# ompBAM API Documentation

#### Alex C H Wong

#### 04/25/2023

Abstract

ompBAM provides C++ header files for developers wishing to create R packages that processes BAM files. ompBAM automates file access, memory management, and handling of multiple threads ‘behind the scenes’, so developers can focus on creating domain-specific functionality. This documentation introduces ompBAM, including a quick-start to create an ompBAM-based R package, step-by-step explanation of the ompBAM example package, and detailed documentation of each function of the two ompBAM objects, ‘pbam_in’ and ‘pbam1_t’. ompBAM package version 1.4.0

# (0) Installation and Quick-Start

### Installation

To install ompBAM, start R (version “4.2”) and enter:

    if (!requireNamespace("BiocManager", quietly = TRUE))
        install.packages("BiocManager")

    BiocManager::install("ompBAM")

To create new packages using ompBAM, install `devtools` and `usethis` packages from CRAN:

    install.packages(c("devtools", "usethis"))

For MacOS users, make sure OpenMP libraries are installed correctly. We recommend users follow this guide, but the quickest way to get started is to install `libomp` via brew:

    brew install libomp

### Creating and testing an example ompBAM-based package

To create a new package (in temporary directory):

    library(ompBAM)
    pkg_path = file.path(tempdir(), "MyPkg")
    use_ompBAM(pkg_path)
    #> ✔ Creating '/tmp/RtmpruF7ss/MyPkg/'
    #> ✔ Setting active project to '/tmp/RtmpruF7ss/MyPkg'
    #> ✔ Creating 'R/'
    #> ✔ Writing 'DESCRIPTION'
    #> Package: MyPkg
    #> Title: What the Package Does (One Line, Title Case)
    #> Version: 0.0.0.9000
    #> Authors@R (parsed):
    #>     * First Last <first.last@example.com> [aut, cre] (YOUR-ORCID-ID)
    #> Description: What the package does (one paragraph).
    #> License: `use_mit_license()`, `use_gpl3_license()` or friends to pick a
    #>     license
    #> Encoding: UTF-8
    #> Roxygen: list(markdown = TRUE)
    #> RoxygenNote: 7.2.3
    #> ✔ Writing 'NAMESPACE'
    #> ✔ Setting active project to '<no active project>'
    #> ✔ Setting active project to '/tmp/RtmpruF7ss/MyPkg'
    #> ✔ Creating 'src/'
    #> ✔ Adding '*.o', '*.so', '*.dll' to 'src/.gitignore'
    #> ✔ Created /tmp/RtmpruF7ss/MyPkg/R/ompBAM_imports.R with roxygen tags
    #> ✔ Created src/Makevars.in and src/Makevars.win
    #> ✔ Created configure scripts
    #> ✔ Created src/ompBAM_example.cpp with idxstats_pbam() function
    #> ✔ Adding Rcpp to Imports field in DESCRIPTION
    #> ✔ Adding zlibbioc to Imports field in DESCRIPTION
    #> ✔ Adding ompBAM to LinkingTo field in DESCRIPTION
    #> ✔ Adding Rcpp to LinkingTo field in DESCRIPTION
    #> ✔ Adding zlibbioc to LinkingTo field in DESCRIPTION
    #> ✔ MyPkg successfully created in /tmp/RtmpruF7ss/MyPkg
    #> Please run roxygen2 using devtools::document() before building the package.

Before this package is functional, it needs to be roxygenised. This will automate the export of the example function `idxstats()`. Run the following:

    devtools::document(pkg_path)
    #> ℹ Updating MyPkg documentation
    #> ℹ Loading MyPkg
    #> Exports from /tmp/RtmpruF7ss/MyPkg/src/ompBAM_example.cpp:
    #>    int idxstats_pbam(std::string bam_file, int n_threads_to_use = 1)
    #>
    #> /tmp/RtmpruF7ss/MyPkg/src/RcppExports.cpp updated.
    #> /tmp/RtmpruF7ss/MyPkg/R/RcppExports.R updated.
    #> ℹ Re-compiling MyPkg (debug build)
    #> ── R CMD INSTALL ───────────────────────────────────────────────────────────────
    #> * installing *source* package ‘MyPkg’ ...
    #> ** using staged installation
    #> ** libs
    #> using C++ compiler: ‘g++ (Ubuntu 11.3.0-1ubuntu1~22.04) 11.3.0’
    #> g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.17-bioc/R/include" -DNDEBUG  -I'/tmp/RtmpM4zqug/Rinst19ebf4319929f0/ompBAM/include' -I'/home/biocbuild/bbs-3.17-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.17-bioc/R/site-library/zlibbioc/include' -I/usr/local/include   -fopenmp  -fpic  -g -O2  -Wall -UNDEBUG -Wall -pedantic -g -O0 -c RcppExports.cpp -o RcppExports.o
    #> g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.17-bioc/R/include" -DNDEBUG  -I'/tmp/RtmpM4zqug/Rinst19ebf4319929f0/ompBAM/include' -I'/home/biocbuild/bbs-3.17-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.17-bioc/R/site-library/zlibbioc/include' -I/usr/local/include   -fopenmp  -fpic  -g -O2  -Wall -UNDEBUG -Wall -pedantic -g -O0 -c ompBAM_example.cpp -o ompBAM_example.o
    #> g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.17-bioc/R/lib -L/usr/local/lib -o MyPkg.so RcppExports.o ompBAM_example.o -fopenmp -L/home/biocbuild/bbs-3.17-bioc/R/lib -lR
    #> installing to /tmp/RtmpruF7ss/devtools_install_19ed2460abe91e/00LOCK-MyPkg/00new/MyPkg/libs
    #> ** checking absolute paths in shared objects and dynamic libraries
    #> * DONE (MyPkg)
    #> Writing 'NAMESPACE'
    #> Writing 'NAMESPACE'

