pipe.RiboClear: Ribo Clearing Pileline Step for NGS Data

In robertdouglasmorrison/DuffyNGS: Duffy Lab NGS Analysis Pipeline for RNA-seq, DNA-seq, ChIP-seq, and RIP-seq

Ribo Clearing Pileline Step for NGS Data

Description

Runs the ribo clearing alignment step to remove unwanted genomic feature reads from FASTQ files, prior to the main genomic alignment and splice alignment steps. Typically invoked on RNA-seq data to remove highly abundant unwanted transcripts such as ribosomal RNA, mitochondrial RNA, albumin, and very high abundance small RNAs; features whose expression abundance might dwarf the expression levels of typical wanted genes.

Usage

pipe.RiboClear(inputFastqFile, sampleID, annotationFile = "Annotation.txt", 
	optionsFile = "Options.txt", asMatePairs = FALSE, verbose = TRUE, 
	rawReadCount = NULL)

Arguments

inputFastqFile	character vector of one or more raw FASTQ files
sampleID	the SampleID for this sample. This SampleID keys for a row of annotation details in the annotation file, for getting sample-specific details. The SampleID is also used as a sample-specific prefix for all files created during the processing of this sample.
annotationFile	the file of sample annotation details, which specifies all needed sample-specific information about the samples under study. See DuffyNGS_Annotation.
optionsFile	the file of processing options, which specifies all processing parameters that are not sample specific. See DuffyNGS_Options.
asMatePairs	logical flag, should the vector of FASTQ be treated as two mate pair files?
rawReadCount	optional argument of the number of reads in the files. By default this gets calculated on the fly.

Details

Aligns the given FASTQ files against the Bowtie2 ribo clearing index specified in the options file. Reads that do align are written to a BAM file in the riboClear results subfolder. Reads that fail to align are written to temporary file(s) in the current working folder for the subsequent genomic alignment step.

The genomic features to be removed are hard-coded into the RrnaMap map and the Bowtie ribo clearing indexes. It is not currently something that can be modified by the end user. See MapSets.

Value

a family of BAM files, FASTQ files, and summary files, written to subfolders under the results.path folder.

Also, a list of alignment counts:

RawReads	the number of reads in the raw FASTQ files
UniqueReads	the number of reads that aligned to exactly one location
MultiReads	the number of reads that aligned to two or more locations
NoHitReads	the number of reads that failed to align
Time	the time usage of this alignment step, as from proc.time

Note

the interplay of paired end strand specific reads and ribo clearing is messy. Typically, ribo clearing finds both multi-hit reads and un-paired alignments where only one read maps. Both of these behavoirs break the expected convention that paired end read files are still perfect mate pairs after the alignment step. The default mode is to turn off paired end strand specific behavior when ribo clearing, but this can be forced using option forcePairedEnd.

Author(s)

Bob Morrison

See Also

pipe.GenomicAlign Genomic alignment against an index of target genome(s). pipe.SpliceAlign Splice junction alignment against an index of standard and alternative splice junctions.

robertdouglasmorrison/DuffyNGS documentation built on March 24, 2024, 4:16 p.m.