Normalization

Proper normalization is important as it ensures that other sources of variability are not mistakenly treated as real differences in datasets. TRANSIT provides various normalization methods, which are briefly described below:

• TTR:

  Trimmed Total Reads (TTR), normalized by the total read-counts (like totreads), but trims top and bottom 5% of read-counts. This is the recommended normalization method for most cases, as it has the benefit of compensating for differences in saturation (which is especially important for resampling).

• nzmean:

  Normalizes datasets to have the same mean over the non-zero sites.

• totreads:

  Normalizes datasets by total read-counts, and scales them to have the same mean over all counts.

• zinfnb:

  Fits a zero-inflated negative binomial model, and then divides read-counts by the mean. The zero-inflated negative binomial model will treat some empty sites as belonging to the “true” negative binomial distribution responsible for read-counts while treating the others as “essential” (and thus not influencing its parameters).

• quantile:

  Normalizes datasets using the quantile normalization method described by Bolstad et al. (2003). In this normalization procedure, datasets are sorted, an empirical distribution is estimated as the mean across the sorted datasets at each site, and then the original (unsorted) datasets are assigned values from the empirical distribution based on their quantiles. This actually doesn’t work well on TnSeq data if a large fraction of TA sites have counts of 0 (ties).

• betageom:

  Normalizes the datasets to fit an “ideal” Geometric distribution with a variable probability parameter p. Specially useful for datasets that contain a large skew.

• nonorm:

  No normalization is performed.

Beta-Geometric Correction (BGC)

If you have a “bad” or poorly-behaving or “skewed” dataset (e.g. with mostly low counts, dominated by a few high counts), right now the only remedy you can try is applying the Beta-Geometric correction (BGC), which is a non-linear adjustment to the insertion counts in a wig file to make them more like an ideal Geometric distribution (DeJesus & Ioerger, 2016).

Note, BGC is the only non-linear normalization available in Transit. All the other normalization methods, like TTR, are linear adjustments (scaling) of counts, and so they can’t correct for skewing.

BGC normalization helps ‘de-skew’ a dataset by bring the highest insertion counts down into the range of a few thousands. This sometimes improves statistical analyses like resampling, etc by reducing noise.

In the GUI, when you are looking at wig files loaded, you can change the normalization (e.g. from TTR to betageom) using the drop-down. Be aware that the Beta-Geometric normalization is computationally-intensive and might take few minutes per sample.

If it looks like it might help (i.e. if the QQ-plot fits the diagonal better using BG normalization), you can create BG-corrected versions of individual wig files by exporting them using the normalize command on the command-line with ‘-n betageom’ to specify normalization.

Command-line

In addition to choosing normalization for various analyses in the GUI, you can also call Transit to normalize wig files from the command-line, as shown in this example:

Example

> python3 src/transit.py normalize --help

usage: python3 src/transit.py normalize <input.wig> <output.wig> [-n TTR|betageom]
   or: python3 src/transit.py normalize -c <combined_wig> <output> [-n TTR|betageom]

> python3 src/transit.py normalize Rv_1_H37RvRef.wig Rv_1_H37RvRef_TTR.wig -n TTR

> python3 src/transit.py normalize Rv_1_H37RvRef.wig Rv_1_H37RvRef_BG.wig -n betageom

The normalize command now also works on combined_wig files too. If the input file is a combined_wig file, indicate it with a ‘-c’ flag.

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