To simulate the compilation and loading of this package (this is almost equivalent to running `R CMD BUILD` then loading the `MyPkg` package:

    devtools::load_all(pkg_path)
    #> ℹ Loading MyPkg

To test the new package works as expected, run the following commands:

    library(MyPkg)

    idxstats(ompBAM::example_BAM("Unsorted"), 2)
    #> /tmp/RtmpM4zqug/Rinst19ebf4319929f0/ompBAM/extdata/THP1_ND_1.bam summary:
    #> Name Length  Number of reads
    #> 1    248956422   1308
    #> 10   133797422   308
    #> 11   135086622   600
    #> 12   133275309   648
    #> 13   114364328   162
    #> 14   107043718   230
    #> 15   101991189   294
    #> 16   90338345    334
    #> 17   83257441    570
    #> 18   80373285    78
    #> 19   58617616    600
    #> 2    242193529   586
    #> 20   64444167    168
    #> 21   46709983    76
    #> 22   50818468    200
    #> 3    198295559   454
    #> 4    190214555   252
    #> 5    181538259   516
    #> 6    170805979   824
    #> 7    159345973   412
    #> 8    145138636   452
    #> 9    138394717   372
    #> MT   16569   288
    #> X    156040895   254
    #> Y    57227415    14
    #> [1] 0

    idxstats(ompBAM::example_BAM("scRNAseq"), 2)
    #> /tmp/RtmpM4zqug/Rinst19ebf4319929f0/ompBAM/extdata/MGE_E35_1.bam summary:
    #> Name Length  Number of reads
    #> 1    195471971   50000
    #> 10   130694993   0
    #> 11   122082543   0
    #> 12   120129022   0
    #> 13   120421639   0
    #> 14   124902244   0
    #> 15   104043685   0
    #> 16   98207768    0
    #> 17   94987271    0
    #> 18   90702639    0
    #> 19   61431566    0
    #> 2    182113224   0
    #> 3    160039680   0
    #> 4    156508116   0
    #> 5    151834684   0
    #> 6    149736546   0
    #> 7    145441459   0
    #> 8    129401213   0
    #> 9    124595110   0
    #> MT   16299   0
    #> X    171031299   0
    #> Y    91744698    0
    #> JH584299.1   953012  0
    #> GL456233.1   336933  0
    #> JH584301.1   259875  0
    #> GL456211.1   241735  0
    #> GL456350.1   227966  0
    #> JH584293.1   207968  0
    #> GL456221.1   206961  0
    #> JH584297.1   205776  0
    #> JH584296.1   199368  0
    #> GL456354.1   195993  0
    #> JH584294.1   191905  0
    #> JH584298.1   184189  0
    #> JH584300.1   182347  0
    #> GL456219.1   175968  0
    #> GL456210.1   169725  0
    #> JH584303.1   158099  0
    #> JH584302.1   155838  0
    #> GL456212.1   153618  0
    #> JH584304.1   114452  0
    #> GL456379.1   72385   0
    #> GL456216.1   66673   0
    #> GL456393.1   55711   0
    #> GL456366.1   47073   0
    #> GL456367.1   42057   0
    #> GL456239.1   40056   0
    #> GL456213.1   39340   0
    #> GL456383.1   38659   0
    #> GL456385.1   35240   0
    #> GL456360.1   31704   0
    #> GL456378.1   31602   0
    #> GL456389.1   28772   0
    #> GL456372.1   28664   0
    #> GL456370.1   26764   0
    #> GL456381.1   25871   0
    #> GL456387.1   24685   0
    #> GL456390.1   24668   0
    #> GL456394.1   24323   0
    #> GL456392.1   23629   0
    #> GL456382.1   23158   0
    #> GL456359.1   22974   0
    #> GL456396.1   21240   0
    #> GL456368.1   20208   0
    #> JH584292.1   14945   0
    #> JH584295.1   1976    0
    #> [1] 0

#### To create an ompBAM-based package from within RStudio:

To create an example package, create a new R project using ompBAM by running the following in the R console:

    library(ompBAM)
    project_path = "\path\to\MyPkg"
    use_ompBAM(project_path)

NB: be sure to replace `project_path` to the actual directory path in which you wish to store your project.

After running the example code above, use RStudio to open the newly created `MyPkg.Rproj` file located at the given path. Then, run the following:

    devtools::document()

This will process the roxygen2 flags and write to the `NAMESPACE` file.

After this, the new package can be built by running Install and Restart from the Build tab.

# (1) Requirements to setting up a new R package to include ompBAM

ompBAM provides a one-step function called `use_ompBAM()` that creates a new package R project in a new directory (or adds requisite files to an existing project). The function is similar to those in the usethis package. The user supplies a directory path and the directory containing the package must also be the name of the new package.

## (1a) Making a new package that compiles with ompBAM

Using the R function `use_ompBAM()`, we can create a new package inside the R-generated temporary directory as an example:

    library(ompBAM)
    pkg_path = file.path(tempdir(), "myPkgName")
    use_ompBAM(pkg_path)
    #> ✔ Creating '/tmp/RtmpruF7ss/myPkgName/'
    #> ✔ Setting active project to '/tmp/RtmpruF7ss/myPkgName'
    #> ✔ Creating 'R/'
    #> ✔ Writing 'DESCRIPTION'
    #> Package: myPkgName
    #> Title: What the Package Does (One Line, Title Case)
    #> Version: 0.0.0.9000
    #> Authors@R (parsed):
    #>     * First Last <first.last@example.com> [aut, cre] (YOUR-ORCID-ID)
    #> Description: What the package does (one paragraph).
    #> License: `use_mit_license()`, `use_gpl3_license()` or friends to pick a
    #>     license
    #> Encoding: UTF-8
    #> Roxygen: list(markdown = TRUE)
    #> RoxygenNote: 7.2.3
    #> ✔ Writing 'NAMESPACE'
    #> ✔ Setting active project to '/tmp/RtmpruF7ss/MyPkg'
    #> ✔ Setting active project to '/tmp/RtmpruF7ss/myPkgName'
    #> ✔ Creating 'src/'
    #> ✔ Adding '*.o', '*.so', '*.dll' to 'src/.gitignore'
    #> ✔ Created /tmp/RtmpruF7ss/myPkgName/R/ompBAM_imports.R with roxygen tags
    #> ✔ Created src/Makevars.in and src/Makevars.win
    #> ✔ Created configure scripts
    #> ✔ Created src/ompBAM_example.cpp with idxstats_pbam() function
    #> ✔ Adding Rcpp to Imports field in DESCRIPTION
    #> ✔ Adding zlibbioc to Imports field in DESCRIPTION
    #> ✔ Adding ompBAM to LinkingTo field in DESCRIPTION
    #> ✔ Adding Rcpp to LinkingTo field in DESCRIPTION
    #> ✔ Adding zlibbioc to LinkingTo field in DESCRIPTION
    #> ✔ myPkgName successfully created in /tmp/RtmpruF7ss/myPkgName
    #> Please run roxygen2 using devtools::document() before building the package.

We recommend the user run this function in a project directory of their choice and NOT use `tempdir()`, as the temp directory is destroyed on each R session restart! We only demonstrate using `tempdir()` here to demonstrate the typical output of the `use_ompBAM()` function.

Users following this vignette should instead use something like:

    use_ompBAM("/path/to/myPkgName")

In the remainder of this section we will examine this newly-created project and explain the importance of the configurations made.

After running ompBAM(), please open the newly-generated `myPkgName.Rproj` from within RStudio.

Note that the newly-created package is designed to run with roxygen2. We recommend users build their package documentation and NAMESPACE using roxygen2. To do this, simply run `devtools::document()` to process roxygen2 tags.

