# SpliceWiz: the cookbook

#### Alex CH Wong

#### 11/01/2022

Abstract

This vignette is a guide containing example code for performing real-life tasks. Importantly, it covers some functionality that were not covered in the Quick-Start vignette (because they are too computationally intensive to be reproducible in a vignette). Version 1.0.0

# Loading SpliceWiz

For instructions on installing and configuring SpliceWiz, please see the Quick-Start vignette.

    library(SpliceWiz)
    #> Loading required package: NxtIRFdata
    #> SpliceWiz package loaded with 2 threads
    #> Use setSWthreads() to set the number of SpliceWiz threads

# Reference Generation

First, define the path to the directory in which the reference should be stored. This directory will be made by SpliceWiz, but its parent directory must exist, otherwise an error will be returned.

    ref_path <- "./Reference"

### Create a SpliceWiz reference from user-defined FASTA and GTF files locally

Note that setting `genome_path = "hg38"` will prompt SpliceWiz to use the default files for nonPolyA and Mappability exclusion references in the generation of its reference. Valid options for `genome_path` are “hg38”, “hg19”, “mm10” and “mm9”.

    buildRef(
        reference_path = ref_path,
        fasta = "genome.fa", gtf = "transcripts.gtf",
        genome_type = "hg38"
    )

### Prepare genome resources and building the reference as separate steps

`buildRef()` first fetches the genome and gene annotations and makes a compressed local copy in the `resources` subdirectory of the given reference path. This can sometimes be a long process (especially when downloading the genome from the internet). Also, one may choose to generate the mappability exclusion regions manually (see `Reference Generation` - `Mappability exclusion generation using Rsubread` section). This needs to occur prior to generation of the SpliceWiz reference.

To separately prepare the annotation data and build the SpliceWiz reference, use the `getResources()` function to specify the FASTA and GTF files. Once `getResources()` has completed successfully, call `buildRef()` and leave the `fasta` and `gtf` arguments blank, as in the example below:

    getResources(
        reference_path = ref_path,
        fasta = "genome.fa",
        gtf = "transcripts.gtf"
    )

    buildRef(
        reference_path = ref_path,
        genome_type = "hg38"
    )

### Overwriting an existing reference, but using the same annotations

To re-build and overwrite an existing reference, using the same resource annotations:

    # Assuming hg38 genome:

    buildRef(
        reference_path = ref_path,
        genome_type = "hg38",
        overwrite = TRUE
    )

### Create a SpliceWiz reference using web resources from Ensembl’s FTP

The following will first download the genome and gene annotation files from the online resource and store a local copy of it in a file cache, facilitated by BiocFileCache. Then, it uses the downloaded resource to create the SpliceWiz reference.

    FTP <- "ftp://ftp.ensembl.org/pub/release-94/"

    buildRef(
        reference_path = ref_path,
        fasta = paste0(FTP, "fasta/homo_sapiens/dna/",
            "Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz"),
        gtf = paste0(FTP, "gtf/homo_sapiens/",
            "Homo_sapiens.GRCh38.94.chr.gtf.gz"),
        genome_type = "hg38"
    )

### Create a SpliceWiz reference using AnnotationHub resources

AnnotationHub contains Ensembl references for many genomes. To browse what is available:

    require(AnnotationHub)
    #> Loading required package: AnnotationHub
    #> Loading required package: BiocGenerics
    #>
    #> Attaching package: 'BiocGenerics'
    #> The following objects are masked from 'package:stats':
    #>
    #>     IQR, mad, sd, var, xtabs
    #> The following objects are masked from 'package:base':
    #>
    #>     Filter, Find, Map, Position, Reduce, anyDuplicated, aperm, append,
    #>     as.data.frame, basename, cbind, colnames, dirname, do.call,
    #>     duplicated, eval, evalq, get, grep, grepl, intersect, is.unsorted,
    #>     lapply, mapply, match, mget, order, paste, pmax, pmax.int, pmin,
    #>     pmin.int, rank, rbind, rownames, sapply, setdiff, sort, table,
    #>     tapply, union, unique, unsplit, which.max, which.min
    #> Loading required package: BiocFileCache
    #> Loading required package: dbplyr

