NxtFilter-class: NxtIRF filters to remove low-abundance alternative splicing...

In alexchwong/NxtIRFcore: Core Engine for NxtIRF: a User-Friendly Intron Retention and Alternative Splicing Analysis using the IRFinder Engine

NxtIRF filters to remove low-abundance alternative splicing and intron retention events

Description

NxtIRF filters to remove low-abundance alternative splicing and intron retention events

Usage

NxtFilter(
  filterClass = c("Data", "Annotation"),
  filterType = c("Depth", "Coverage", "Consistency", "Protein_Coding", "NMD", "TSL",
    "Terminus", "ExclusiveMXE"),
  pcTRUE = 100,
  minimum = 20,
  maximum = 1,
  minDepth = 5,
  condition = "",
  minCond = -1,
  EventTypes = c("IR", "MXE", "SE", "A3SS", "A5SS", "AFE", "ALE", "RI")
)

Arguments

filterClass	Must be either "Data" or "Annotation". See details
filterType	If filterClass = "Data", then must be one of c("Depth", "Coverage", "Consistency"). If filterClass = "Annotation", must be one of c("Protein_Coding", "NMD", "TSL"). See details
pcTRUE	If conditions are set, what percentage of all samples in each of the condition must the filter be satisfied for the event to pass the filter check. Must be between 0 and 100 (default 100)
minimum	Filter-dependent argument. See details
maximum	Filter-dependent argument. See details
minDepth	Filter-dependent argument. See details
condition	(default "") If set, must match the name of an experimental condition in the NxtSE object to be filtered, i.e. a column name in colData(se). Leave blank to disable filtering by condition
minCond	(default -1) If condition is set, how many minimum number of conditions must pass the filter criteria. For example, if condition = "Batch", and batches are "A", "B", or "C", setting minCond = 2 with pcTRUE = 100 means that all samples belonging to two of the three types of Batch must pass the filter criteria. Setting -1 means all elements of condition must pass criteria. Set to -1 when the number of elements in the experimental condition is unknown. Ignored if condition is left blank.
EventTypes	What types of events are considered for filtering. Must be one of c("IR", "MXE", "SE", "A3SS", "A5SS", "AFE", "ALE", "RI"). Events not specified in EventTypes are not filtered (i.e. they will pass the filter without checks)

Details

Annotation Filters

• Protein_Coding: Filters for alternative splicing or IR events involving protein-coding transcripts. No additional parameters required.

• NMD: Filters for events in which one isoform is a predicted NMD substrate.

• TSL: filters for events in which both isoforms have a TSL level below or equal to minimum

• Terminus (New as of version 1.1.1): In alternate first exons, the splice junction must not be shared with another transcript for which it is not its first intron. For alternative last exons, the splice junction must not be shared with another transcript for which it is not its last intron

• ExclusiveMXE (New as of version 1.1.1): For MXE events, the two alternate casette exons must not overlap in their genomic regions

Data Filters

• Depth: Filters IR or alternative splicing events of transcripts that are "expressed" with adequate Depth as calculated by the sum of all splicing and IR reads spanning the event. Events with Depth below minimum are filtered out

• Coverage: Coverage means different things to IR and alternative splicing.
  
  For IR, Coverage refers to the percentage of the measured intron covered with reads. Introns of samples with an IntronDepth above minDepth are assessed, with introns with coverage below minimum are filtered out.
  
  For Alternative Splicing, Coverage refers to the percentage of all splicing events observed across the genomic region that is compatible with either the included or excluded event. This prevents NxtIRF from doing differential analysis between two minor isoforms. Instead of IntronDepth, in AS events NxtIRF considers events where the spliced reads from both exonic regions exceed minDepth. Then, events with a splicing coverage below minimum are excluded.
  
  We recommend testing IR events for > 90% coverage and AS events for > 60% coverage as given in the default filters which can be accessed using get_default_filters
  
  

• Consistency: Skipped exons (SE) and mutually exclusive exons (MXE) comprise reads aligned to two contiguous splice junctions. Most algorithms take the average counts from both junctions. This will inadvertently include transcripts that share one but not both splice events. To check that this is not happening, we require both splice junctions to have comparable counts. This filter checks whether reads from each splice junction comprises a reasonable proportion of the sum of these reads.
  
  Events are excluded if either of the upstream or downstream event is lower than total splicing events by a log-2 magnitude above maximum. For example, if maximum = 2, we require both upstream and downstream events to represent at least 1/(2^2) = 1/4 of the sum of upstream and downstream event. If maximum = 3, then each junction must be at least 1/8 of total, etc. This is considered for each isoform of each event, as long as the total counts belonging to the considered isoform is above minDepth.
  
  IR-events are also checked. For IR events, the upstream and downstream exon-intron spanning reads must comprise a reasonable proportion of total exon-intron spanning reads.

We highly recommend using the default filters, which can be acquired using get_default_filters

Value

A NxtFilter object with the specified parameters

Functions

• NxtFilter: Constructs a NxtFilter object

See Also

Run_NxtIRF_Filters

Examples

# Create a NxtFilter that filters for protein-coding ASE
f1 <- NxtFilter(filterClass = "Annotation", filterType = "Protein_Coding")

# Create a NxtFilter that filters for Depth >= 20 in IR events
f2 <- NxtFilter(
    filterClass = "Data", filterType = "Depth",
    minimum = 20, EventTypes = c("IR", "RI")
)

# Create a NxtFilter that filters for Coverage > 60% in splice events
# that must be satisfied in at least 2 categories of condition "Genotype"
f3 <- NxtFilter(
    filterClass = "Data", filterType = "Coverage",
    minimum = 60, EventTypes = c("MXE", "SE", "AFE", "ALE", "A3SS", "A5SS"),
    condition = "Genotype", minCond = 2
)

# Create a NxtFilter that filters for Depth > 10 in all events
# that must be satisfied in at least 50% of each gender
f4 <- NxtFilter(
    filterClass = "Data", filterType = "Depth",
    minimum = 10, condition = "gender", pcTRUE = 50
)

# Get a description of what these filters do:
f1
f2
f3
f4

alexchwong/NxtIRFcore documentation built on Oct. 31, 2022, 9:14 a.m.