## (1b) Dependencies required to compile with ompBAM

To compile with ompBAM capabilities, the package must import both Rcpp and zlibbioc. Check that the following has been added to the DESCRIPTION file:

    Imports: Rcpp, zlibbioc
    LinkingTo: Rcpp, zlibbioc, ompBAM

Also, `use_ompBAM()` creates a “wrapper” function for the C++ function `idxstats_pbam()` function. This is a simple R function `idxstats()` which in turn calls the C++ function. We export this function with a roxygen2 tag so that `idxstats()` can be called once the `MyPkgName` package is loaded.

Open this file to verify that the following has been added:

    #' @useDynLib myPkgName, .registration = TRUE
    #' @import Rcpp
    #' @import zlibbioc
    NULL

    #' @export
    idxstats <- function(bam_file, n_threads) {
        idxstats_pbam(bam_file, n_threads)
    }

To make sure your roxygen2 setup works, make sure the new package is your active project by opening the project within RStudio. Then run `devtools::document()` to allow roxygen2 to do its magic. When it is done, the NAMESPACE file should contain the following:

    # Generated by roxygen2: do not edit by hand

    export(idxstats)
    import(Rcpp)
    import(zlibbioc)
    useDynLib(myPkgName, .registration = TRUE)

If for whatever reason roxygen2 says it did not edit the NAMESPACE file because it was not created by roxygen2, we suggest deleting the NAMESPACE file, then run `devtools::document()` again.

Also, you may have noticed, the included source code may have been prompted to compile. Don’t worry about this, we will explain it in detail in the next section.

## (1c) Make Files

use_ompBAM() has created two `make` files that instructs the compiler to link with OpenMP and the zlib library. You can view these files from within the `src/` directory.

Make files instruct the C++ compiler to link to the appropriate libraries at compile-time. For R packages, these are called `Makevars` and are streamlined `make` files. For more information, refer to (Writing R Extensions - Using MakeVars) [https://cran.r-project.org/doc/manuals/r-release/R-exts.html#Using-Makevars]

`Makevars.in` is a template `make` file used by Linux and MacOS, and should contain the following:

    PKG_CXXFLAGS = $(OMPBAM_PKG_CXXFLAGS)
    PKG_LIBS = $(OMPBAM_PKG_LIBS)

Additionally, a `configure` script is added to the root project directory. This is a shell script run when the package is built from source. It detects whether the operating system is Linux or MacOS, and whether OpenMP libraries are available for MacOS. It assigns the correct compile and linker flags to the `OMPBAM_PKG_CXXFLAGS` and `OMPBAM_PKG_LIBS` variables in the template `make` file. The contents of the `configure` script is beyond the scope of this documentation, but feel free to look at it. It is based on the `configure` script for the data.table R package on CRAN.

For Windows installations, a second `make` file called “Makevars.win” is required. This is because zlib libraries are linked dynamically (see the zlibbioc vignette for more information). In windows systems, “Makevars.win” is used to build your package, and contains the following:

    PKG_CXXFLAGS = $(SHLIB_OPENMP_CXXFLAGS)
    PKG_LIBS = $(SHLIB_OPENMP_CXXFLAGS)

    ZLIB_CFLAGS+=$(shell echo 'zlibbioc::pkgconfig("PKG_CFLAGS")'|\
        "${R_HOME}/bin/R" --vanilla --slave)
    PKG_LIBS+=$(shell echo 'zlibbioc::pkgconfig("PKG_LIBS_shared")' |\
        "${R_HOME}/bin/R" --vanilla --slave)

This will ensure that the zlib libraries are linked correctly to Windows systems.

## (1d) OpenMP compatibility

Windows and Linux systems should support OpenMP natively. MacOS, however, does not. In order to set up OpenMP for MacOS, we recommend following this guide. In short, to utilise OpenMP on MacOS users must first install the correct OpenMP libraries. Secondly, specific compiler and linker flags must be set. This has already been done using the `configure` script and template `Makevars.in` file as described in section (1c).

To simplify the installation of OpenMP libraries on MacOS, instruct package users to run the following:

    brew install libomp

As can be seen, OpenMP for MacOS will continue to be a difficult issue. We recommend writing C++ code that is compatible for both OpenMP and non-OpenMP systems. For non-OpenMP systems, multi-threading can still be implemented via `BiocParallel::MulticoreParam()` but the session memory will be multiplied over the number of cores used.

In C++ code, to check whether OpenMP is installed in a system, use the `#ifdef` and `#ifndef` directives. An example below:

    #ifdef _OPENMP
      // Add code here for programs compiled with OpenMP
    #else
      // Add code here for programs not using OpenMP
    #endif

# (2) Step-by-step guide to creating your first ompBAM-powered package

use_ompBAM() creates source code contained within `src/ompBAM_example.cpp` which contains a “Hello world!” equivalent function that is ready to be compiled. This function, called `idxstats_pbam()`, replicates the idxstats function of Samtools. It reports the chromosome names and lengths of the genome to which the sequences in the BAM file is aligned, as well as the number of reads aligned to each chromosome. See the idxstats documentation for more details

In this section, we will examine the `src/ompBAM_example.cpp` source code in detail and explain how we used ompBAM to re-create the idxstats function.

First, lets see how this function works. Make sure you have created the example project using the steps as described in Section (0) - Installation and Quick-Start. Make sure you can reproduce all the example output in that section.

Lets look at the line used to run the ompBAM-based idxstats function:

    idxstats(ompBAM::example_BAM("Unsorted"), 2)

Like the Samtools idxstats, this function counts the number of reads aligned to each chromosome in the genome. However, unlike idxstats, this function can be run on BAM files that are not sorted by chromosome and not indexed.

In the following guide, we will go through the `src/ompBAM_example.cpp` in the example package, step by step, explaining the code behind this simple function.

## (2a) Headers and Includes

To add the ompBAM header files into your source code, `src/ompBAM_example.cpp` contains the following:

    #include "Rcpp.h"
    using namespace Rcpp;

    // Required to print cout output generated by ompBAM
    #define cout Rcpp::Rcout

    // [[Rcpp::depends(ompBAM)]]
    #include <ompBAM.hpp>

ompBAM headers must be added AFTER Rcpp. This is because ompBAM uses `cout` function for console output. The `cout` C++ function is not supported in R, and must replaced by `Rcout` in Rcpp.

The line `#define cout Rcpp::Rcout` ensures that `cout` in all ompBAM headers are interpreted by the compiler as `Rcpp::Rcout`.

## (2b) Sanity check for number of threads

`src/ompBAM_example.cpp` contains a simple internal function `use_threads()` which takes an `int` parameter and returns an `unsigned int` parameter:

    unsigned int use_threads(int n_threads = 1) {
      #ifdef _OPENMP
      if(n_threads <= 1) return(1);
      if(n_threads > omp_get_max_threads()) {
        return((unsigned int)omp_get_max_threads());
      }
      return((unsigned int)n_threads);
      #else
      return(1);
      #endif
    }

This function takes a user request for the number of threads (CPU workers) to be used. Sometimes these requests may not be appropriate (e.g. requesting more threads than available in the system). First, notice that we use the `#ifdef` directives to tell the compiler which block of code to compile, based on whether OpenMP is available on the system. If not compiled with OpenMP , this function returns `1` (single core). If compiled with OpenMP, the function checks the maximum available threads and returns this number if the user requests more threads than is available.