    ah <- AnnotationHub()
    #> snapshotDate(): 2022-10-26
    query(ah, "Ensembl")
    #> AnnotationHub with 33426 records
    #> # snapshotDate(): 2022-10-26
    #> # $dataprovider: Ensembl, FANTOM5,DLRP,IUPHAR,HPRD,STRING,SWISSPROT,TREMBL,E...
    #> # $species: Homo sapiens, Mus musculus, Danio rerio, Rattus norvegicus, Pan ...
    #> # $rdataclass: TwoBitFile, GRanges, EnsDb, SQLiteFile, data.frame, OrgDb, list
    #> # additional mcols(): taxonomyid, genome, description,
    #> #   coordinate_1_based, maintainer, rdatadateadded, preparerclass, tags,
    #> #   rdatapath, sourceurl, sourcetype
    #> # retrieve records with, e.g., 'object[["AH5046"]]'
    #>
    #>              title
    #>   AH5046   | Ensembl Genes
    #>   AH5160   | Ensembl Genes
    #>   AH5311   | Ensembl Genes
    #>   AH5434   | Ensembl Genes
    #>   AH5435   | Ensembl EST Genes
    #>   ...        ...
    #>   AH107263 | LRBaseDb for Taeniopygia guttata (Zebra finch, v004)
    #>   AH107264 | LRBaseDb for Takifugu rubripes (Fugu, v004)
    #>   AH107265 | LRBaseDb for Ursus maritimus (Polar bear, v004)
    #>   AH107266 | LRBaseDb for Vulpes vulpes (Red fox, v004)
    #>   AH107267 | LRBaseDb for Xenopus tropicalis (Tropical clawed frog, v004)

For a more specific query:

    query(ah, c("Homo Sapiens", "release-94"))
    #> AnnotationHub with 9 records
    #> # snapshotDate(): 2022-10-26
    #> # $dataprovider: Ensembl
    #> # $species: Homo sapiens
    #> # $rdataclass: TwoBitFile, GRanges
    #> # additional mcols(): taxonomyid, genome, description,
    #> #   coordinate_1_based, maintainer, rdatadateadded, preparerclass, tags,
    #> #   rdatapath, sourceurl, sourcetype
    #> # retrieve records with, e.g., 'object[["AH64628"]]'
    #>
    #>             title
    #>   AH64628 | Homo_sapiens.GRCh38.94.abinitio.gtf
    #>   AH64629 | Homo_sapiens.GRCh38.94.chr.gtf
    #>   AH64630 | Homo_sapiens.GRCh38.94.chr_patch_hapl_scaff.gtf
    #>   AH64631 | Homo_sapiens.GRCh38.94.gtf
    #>   AH65744 | Homo_sapiens.GRCh38.cdna.all.2bit
    #>   AH65745 | Homo_sapiens.GRCh38.dna.primary_assembly.2bit
    #>   AH65746 | Homo_sapiens.GRCh38.dna_rm.primary_assembly.2bit
    #>   AH65747 | Homo_sapiens.GRCh38.dna_sm.primary_assembly.2bit
    #>   AH65748 | Homo_sapiens.GRCh38.ncrna.2bit

We wish to fetch “AH65745” and “AH64631” which contains the desired FASTA and GTF files, respectively. To build a reference using these resources:

    buildRef(
        reference_path = ref_path,
        fasta = "AH65745",
        gtf = "AH64631",
        genome_type = "hg38"
    )

`Build-Reference-methods` will recognise the inputs of `fasta` and `gtf` as AnnotationHub resources as they begin with “AH”.

### Create a SpliceWiz reference from species other than human or mouse

For human and mouse genomes, we highly recommend specifying `genome_type` as the default mappability file is used to exclude intronic regions with repeat sequences from intron retention analysis. For other species, one could generate a SpliceWiz reference without this reference:

    buildRef(
        reference_path = ref_path,
        fasta = "genome.fa", gtf = "transcripts.gtf",
        genome_type = ""
    )

# STAR reference generation

### Checking if STAR is installed

To use `STAR` to align FASTQ files, one must be using a system with `STAR` installed. This software is not available in Windows. To check if `STAR` is available:

    STAR_version()
    #> Nov 01 19:07:34 STAR is not installed

### Building a STAR reference

    ref_path = "./Reference"

