (or terminiation site),
  • read header of the read that the call came from (similar to those in the output file of DNAscent detect).

Note that the “resolution” of the calls (i.e., the third column minus the second column) will depend on your experimental setup. In synchronised early S-phase cells, this difference for origin calls is likely to be small as the leftward- and rightward-moving forks from a fired origin are nearby one another. In asynchronous or mid/late S-phase cells, the difference is likely to be larger as the forks from a single origin will have travelled some distance before the BrdU pulse. The bed files only specify the region between matching leftward- and rightward-moving forks. Any subsequent assumptions (such as assuming uniform fork speed and placing the origin in the middle of that region) are left to the user.

The output of DNAscent forkSense is a file with similar formatting to that of DNAscent detect. The format for the read headers is the same. From left to right, the tab-delimited columns indicate:

  • the coordinate on the reference,
  • probability that a leftward-moving fork passed through that coordinate during a BrdU pulse,
  • probability that a rightward-moving fork passed through that coordinate during a BrdU pulse.

A low probability in both the second and third columns suggests it was unlikely that a fork passed through that position during the pulse.

The following example output shows the end of a read that was passed through by a leftward-moving fork:

>22c8a674-ed0e-475f-9c54-cb185299d923 chrII 173332 210452 fwd
173339  0.687217        0.062620
173341  0.687217        0.062620
173342  0.687217        0.062620
173345  0.687217        0.062620
173347  0.687217        0.062620
173348  0.687217        0.062620
173349  0.743986        0.045767
173358  0.743986        0.045767
173375  0.743986        0.045767
173377  0.743986        0.045767
173378  0.743986        0.045767
173381  0.743986        0.045767
173382  0.806924        0.038138
173383  0.806924        0.038138
173387  0.806924        0.038138
173390  0.806924        0.038138
173392  0.806924        0.038138
173393  0.806924        0.038138
173398  0.846875        0.032027
173402  0.846875        0.032027
173404  0.846875        0.032027
173406  0.846875        0.032027
173407  0.846875        0.032027
173417  0.846875        0.032027
173418  0.906748        0.028587
173419  0.906748        0.028587
173423  0.906748        0.028587
173425  0.906748        0.028587
173426  0.906748        0.028587
173428  0.906748        0.028587
173441  0.909755        0.029341
173445  0.909755        0.029341
173446  0.909755        0.029341
173449  0.909755        0.029341
173450  0.909755        0.029341
173451  0.909755        0.029341
173454  0.907803        0.029983