We recommend users include a similar function to make sure their program knows how many threads to run, as they will be running an OpenMP-based loop in their functions.

## (2c) Opening BAM files with ompBAM

Now, lets return to the `idxstats_pbam()` function. We will show a subset of this function below:

    // [[Rcpp::export]]
    int idxstats_pbam(std::string bam_file, int n_threads_to_use = 1){

      // Ensure number of threads requested < number of system threads available
      unsigned int n_threads_to_really_use = use_threads(n_threads_to_use);

      pbam_in inbam;
      inbam.openFile(bam_file, n_threads_to_really_use);

      /*
        lots of code
      */

      return(0);
    }

#### Note 3 things:

1. The line `// [[Rcpp::export]]` tells Rcpp to export this function so that R can call these functions directly. They will not be exported functions so you will still need to create wrapper functions from within R, and export them, for example:

    # this is added to R/ompBAM_imports.R by use_ompBAM()

    #' @export
    idxstats <- function(bam_file, n_threads) {
      idxstats_pbam(bam_file, n_threads)
    }

2. `idxstats_pbam()` takes two inputs. The first, `bam_file`, takes a string input which is the file name of the BAM file. The second, `n_threads_to_use` will take a user input to request the number of threads. We sanitise this number as discussed in the previous section, to make sure the number of threads in this system is not above that available to the system.

3. We create a `pbam_in` object called `inbam`. The `pbam_in` class is described in the `pbam_in.hpp` file which is included in `ompBAM.hpp`. Users can find the header files in the `include/` directory within the installation path of ompBAM, but we endeavour to explain as much as you need to know in this document. For now, we call `pbam_in::openFile()` which directs the `pbam_in` object `inbam` to open and examine the given BAM file. `openFile()` takes a `std::string` for the path to the BAM file, and an `unsigned int` to specify the number of threads to initialize pbam_in for parallel file reading and decompression.

## (2d) Obtaining chromosome names and lengths

Once `pbam_in` opens a BAM file, it will automatically check the BAM file for errors. After noting the file size, it will check whether the BAM file is truncated (by verifying whether a BGZF EOF end of file marker is set at the end of the file). It will then read the BAM header and extract the chromosome names and their genome lengths.

To obtain the chromosome names and lengths, and to check that the BAM file is indeed valid:

    std::vector<std::string> s_chr_names;
    std::vector<uint32_t> u32_chr_lens;
    int chrom_count = inbam.obtainChrs(s_chr_names, u32_chr_lens);

Here, we create a string vector to store the chromosome names, and an unsigned 32-bit integer vector for chromosome lengths. These vectors are passed by reference to the `pbam_in::obtainChrs()` function which will fill these vectors with the chromosome names and lengths. `obtainChrs()` will return a negative number if the BAM header read fails, and will return the number of chromosomes if the function is run successfully. We can check for this with the following:

    // tells idxstats_pbam to return -1 to exit the function if the BAM file
    // has zero chromosomes or is not a valid BAM file
    if(chrom_count <= 0) return(-1);

## (2e) Constructing a loop to read the BAM file using ompBAM

ompBAM is designed to read an entire BAM file using multiple threads. This functionality is ideal for programs that perform whole-transcriptome calculations. Programs that interrogate small genomic regions are better off using htslib and interrogating sorted BAM files.

To read an entire BAM file, we need to construct code contained within a loop. This is because we can conserve RAM by not committing the entire decompressed BAM to memory. Instead, we read a small portion of the BAM file sequentially, process all the aligned reads therein, before reading / decompressing more of the BAM file again.

First, lets declare a vector to store the per-chromosome reads so we can count these reads:

    std::vector<uint32_t> total_reads(chrom_count);

To sequentially read a “small” portion of the BAM file, we call `pbam_in::fillReads()` and contain this within a `while` loop:

    while(0 == inbam.fillReads()) {
      // Some code here
    }

`pbam_in::fillReads()` returns `0` if the BAM file successfully extracts some reads and we are not at the end of file. If all the reads have already been extracted from the BAM file and no data was decompressed, `fillReads()` returns `1`, and if the BAM file is corrupt or the decompression returns an error, `fillReads()` returns `-1`.

Note that `fillReads()` will also return an `-1` error if the program has not processed all the reads decompressed by the previous call to `fillReads()`. We will explain this further in the next section

## (2f) Processing BAM reads using an OpenMP parallel FOR loop

OpenMP provides an easy-to-use parallelism which we can use to run each iteration of a `FOR` loop in parallel. That is, each iteration of the `FOR` loop is run simultaneously, each iteration being processed by a separate thread. We can set up this parallel `FOR` loop in the following code:

    #ifdef _OPENMP
    #pragma omp parallel for num_threads(n_threads_to_really_use) schedule(static,1)
    #endif
    for(unsigned int i = 0; i < n_threads_to_really_use; i++) {
      std::vector<uint32_t> read_counter(chrom_count);
      // Process thread-specific BAM reads here
    }

Note the line beginning with `#pragma omp parallel for`. This line simply specifies that the `FOR` loop declared after this line should be run in parallel. `num_threads(n_threads_to_really_use)` declares that we want to use the specified number of threads, as stored in the `n_threads_to_really_use` variable. `schedule(static,1)` declares that one thread should be used to run each `FOR` iteration.

Also note that this line is enclosed within an `#ifdef _OPENMP`. If OpenMP is not available at compile, the compiler simply ignores the `#pragma` and will run the `FOR` loop sequentially.

Within this loop, we are free to declare any thread-specific variables. Variables declared within the parallel FOR loop are thread-specific, meaning their contents cannot be accessed by other threads processing other iterations. So within the `FOR` loop, we declare a `read_counter` vector which will count reads processed by each thread.

## (2g) Getting thread-specific reads from ompBAM

To obtain a single aligned read from a thread-specific buffer of reads processed by ompBAM, we declare a `pbam1_t` object and use `pbam_in::supplyRead()` to get a single read from the thread-specific buffer:

    pbam1_t read(inbam.supplyRead(i));

As the above code is contained within the parallel `FOR` loop, `i` here refers to the thread ID. For n threads, `i` will take any integer between `0` and `n-1`. `supplyRead()` will return the next read in the thread-specific buffer which we receive by calling it within the `pbam1_t` constructor at declaration.

