Package: sep241
2for1separator2for1 separator is an algorithm to deconvolve CUT&Tag2for1 data.  It uses
differences in the fragment length distributions of the two targets and the
proximity of chromatin cuts to estimate the probability for each cut to
originate from one target or the other. The result is a set of cut density
tracks that represent the estimated number of cuts induced by the two
antibodies used in the CUT&Tag2for1 experiment.InstallationUsing condaTBAUsing pipPlease make surepythonpoints to a Python 3.9+ interpreter
andlibcurlis installed.
We highly recommed to install installscikit-sparseto significantly reduce memory demand and runtime.
Finally, inslatt 2for1separator withpipinstallsep241From sourceTo install from source you can run:gitclonehttps://github.com/settylab/2for1separatorcd2for1separator
pipinstall.UsageBefore the deconvolution, the data has to be split up into manageable chunks:sep241prep [bed files] --out [jobdata pkl file] --memory [max memory target in GB]It is recommended to use approximately20GB or more for--memory.
This specifies the targeted memory demand during the subsequent deconvolution.The output of the function reports the number of separate work chunks
and suggests subsequent calls for the deconvolution.
The number of slurm jobs is also stored in an additional output file named
with[jobdata pkl file].njobs.
Note that if memory resources are exhausted, deconvolution jobs
may be cancled by slurm or the operating system and subsequent
results will be missing. The downstream scripts will look for missing
results and report a comma sperated list of respective work chunk
numbers. If you are using slurm you can rerun the slurm jobs of only
the specified jobs by passing the list with the--array=parameter
of thesbatchcommand.Exporting ResultsIf not specified otherwise through the--outargument, all outputs are placed
into the same directory with the[jobdata pkl file]. Most outputs containc1orc2in their file name, which stand forchannel 1orchannel 2,
and represent the two constituent parts of the data that were
induced by the two different targets and that were
reconstructed through the deconvolution.To produce bigwig files from the deconvolution results runsep241mkbw [jobdata pkl file] [chrom sizes file]The chomosome sizes file needs to have two columns withchromosome nameandsize in bases(seebigWIG format).The produced bigWIG files may be used for downstream analysis such as peak
calling. To use the 2for1seperator cut-likelihood-based peak calling
with overlap identification run:sep241peakcalling [jobdata pkl file]Note, thatsep241peakcallingdoes not require the prior conversion
to bigWIG but instead uses the raw deconvolution output.To write out the target specific likelihoods of each genomic cut you can runsep241events [jobdata pkl file]For more information pass--helpto the respective commands.VisualizationOutput files are bigwigs and bed-files. These can be visualised
with software tools such as theIGV BrowserorJBrowser 2.
Visualization of intermediate results are currently only possible
if intermediate function calls within the supplied scripts are
reproduced in a python environment.CitationJanssens, D.H., Otto, D.J., Meers, M.P. et al. CUT&Tag2for1: a modified method
for simultaneous profiling of the accessible and silenced regulome in single
cells. Genome Biol 23, 81 (2022).https://doi.org/10.1186/s13059-022-02642-w