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EZbakRKinetics helper function for users

EZbakRKinetics.Rd

EZbakRKinetics creates an object of class EZbakRKinetics and checks the validity of the provided input.

Usage

EZbakRKinetics(kinetics, metadf, features, name = NULL, character_limit = 20)

Arguments

kinetics

Data frame with the following columns:

  • sample: Name given to particular sample from which data was collected.

  • features: Any columns that cannot be interpreted as a mutation count or base nucleotide count (and that aren't named sample or n) will be interpreted as an ID for a genomic "feature" from which a read originated. Common examples of features and typical column names for said features include:

    • Genes; common column names: gene, gene_id, gene_name, GF

    • Genes-exonic; common column names: gene_exon, gene_id_exon, gene_name_exon, XF

    • Transcripts; common column names: transcripts, TF

    • Exonic bins; common column names: exonic_bins, EF, EB

    • Exons; common column names: exons, exon_ids

    In some cases, a read will often map to multiple features (e.g., exons). Many functions in bakR expect each of the feature IDs in these cases to be separated by +. For example, if a read overlaps with two exons, with IDs exon_1 and exon_2, then the corresponding entry in a column of exonic assignments would be "exon_1+exon_2". The default expectation can be overwritten though and is thus not strictly enforced.

  • n: Number of reads with identical values for all other columns.

  • kinetic parameter estimates: These can be named whatever you would like as long as they do not start with the string "se_". This should be reserved for kinetic parameter uncertainties, if provided.

  • kinetic parameter uncertainties: Uncertainty in your kinetic parameter estimates. These should be named "se_" followed by the kinetic parameter as its name appears in the relevant column name of the kinetics table.

metadf

Data frame detailing various aspects of each of the samples included in the kinetics data frame. This includes:

  • sample: The sample ID, which should correspond to a sample ID in the provided kinetics data frame.

  • tl: Metabolic label time. There are several edge cases to be aware of:

    • If more than one metabolic label was used in the set of samples described by the metadf (e.g., s4U and s6G were used), then the tl column should be replaced by tl_<muttype>, where <muttype> represents the corresponding mutation type referenced in the fractions that the label whose incubation time will be listed in this column. For example, if feeding with s4U in some samples and s6G in others, then performing standard nucleotide recoding chemistry, you will include tl_TC and tl_GA columns corresponding to the s4U and s6G label times, respectively.

    • If a pulse-chase experimental design was used (!!this is strongly discouraged unless you have a legitimate reason to prefer this design to a pulse-label design!!), then you should have columns named tpulse and tchase, corresponding to the pulse and chase times respectively. The same _ convention should be used in the case of multi-label pulse-chase designs.

  • sample characteristics: The remaining columns can be named whatever you like and should include distinguishing features of groups of samples. Common columns might include:

    • treatment: The experimental treatment applied to a set of samples. This could represent things like genetic knockouts or knockdowns, drug treatments, etc.

    • batch: An ID for sets of samples that were collected and/or processed together. Useful for regressing out technical batch effects

  • assay: This optional column should include a string that describes the type of experiment that was done so as to influence how EZbakR analyzes and interprets the data from those samples. Possible values for assay currently include:

    • standard: Refers to the "standard" nucleotide recoding RNA-seq methods (e.g., TimeLapse-seq, SLAM-seq, TUC-seq, etc.), in which cells are fed with a single metabolic label, RNA is extracted and sequenced, and mutations of a particular type are counted

    • STL: Refers to Start-TimeLapse-seq, a method combining Start-seq (developed by Karen Adelman's lab) with TimeLapse-seq. Used to infer the kinetics of transcription initiation and promoter-proximal pause site departure.

    • TT: Refers to Transient-Transcriptome NR-seq, a method combining TT-seq (developed by Patrick Cramer's lab) with NR-seq. TT-seq involves biochemically enriching for labeled RNA. By combining this method with nucleotide recoding chemistry (as was first done by the Simon lab with TT-TimeLapse-seq and has since been done with SLAM chemistry, often referred to as TTchem-seq), it is possible to bioinformatically filter out reads coming from unlabeled RNA background.

    • TILAC: Refers to TILAC, a method developed by the Simon lab to achieve spike-in free normalization of RNA-seq data through the use of a dual labeling approach inspired by the proteomic method SILAC.

    • subcellular: Refers to techniques such as subcellular TimeLapse-seq (developed by Stirling Churchman's lab) which combine subcellular fractionation with NR-seq to infer additional kinetic parameters.

    • sc: Refers to single-cell RNA-seq implementations of NR-seq.

features

Features tracked in kinetics data frame. Needs to be specified explicitly as it cannot be automatically inferred.

name

Optional; name to give to fractions table.

character_limit

If name is chosen automatically, limit on the number of characters in said name. If default name yields a string longer than this, then kinetics table will be named kinetics1

Value

An EZbakRKinetics object. This is simply a list of the provided kinetics and metadf with class EZbakRKinetics