Note that the above code is equivalent to:

    pbam1_t read;
    read = inbam.supplyRead(i);

### (2g:i) Checking “validity” of reads and “realizing” reads

`pbam1_t` is an ompBAM object that is designed to store and get values contained in a single aligned BAM read. We will discuss its full functionality in section (4). For now, the most important function is `pbam1_t::validate()`, which will check whether the obtained read actually contains any data.

A `pbam1_t` can be “invalid” for several reasons. The most important reason is when `supplyRead()` has reached the end of its buffer. When this occurs, `supplyRead()` will simply return an empty read, which will not validate.

So, to profile all the reads in a single thread, we simply call `supplyRead()` for thread `i` until it returns an invalid read, using the following:

    // Gets the first read from the thread-specific buffer
    pbam1_t read(inbam.supplyRead(i));

    while(read.validate()) {
      // Do stuff with this valid read

      // Gets the next read
      read = inbam.supplyRead(i);
    }

`pbam1_t::validate()` will return `false` for two other reasons.

1. If the BAM contains corrupt data, `validate()` will return false. `validate()` checks the first 4 bytes of the read which returns the size of the read (in bytes). It then checks whether this is larger than the size of the individual components, including read name, cigar, sequence and quality scores. If there is corrupt data, we might expect that this will trigger a failure and safely abort the program, as the next call to `fillReads()` will trigger an error notifying that not all reads were processed.

2. `pbam1_t` optimises performance by storing pointers to the memory that contains decompressed BAM data, rather than creating separate memory containers. Therefore, copying a `pbam1_t` read will only copy its memory address, not the data contained in the read. We refer to these as “virtual” `pbam1_t` reads. The consequence is, the next time `fillReads()` is called, these reads will point to an invalid memory address and will become invalid.

In some situations (e.g. for paired RNA-seq reads), it is desirable to keep some reads around in memory. To do this we can do something like the following:

    std::vector<pbam1_t> read_storage;
    pbam1_t real_read = read;
    real_read.realize();

    read_storage.push_back(real_read);

In the above code, we call `pbam1_t::realize()`. This function creates a read- specific data buffer and copies the data from the thread-specific buffer. A “real” `pbam1_t` read simply means it has its own data storage and is thus “persistent”, i.e. it will still store the read after the next call to `fillReads()`. In other words:

    pbam1_t virtual_read = inbam.supplyRead(i);
    pbam1_t real_read = inbam.supplyRead(i);
    pbam1_t read = inbam.supplyRead(i);
    while(read.validate()){
      // consumes the remainder of reads
      read = inbam.supplyRead(i);
    }

    real_read.realize();    // make this read 'real'

    virtual_read.isReal();  // returns false
    real_read.isReal();     // returns true

    inbam.fillReads();

    virtual_read.validate();  // returns false
    real_read.validate();     // returns true

NB: the `pbam1_t::realize()` and `pbam1_t::isReal()` functions are not demonstrated in the included example code created by `use_ompBAM()`. For more information about these functions, refer to Section (4c)

## (2h) Counting chromosome-specific reads

Now that we have discussed the most important aspects of `pbam1_t` as well as the `fillReads()` and `supplyRead()` functions of `pbam_in`, we will now count the reads within the OpenMP parallel FOR loop. The entire processing loop is included below and we will discuss the remaining lines in detail:

    // Creates a data structure that stores per-chromosome read counts
    std::vector<uint32_t> total_reads(chrom_count);

    while(0 == inbam.fillReads()) {
      // OpenMP parallel FOR loop, each thread runs 1 loop simultaneously.
      #ifdef _OPENMP
      #pragma omp parallel for num_threads(n_threads_to_really_use) schedule(static,1)
      #endif
      for(unsigned int i = 0; i < n_threads_to_really_use; i++) {
        std::vector<uint32_t> read_counter(chrom_count);

        // Gets the first read from the thread read storage buffer
        pbam1_t read(inbam.supplyRead(i));
        // Keep looping while reads are valid
        while(read.validate()) {
          // Counts the read if it is mapped to a chromosome
          if(read.refID() >= 0 && read.refID() < chrom_count) {
            read_counter.at(read.refID())++;
          }

          // Gets the next read
          read = inbam.supplyRead(i);
        }
        // Adds the counted reads to the final count
        // #pragma omp critical ensures only 1 thread at a time runs the following
        // block of code.
        #ifdef _OPENMP
        #pragma omp critical
        #endif
        for(unsigned int j = 0; j < (unsigned int)chrom_count; j++) {
          total_reads.at(j) += read_counter.at(j);
        }
      }
      // At this stage, all threads would have read all their thread-specific reads
      // At the next call to pbam_in::fillReads(), if any reads were not read, it
      // will throw an error and fillReads() will return -1.
      // If we have finished reading the BAM file, fillReads() will return 1.
    }

If you have read all the sections until now, you will understand most of the code in this loop. We will discuss the things we have not already addressed.

### (2h:i) Obtaining the chromosome ID of each aligned read

The `pbam1_t::refID()` returns the `refID` of the aligned read. Rather than the name of the chromosome, it returns the ID as a number from `0` to `k-1` for an `k`-chromosome genome. These are in the same order as described in the BAM header. So here, we simply check whether the read is mapped to a valid chromosome refID, and add it to the thread-specific read container vector:

    if(read.refID() >= 0 && read.refID() < chrom_count) {
      read_counter.at(read.refID())++;
    }

For other “getter” functions of pbam1_t, refer to Section (4e-h).

### (2h:ii) Tallying reads to a variable external to the parallel FOR loop

Any variables declared outside the OpenMP `FOR` loop is accessible to code within the parallel loop. This can be both a blessing and a curse. It can cause problems when multiple threads write to the variable at the same time (known as a “race condition”). To prevent this, we declare that only 1 thread can run the code that is responsible for accessing the external variable `total_reads`, using the `#pragma omp critical` directive. This directive instructs the following block of code can only be run by a single thread at any one time. Of course, we declare this within an `#ifdef _OPENMP` directive so that compilers without OpenMP will ignore it.

## (2i) Closing the BAM file

After we have read the BAM file, we will close the file.

    inbam.closeFile();

This will instruct `pbam_in` to close the file that it opened internally using the ifstream object contained within. It will also destroy all memory associated with its file and decompressed data buffers, thereby safely freeing up memory. This is also called on destruction of the `pbam_in` object, so files are safely closed when the `pbam_in` object goes out of scope. Of course, `pbam1_t` reads will invalidate once these buffers are cleared, except “real” `pbam1_t` reads created via `pbam1_t::realize()`.

## (2j) Summarizing the tallied reads

For the output of this function, we use the function Rcout (from Rcpp) to print out the chromosome names, lengths and read counts, just like the samtools idxstats function:

      Rcout << bam_file << " summary:\n" << "Name\tLength\tNumber of reads\n";
      for(unsigned int j = 0; j < (unsigned int)chrom_count; j++) {
        Rcout << s_chr_names.at(j) << '\t' << u32_chr_lens.at(j) << '\t'
          << total_reads.at(j) << '\n';
      }

## (2k) Adding a progress bar

BAM files are very large files (or we wouldn’t use multi-threading to read them). Sometimes a progress bar is very useful in this regard.

