Notes on the differences between DiffBind 3.0 and previous versions, and how run in a "backward compatible" manner.
Beginning with version 3.0,
substantial updates and new features that may cause scripts
written for earlier versions to function differently
(or not at all), as well as altering the results.
This page givens details on these changes,
and how to approximate results computed
with earlier version if desired.
The major change in version 3.0 is in how the data are modeled. In previous versions, a separate model was derived for each contrast, including data only for those samples present in the contrast. Model design options were implicit and limited to either a single factor, or a subset of two-factor "blocked" designs. Starting in version 3.0, the default mode is to include all the data in a single model, allowing for any allowable design formula and any set of allowable contrasts.
Another change starting from version 3.0 is in how normalization is done. There are more normalization options, and more explicit control over them. The default normalization options have also changed, so reproducing a pre-3.0 analysis requires that normalization parameters to be specified.
It is recommended that existing analyses be re-run with the current software.
Existing scripts should execute (with the exception of two
normalization parameters which have been moved from
to the new interface function
DiffBind vignette for more information on
processing and analyzing ChIP-seq (and ATAC-seq) experiments.
blacklist is applied by default, if available, using automatic detection of reference genome.
greylists are generated from controls and applied by default.
minimum read counts are now 0 instead of being rounded up to 1 (this is now controllable).
centering peaks around summits is now done by default using 401-bp wide peaks (recommend to use 'summits=100' for ATAC-seq).
read counting is now performed by 'summarizeOverlaps()' by default, with single-end/paired-end counting automatically detected.
filtering is performed by default; consensus peaks where no peak has and RPKM value of at least 1 in any sample are filtered.
control read subtraction is now turned off by default if a greylist is present
normalization is based on full library sizes by default for both 'edgeR' and 'DESeq2'analyses.
score is set to normalized values by default.
DiffBind scripts and saved objects will
run correctly using version 3.0, but there may be differences
in the results.
This section describes how to approximate earlier results for existing scripts and objects.
DBA object was created with an earlier version of
DiffBind, and saved using the
function, and loaded using the
all settings should be preserved, such that running the analysis anew
will yield the same results.
In order to re-run the analysis using the post-version 3.0 settings,
the original script should be used to re-create the
By default, if you re-run a
DiffBind script, it will use the
new defaults from version 3.0 and beyond.
In order to re-analyze an experiment in the pre-version 3.0 mode,
a number of defaults need to be changed.
dba.count, the following defaults are changed:
summits: This parameter is now set by default. Setting
summits=FALSE will preempt re-centering each
peak interval around its point of highest pileup.
filter: The new default for this parameter is
and is based on
RPKM values; previously
it was set to
filter=0 and was based on read counts.
minCount: This is a new parameter representing a minimum read count value.
It now default to
to get the previous behavior, set
The easiest way to perform subsequent processing in a pre-version 3.0 manner is to set a configuration option:
DBA$config$design <- FALSE
This will result in the appropriate defaults being set for
the new interface function,
does not need to be invoked explicitly.)
The pre-version 3.0 settings for
are as follows:
Note that two parameters that used to be available when calling
dba.analyze have been moved:
bSubControl: now integrated into
FALSE by default (unless a greylist has
been added using
bFullLibrarySize: now integrated into
dba.normalize as an option for the
library=DBA_LIBSIZE_FULL is equivalent to
library=DBA_LIBSIZE_PEAKREADS is equivalent to
DiffBind vignette has been updated to show how to
analyze experiments using version 3.0.
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