    # Ensure genome resources are prepared from genome FASTA and GTF file:

    if(!dir.exists(file.path(ref_path, "resource"))) {
        getResources(
            reference_path = ref_path,
            fasta = "genome.fa",
            gtf = "transcripts.gtf"
        )
    }

    # Generate a STAR genome reference:
    STAR_BuildRef(
        reference_path = ref_path,
        n_threads = 8
    )

### Building a STAR reference alongside the SpliceWiz reference

If `STAR` is available on the same computer or server where R/RStudio is being run, we can use the one-line function `buildFullRef`. This function will: * Prepare the resources from the given FASTA and GTF files * Generate a STAR genome * Use the STAR genome and the FASTA file to de-novo calculate and define low mappability regions * Build the SpliceWiz reference using the genome resources and mappability file

    buildFullRef(
        reference_path = ref_path,
        fasta = "genome.fa", gtf = "transcripts.gtf",
        genome_type = "",
        use_STAR_mappability = TRUE,
        n_threads = 4
    )

`n_threads` specify how many threads should be used to build the STAR reference and to calculate the low mappability regions

### Mappability exclusion generation using Rsubread

Finally, if `STAR` is not available, `Rsubread` is available on Bioconductor to perform mappability calculations. The example code in the manual is displayed here for convenience, to demonstrate how this would be done:

    # (1a) Creates genome resource files

    ref_path <- file.path(tempdir(), "Reference")

    getResources(
        reference_path = ref_path,
        fasta = chrZ_genome(),
        gtf = chrZ_gtf()
    )

    # (1b) Systematically generate reads based on the SpliceWiz example genome:

    generateSyntheticReads(
        reference_path = ref_path
    )

    # (2) Align the generated reads using Rsubread:

    # (2a) Build the Rsubread genome index:

    setwd(ref_path)
    Rsubread::buildindex(basename = "./reference_index",
        reference = chrZ_genome())

    # (2b) Align the synthetic reads using Rsubread::subjunc()

    Rsubread::subjunc(
        index = "./reference_index",
        readfile1 = file.path(ref_path, "Mappability", "Reads.fa"),
        output_file = file.path(ref_path, "Mappability", "AlignedReads.bam"),
        useAnnotation = TRUE,
        annot.ext = chrZ_gtf(),
        isGTF = TRUE
    )

    # (3) Analyse the aligned reads in the BAM file for low-mappability regions:

    calculateMappability(
        reference_path = ref_path,
        aligned_bam = file.path(ref_path, "Mappability", "AlignedReads.bam")
    )

    # (4) Build the SpliceWiz reference using the calculated Mappability Exclusions

    buildRef(ref_path)

# Aligning Raw RNA-seq data using SpliceWiz’s STAR wrappers

First, remember to check that STAR is available via command line:

    STAR_version()
    #> Nov 01 19:07:34 STAR is not installed

### Aligning a single sample using STAR

    STAR_alignReads(
        STAR_ref_path = file.path(ref_path, "STAR"),
        BAM_output_path = "./bams/sample1",
        fastq_1 = "sample1_1.fastq", fastq_2 = "sample1_2.fastq",
        n_threads = 8
    )

### Aligning multiple samples using STAR

    Experiment <- data.frame(
        sample = c("sample_A", "sample_B"),
        forward = file.path("raw_data", c("sample_A", "sample_B"),
            c("sample_A_1.fastq", "sample_B_1.fastq")),
        reverse = file.path("raw_data", c("sample_A", "sample_B"),
            c("sample_A_2.fastq", "sample_B_2.fastq"))
    )

    STAR_alignExperiment(
        Experiment = Experiment,
        STAR_ref_path = file.path("Reference_FTP", "STAR"),
        BAM_output_path = "./bams",
        n_threads = 8,
        two_pass = FALSE
    )

To use two-pass mapping, set `two_pass = TRUE`. We recommend disabling this feature, as one-pass mapping is adequate in typical-use cases.