`pbam_in` has two functions that provide useful data that can be parsed to the RcppProgress progress bar.

- `pbam_in::GetFileSize()` will return the file size (in bytes) of the opened BAM file.
- `pbam_in::IncProgress()` will return the number of bytes processed (read and decompressed) since the last call to `IncProgress()` was made

These functions are demonstrated in the example code contained within the ompBAMExample package contained within the `examples/` folder of the ompBAM installation path. Readers are welcome to peruse this example package which contains the `idxstats_pbam` function with the addition of a RcppProgress bar.

The path to the source code can be returned using the following in R:

    source_path = system.file("examples", "ompBAMExample", "src",
        package = "ompBAM")

Alternatively, refer to Section (3i) for an example RcppProgress bar

# (3) pbam_in function documentation

The `pbam_in` object is responsible for opening BAM files for sequential multi-threaded BAM processing.

Internally, `pbam_in` checks BAM files for truncation, data corruption and other issues. Then it reads and decompresses BAM files using multiple threads. Applications using ompBAM simply have to retrieve reads from the `pbam_in` object using the `supplyRead()` function, after “priming the pump” to fill the reads buffer using the `fillReads()` function. `supplyRead()` is considered “thread-safe”, meaning that applications using ompBAM can run `supplyRead()` within multi-threaded blocks of code. This allows applications using ompBAM to process reads using multiple threads.

It is highly recommend to read the previous section (2) - “Step-by-step guide to creating your first ompBAM-powered package” before scrutinising this section. The “Step-by-step guide” provides the context behind all the functions mentioned in this section.

Please note that currently, ompBAM only supports BAM file reading. BAM writing may be supported in later versions of ompBAM.

## (3a) Constructor

Creates a `pbam_in` object.

#### Usage

    // Empty constructor with defaults
    pbam_in();

    // Constructor with custom settings (for advanced users only)
    pbam_in(
      const size_t file_buffer_cap,
      const size_t data_buffer_cap,
      const unsigned int chunks_per_file_buffer,
      const bool read_file_using_multiple_threads = true
    );

#### Parameters

- `const size_t file_buffer_cap`: (default 500 Mb) The size (in bytes) of each of the two file buffers
- `const size_t data_buffer_cap`: (default 1 Gb) The size (in bytes) of the data buffer containing uncompressed data
- `const unsigned int chunks_per_file_buffer`: (default 5) How many chunks should the file buffer be divided. See details
- `const bool read_file_using_multiple_threads` (default true): Whether to use multiple threads to read compressed data from file.

#### Details

`pbam_in` reads a set amount of data from the BAM file to efficiently process reads. Internally, it allocates two “file” buffers to store compressed data, each of size `file_buffer_cap`, as well as a larger “data” buffer to store uncompressed data, of size `data_buffer_cap`.

The first call to `pbam_in::fillReads()`, the function that decompresses the BAM file to extract BAM reads, will fill the first file buffer with data, as well as a fraction (one “chunk”) of data to fill the second file buffer. This chunk size is determined by `chunks_per_file_buffer`. For example, if `file_buffer_cap = 5e8` i.e. 500 Mb and `chunks_per_file_buffer = 5`, then the chunk size is 100 Mb.

After importing compressed data, `pbam_in` will use multiple threads to decompress enough data to either fill up the data buffer, or decompress a portion of the file buffer (as determined by chunk size), whichever occurs first.

Subsequent calls to `pbam_in::fillReads()` will check whether the number of chunks stored in the second file buffer exceeds the number of chunks already processed in the first file buffer. Should this occur, it will read compressed data from the BAM file and place this in the second file buffer. When the amount of unprocessed data in the first file buffer drop below one chunk size amount, a “buffer swap” occurs where the data is copied from the second file buffer to the first. Thereafter, the process continues until the entire BAM file has been read and decompressed.

As can be seen, the optimal values of `file_buffer_cap`, `data_buffer_cap` and `chunks_per_file_buffer` will depend on the compression ratio of the BAM file, the I/O speed of the storage device, as well as the available memory. We believe the default settings is a good starting point and will consume approximately 2 Gb of RAM.

Storage on hard disk drives (with spinning components) typically found on traditional desktop computers may experience lower file I/O in multi-threaded applications. Solid-state drives (typically found in modern notebook laptops) and some RAID setups may favour multi-threaded file input. To instruct `pbam_in` to read the file using a single thread and decompress with the remaining cores, set `read_file_using_multiple_threads = false`.

Note that copy constructor and copy assignment operators are disabled for `pbam_in`. Thus, code containing things like the following will fail at compile-time:

    pbam_in bam1;           // Default constructor (will compile)
    pbam_in bam2 = bam1;    // copy assignment operation (will FAIL)
    pbam_in bam3(bam1);     // copy constructor (will FAIL)

#### Examples

    // Creates a pbam_in object with default settings
    pbam_in default_pbam;

    // Creates a pbam_in object with two 0.5Gb buffers, one 2 Gb data buffer, and
    // will prompt file access when the last chunk of the first 10-chunk buffer is
    // being decompressed. Set read_file_using_multiple_threads = true to enable
    // multi-threaded file read access.
    pbam_in custom_pbam(5e8, 2e9, 10, true);

## (3b) openFile()

Opens a BAM file given the file path, and the requested number of threads to be used. Also checks the BAM file and reads the BAM header (silently).

#### Usage

    int openFile(
      std::string filename,
      const unsigned int n_threads
    );

#### Parameters

- `std::string filename`: The path to the BAM file to be opened
- `const unsigned int n_threads`: The number of threads to use to read the BAM file

#### Examples

    std::string bam_file = "example.bam";

    pbam_in inbam;
    inbam.openFile(bam_file, 4);    // Accesses the BAM file using 4 threads

## (3c) SetInputHandle()

Assigns an `istream` handle to `pbam_in`. Also defines how many threads to use to read the BAM file. This is used as an alternative to `pbam_in::openFile()`.

#### Usage

    int SetInputHandle(
      std::istream *in_stream,
      const unsigned int n_threads
    );

#### Parameters

- `std::istream *in_stream`: The handle of an ifstream that has opened a BAM file using input binary access
- `const unsigned int n_threads`: The number of threads to use to read the BAM file

#### Details

Frankly this is superseded by `openFile()`.

NB: multi-threaded read access is disabled when `pbam_in` accesses a BAM file via this method, because it does not know the BAM file path.

