windows_pipeline_quantification {FLAMES} | R Documentation

## Windows Pipeline - Quantification

### Description

This is the final step in the 3 step Windows FLAMES pipeline. This should be run after read realignment is performed, following `windows_pipeline_isoforms`.

### Usage

    windows_pipeline_quantification(pipeline_vars)

### Arguments

`pipeline_vars` | the list returned from `windows_pipeline_isoforms`, containing the information required to perform the final step, quantification.

### Value

`windows_pipeline_quantification` returns a SummarizedExperiment object, or a SingleCellExperiment in the case of this function being used for the FLAMES single cell pipeline, containing a count matrix as an assay, gene annotations under metadata, as well as a list of the other output files generated by the pipeline. The pipeline also outputs a number of output files into the given `outdir` directory. These output files generated by the pipeline are:

- transcript_count.csv.gz - a transcript count matrix (also contained in the SummarizedExperiment)

- isoform_annotated.filtered.gff3 - isoforms in gff3 format (also contained in the SummarizedExperiment)

- transcript_assembly.fa - transcript sequence from the isoforms

- align2genome.bam - sorted BAM file with reads aligned to genome

- realign2transcript.bam - sorted realigned BAM file using the transcript_assembly.fa as reference

- tss_tes.bedgraph - TSS TES enrichment for all reads (for QC)

### Examples

    ## example windows pipeline for BULK data. See Vignette for single cell data.

    # download the two fastq files, move them to a folder to be merged together
    temp_path <- tempfile()
    bfc <- BiocFileCache::BiocFileCache(temp_path, ask=FALSE)
    file_url <-
        "https://raw.githubusercontent.com/OliverVoogd/FLAMESData/master/data"
    # download the required fastq files, and move them to new folder
    fastq1 <- bfc[[names(BiocFileCache::bfcadd(bfc, "Fastq1", paste(file_url, "fastq/sample1.fastq.gz", sep="/")))]]
    fastq2 <- bfc[[names(BiocFileCache::bfcadd(bfc, "Fastq2", paste(file_url, "fastq/sample2.fastq.gz", sep="/")))]]
    fastq_dir <- paste(temp_path, "fastq_dir", sep="/") # the downloaded fastq files need to be in a directory to be merged together
    dir.create(fastq_dir)
    file.copy(c(fastq1, fastq2), fastq_dir)
    unlink(c(fastq1, fastq2)) # the original files can be deleted

    # run the FLAMES bulk pipeline setup
    #pipeline_variables <- bulk_windows_pipeline_setup(annot=system.file("extdata/SIRV_anno.gtf", package="FLAMES"),
    #                   fastq=fastq_dir,
    #                   outdir=tempdir(), genome_fa=system.file("extdata/SIRV_genomefa.fasta", package="FLAMES"),
    #                   config_file=system.file("extdata/SIRV_config_default.json", package="FLAMES"))
    # read alignment is handled externally (below downloads aligned bam for example)
    # genome_bam <- paste0(temp_path, "/align2genome.bam")
    # file.rename(bfc[[names(BiocFileCache::bfcadd(bfc, "Genome BAM", paste(file_url, "align2genome.bam", sep="/")))]], genome_bam)
    #
    # genome_index <- paste0(temp_path, "/align2genome.bam.bai")
    # file.rename(bfc[[names(BiocFileCache::bfcadd(bfc, "Genome BAM Index", paste(file_url, "align2genome.bam.bai", sep="/")))]], genome_index)
    # pipeline_variables$genome_bam = genome_bam
    #
    # # run the FLAMES bulk pipeline find isoforms step
    # pipeline_variables <- windows_pipeline_isoforms(pipeline_variables)
    #
    # # read realignment is handled externally
    # realign_bam <- paste0(temp_path, "/realign2genome.bam")
    # file.rename(bfc[[names(BiocFileCache::bfcadd(bfc, "Realign BAM", paste(file_url, "realign2transcript.bam", sep="/")))]], realign_bam)
    #
    # realign_index <- paste0(temp_path, "/realign2genome.bam.bai")
    # file.rename(bfc[[names(BiocFileCache::bfcadd(bfc, "Realign BAM Index", paste(file_url, "realign2transcript.bam.bai", sep="/")))]], realign_index)
    # pipeline_variables$realign_bam <- realign_bam
    #
    # # finally, quantification, which returns a Summarized Experiment object
    # se <- windows_pipeline_quantification(pipeline_variables)

[Package FLAMES version 1.0.2 Index]