### Finding FASTQ files recursively from a given directory

SpliceWiz can identify sequencing FASTQ files recursively from a given directory. It assumes that forward and reverse reads are suffixed as `_1` and `_2`, respectively. Users can choose to identify such files using a specified file extension. For example, to recursively identify FASTQ files of the format `{sample}_1.fq.gz` and `{sample}_2.fq.gz`, use the following:

    # Assuming sequencing files are named by their respective sample names
    fastq_files <- findFASTQ("./sequencing_files", paired = TRUE,
        fastq_suffix = ".fq.gz", level = 0)

Then, these can be aligned as follows:

    STAR_alignExperiment(
        Experiment = fastq_files,
        STAR_ref_path = file.path("Reference_FTP", "STAR"),
        BAM_output_path = "./bams",
        n_threads = 8,
        two_pass = FALSE
    )

# Running processBAM on BAM files

To conveniently find all BAM files recursively in a given path:

    bams <- findBAMS("./bams", level = 1)

This convenience function returns the putative sample names, either from BAM file names themselves (`level = 0`), or from the names of their parent directories (`level = 1`).

To run `processBAM()` using 4 OpenMP threads:

    # assume SpliceWiz reference has been generated in `ref_path`

    processBAM(
        bamfiles = bams$path,
        sample_names = bams$sample,
        reference_path = ref_path,
        output_path = "./pb_output",
        n_threads = 4,
        useOpenMP = TRUE
    )

### Creating COV files from BAM files without running processBAM

Sometimes one may wish to create a COV file from a BAM file without running `processBAM()`. One reason might be because a SpliceWiz reference is not available.

To convert a list of BAM files, run `BAM2COV()`. This is a function structurally similar to `processBAM()` but without the need to give the path to the SpliceWiz reference:

    BAM2COV(
        bamfiles = bams$path,
        sample_names = bams$sample,
        output_path = "./pb_output",
        n_threads = 4,
        useOpenMP = TRUE
    )

# Collating the experiment

Assuming the SpliceWiz reference is in `ref_path`, after running `processBAM()` as shown in the previous section, use the convenience function `findSpliceWizOutput()` to tabulate a list of samples and their corresponding `processBAM()` outputs:

    expr <- findSpliceWizOutput("./pb_output")

This data.frame can be directly used to run `collateData`:

    collateData(
        Experiment = expr,
        reference_path = ref_path,
        output_path = "./NxtSE_output"
    )

- NB: Novel splicing detection can be enabled by setting `novelSplicing = TRUE`. See the SpliceWiz Quick-Start vignette for more details.

Then, the collated data can be imported as a `NxtSE` object, which is an object that inherits `SummarizedExperiment` and has specialized containers to hold additional data required by SpliceWiz.

    se <- makeSE("./NxtSE_output")

# Downstream analysis using SpliceWiz

Please refer to SpliceWiz: Quick-Start vignette for worked examples using the example dataset.

# SessionInfo

    sessionInfo()
    #> R version 4.2.1 (2022-06-23)
    #> Platform: x86_64-pc-linux-gnu (64-bit)
    #> Running under: Ubuntu 20.04.5 LTS
    #>
    #> Matrix products: default
    #> BLAS:   /home/biocbuild/bbs-3.16-bioc/R/lib/libRblas.so
    #> LAPACK: /home/biocbuild/bbs-3.16-bioc/R/lib/libRlapack.so
    #>
    #> locale:
    #>  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C
    #>  [3] LC_TIME=en_GB              LC_COLLATE=C
    #>  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8
    #>  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C
    #>  [9] LC_ADDRESS=C               LC_TELEPHONE=C
    #> [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
    #>
    #> attached base packages:
    #> [1] stats     graphics  grDevices utils     datasets  methods   base
    #>
    #> other attached packages:
    #> [1] AnnotationHub_3.6.0 BiocFileCache_2.6.0 dbplyr_2.2.1
    #> [4] BiocGenerics_0.44.0 SpliceWiz_1.0.0     NxtIRFdata_1.3.2
    #>
    #> loaded via a namespace (and not attached):
    #>   [1] lazyeval_0.2.2                shinydashboard_0.7.2
    #>   [3] splines_4.2.1                 BiocParallel_1.32.0
    #>   [5] GenomeInfoDb_1.34.0           ggplot2_3.3.6
    #>   [7] digest_0.6.30                 ca_0.71.1
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