#### Examples

    std::string bam_file = "example.bam";
    std::ifstream inbam_stream;

     // Opens the BAM file for read only binary access
    inbam_stream.open(bam_file, std::ios::in | std::ios::binary);

    pbam_in inbam;

    // Accesses the BAM file using 4 threads
    inbam.SetInputHandle(&inbam_stream, 4);

## (3d) closeFile()

Closes the BAM file. Also deallocates all memory contained in the `pbam_in` object, essentially resetting this object.

#### Usage

    int closeFile();

#### Parameters

None

#### Details

Note that `closeFile()` internally calls `reset()`, which is also called at object destruction. Thus, any open BAM files are closed when the `pbam_in` object goes out of scope. This function is provided if closing the `ifstream` associated with the BAM file, and/or freeing up RAM associated with `pbam_in`, is desired.

#### Examples

    std::string bam_file = "example.bam";

    pbam_in inbam;
    inbam.openFile(bam_file, 4);    // Accesses the BAM file using 4 threads

    inbam.closeFile();              // Closes the file associated with the pbam_in

## (3e) obtainChrs()

Retrieves chromosome names and lengths from an opened BAM file

#### Usage

    int obtainChrs(
      std::vector<std::string> & s_chr_names,
      std::vector<uint32_t> & u32_chr_lens
    );

#### Parameters

- `std::vector<std::string> & s_chr_names` A reference to a string vector to contain chromosome names
- `std::vector<uint32_t> & u32_chr_lens` A reference to an uint32_t vector to contain chromosome lengths

#### Return value

(int) The number of chromosomes in the genome the BAM file is aligned to. Chromosome names and lengths are returned to the two vectors supplied by reference.

If `pbam_in` has not opened a valid BAM file, this function returns `-1`

#### Details

When a BAM file is opened via `openFile()` or `SetInputHandle()`, pbam_in will automatically read and store the header data, namely chromosome names and lengths. `obtainChrs()` can be called to retrieve this data.

#### Examples

    std::string bam_file = "example.bam";

    pbam_in inbam;
    inbam.openFile(bam_file, 4);

    std::vector<std::string> s_chr_names; // A vector to contain chromosome names
    std::vector<uint32_t> u32_chr_lens;   // A vector to contain chromosome lengths
    int chrom_count = inbam.obtainChrs(s_chr_names, u32_chr_lens);

## (3f) fillReads()

Reads from the BAM file, decompresses the file buffer to extract the reads.

#### Usage

    int fillReads();

#### Parameters

None

#### Return value

Returns `0` if successful, `-1` if error, and `1` if end of file is already reached.

#### Details

A loop is required to read the BAM file until finished. This can be set up by checking the return value of fillReads() and making sure it is zero. If it is non-zero, abort the loop as there is either an error with the file, or end of file was reached in the last call to fillReads(). See example below.

NB1: `fillReads()` should only be called by the main thread (i.e. it should not be called from within child threads). `fillReads()` contain OpenMP parallel `FOR` loops that perform file reads and decompressions using multi-threading.

NB2: Applications must ensure that all reads decompressed by `fillReads()` are obtained from every thread via `pbam_in::supplyRead()`. If some reads are not obtained, the next call to `fillReads()` will return an error.

#### Examples

    std::string bam_file = "example.bam";

    pbam_in inbam;
    inbam.openFile(bam_file, 4);

    while(0 == inbam.fillReads()) {
      // Process reads here
    }

## (3g) supplyRead()

Reads the thread-specific data buffer to supply a single aligned read from the BAM file.

#### Usage

    pbam1_t supplyRead(const unsigned int thread_id = 0);

#### Parameters

- `const unsigned int thread_id` The index of the thread-specific buffer from which to retrieve the read.

#### Return value

`pbam1_t` A `pbam1_t` object containing the data from the aligned read.

#### Details

After `fillReads()` is called and the data buffer is filled, the reads are split into equal portions of decompressed data. Each chunk of data is processed by a single thread. This allows developers to set up an OpenMP for-loop such that reads from each chunk are processed exclusively by each thread.

Reads are divided into contiguous blocks. E.g., thread `i=0` contains a contiguous block of reads, and that the first read in thread `i=1` contains the read following the last read from thread `i=0`.

Setting `i` to the thread-ID makes sure `supplyRead()` is thread-safe, ensuring that we don’t have a situation where multiple threads are accessing the same data (i.e. a “race condition”).

When the end of the thread-specific buffer is reached, `fillReads()` will return an empty read (which will not validate using `pbam1_t::validate()`. This is useful to check whether the thread-specific buffer is exhausted.

#### Examples

    std::string bam_file = "example.bam";

    pbam_in inbam;
    inbam.openFile(bam_file, 4);

    inbam.fillReads()

    // Presuming we are in an OpenMP parallel for loop, in thread `i`
    pbam1_t read;
    read = inbam.supplyRead(i);

## (3h) remainingThreadReadsBuffer();

Queries how much decompressed data is available to be read by the thread- specific buffer

#### Usage

    size_t remainingThreadReadsBuffer(
        const unsigned int thread_id = 0
    );

#### Parameters

- `const unsigned int thread_id` (default 0) The index of the thread- specific buffer from which to retrieve the read.

#### Return value

Returns (in bytes) the amount of aligned reads available to be returned by `supplyReads()`

#### Details

This function is useful to distinguish between whether an invalid read returned by `supplyReads()` is because all the data from the thread-specific buffer has been process, or whether corrupt data is detected.

Frankly, it is sufficient to check for errors at `fillReads()`, because it is probably inappropriate to keep reading the BAM file if some reads are corrupt.

#### Examples

    std::string bam_file = "example.bam";

    pbam_in inbam;
    inbam.openFile(bam_file, 4);

    inbam.fillReads()

    if(0 != inbam.remainingThreadReadsBuffer(i)) {
      Rcpp::Rcout << "Thread " << i << " still has reads remaining!\n";
    }

## (3i) Progress and File Size Functions

Returns data regarding file size, and progress

#### Usage

    size_t GetFileSize();
    size_t GetProgress();
    size_t IncProgress();

#### Return value

- `GetFileSize()` Returns the file size of the BAM file
- `GetProgress()` Returns the total number of (compressed) bytes in the BAM file that has been processed (i.e. read and decompressed)
- `IncProgress()` Returns the number of bytes processed since the last call to `IncProgress()`

#### Details

These functions are useful for addition of progress bars. The following example demonstrates a simple method of setting up an `RcppProgress` progress bar.

#### Examples

    #include "Rcpp.h"
    using namespace Rcpp;

    // Required to print cout output generated by ompBAM
    #define cout Rcpp::Rcout

    // [[Rcpp::depends(ompBAM)]]
    #include <ompBAM.hpp>

    // [[Rcpp::depends(RcppProgress)]]
    #include <progress.hpp>

    int main() {
      std::string bam_file = "example.bam";

      pbam_in inbam;
      inbam.openFile(bam_file, 4);

      size_t file_size = inbam.GetFileSize();
      size_t bytes_read = inbam.GetProgress();

      // Initialize the RcppProgress bar with "100%" set as the size of the BAM file
      Progress p(inbam.GetFileSize());
      while(0 == inbam.fillReads()) {
        // Increment the progress bar by the number of bytes incrementally processed
        p.increment(inbam.IncProgress());

        #ifdef _OPENMP
        #pragma omp parallel for num_threads(4) schedule(static,1)
        #endif
        for(unsigned int i = 0; i < 4; i++) {
          pbam1_t read(inbam.supplyRead(i));
          // Keep looping while reads are valid
          while(read.validate()) {
            read = inbam.supplyRead(i);
          }
        }
      }
      // Final increment to RcppProgress bar to fill it to 100%
      p.increment(inbam.IncProgress());

      return(0);
    }

# (4) pbam1_t function documentation

The `pbam1_t` object is used to retrieve data from a single aligned read.

`pbam1_t` object is returned from the `supplyRead()` function of `pbam_in` and is the primary way in which ompBAM provides access to the alignment data in BAM files. `pbam1_t` contain many useful functions that make it easy to access data from read alignments. In the documentation below, “reads” and “read alignments” are use interchangeably and are equivalent.

NB: In all the examples below, it is assumed that a `pbam_in` object, named `inbam`, has been initialized, and the first `fillReads()` has been called using the following code:

    #include "Rcpp.h"
    using namespace Rcpp;

    // Required to print cout output generated by ompBAM
    #define cout Rcpp::Rcout

    // [[Rcpp::depends(ompBAM)]]
    #include <ompBAM.hpp>

    std::string bam_file = "example.bam";

    pbam_in inbam;
    inbam.openFile(bam_file, 4);
    inbam.fillReads();

    int i = 0; // Thread 0

## (4a) Constructor

Creates a `pbam1_t` object

#### Usage

    // Empty constructor with defaults
    pbam1_t();

    // Used by pbam_in::supplyRead()
    pbam1_t(char * src, const bool realize = false);

    // Copy constructor
    pbam1_t(const pbam1_t &t);

    // Copy assignment operator
    pbam1_t & operator = (const pbam1_t &t);

#### Parameters

- `char * src` A raw pointer to the char* in the `pbam_in` data buffer containing the read.
- `const bool realize` Whether to “realize” the read.

#### Details

As explained in the previous section (2g:i Checking “validity” of reads and “realizing” reads"), `pbam1_t` does not initially contain dedicated memory space to store the data in a BAM read. Rather, it is a “virtual” read, in which it contain pointers to the corresponding data on the data buffer in the `pbam_in` object. This allows rapid processing of the BAM file and spares unnecessary `memcpy` operations which will increase RAM usage and slow the program.

However it can be converted into a “real” read, whereby `pbam1_t` will allocate the required memory space and copies the data from the `pbam_in` data buffer. When reads are “realized”, subsequent calls to `pbam_in::fillReads()` will not result in invalidation of such reads. This is important when data persistence is required, e.g. when matching paired reads in coordinate-sorted paired-end RNA-seq data.

NB: In typical usage, users are not expected to use the constructor to create “real” reads. Instead, it is better to first obtain the virtual read using `pbam_in::supplyRead()`, and subsequently “realize” the read via `pbam1_t::realize()`.

#### Examples

    // Creates a pbam1_t object
    pbam1_t read;

    // Construct and "realize" read based on a known location
    // in a buffer given by pointer (char* dest)
    pbam1_t read(dest, true)

    // Use copy constructor to get a read from the pbam_in object `inbam`
    pbam1_t read(inbam::supplyRead(i));

    // Use copy assignment to get a read from the pbam_in object `inbam`
    pbam1_t read = inbam::supplyRead(i);

## (4b) validate()

Checks whether the read contained within the `pbam1_t` object contains valid data for a BAM read.

#### Usage

    bool validate() const;

#### Parameters

None

#### Return value

Returns `true` if the read contains valid data, and `false` otherwise

#### Details

`validate()` makes the following checks, and returns false quickly if any of these checks fail:

- whether a valid pointer to data exists.
- whether the block_size as given by the first 4 bytes of the buffer matches the internally stored value of the read block_size calculated at its construction
- whether the size of the individual components (cigar, sequence, quality scores, and size of additional tag data) totals the given block_size.

It is most often used to check whether `pbam_in::supplyRead()` has supplied an empty read signifying end of the thread-specific buffer. It can also check for invalid data pointers if users call `pbam_in::fillReads()` before “realizing” the reads to make the `pbam1_t` data persist. As explained previously in Section (2g:i), `pbam_in::fillReads()` will invalidate virtual reads as their data would have been recycled; however, realized reads will be persistent and continue to validate.

Internally, `validate()` is called prior to returning any values from the `pbam_1` “getter” functions.

#### Examples

    pbam1_t read = inbam.supplyRead(i);
    while(read.validate()){
      // keep getting reads from pbam_in::supplyRead()
      // until it returns an empty, invalid read
      read = inbam.supplyRead(i);
    }

## (4c) realize()

Converts a virtual read into a real read, thereby allowing data persistence by allocating specific memory space to store the data.

#### Usage

    int realize();

#### Parameters

None

#### Return value

Returns `0` if the `pbam1_t` was successfully converted to a “real” read, and `-1` if the given read was invalid or if this function fails.

#### Details

`realize()` first checks if the `pbam1_t` was already a real read (i.e. it is contained within a dedicated persistent data buffer). If it is not, it allocates a buffer using `malloc`, and copies the data from the buffer pointed to by the previously-virtual read, using `memcpy`. It then rechecks validity.

This function is used to convert reads such that they no longer point to memory initiated by `pbam_in`, but instead have their own dedicated memory buffers (i.e. their memory becomes persistent)

#### Examples

    // Stores every read in a vector of `pbam1_t` objects
    std::vector<pbam1_t> read_container;

    pbam1_t read = inbam.supplyRead(i);
    while(read.validate()){
      read.realize();
      read_container.push_back(read);

      // keep getting reads from pbam_in::supplyRead()
      // until it returns an empty, invalid read
      read = inbam.supplyRead(i);
    }
    // reads stored inside read_container should now persist beyond the next call
    // to pbam_in::fillReads()

## (4d) isReal()

Checks whether the pbam1_t is persistent (real) or points to the pbam_in memory (virtual)

#### Usage

    bool isReal() const;

#### Parameters

None

#### Return value

Returns `true` if the read is real, and `false` if it is virtual.

#### Details

See Section (4c) `pbam1_t::realize()` for detailed explanation of “real” vs “virtual” reads

#### Examples

    pbam1_t read = inbam.supplyRead(i);
    read.isReal();  // Returns false

    read.realize();
    read.isReal();  // Returns true

## (4e) Getters of Read Core Data

The core is the first 36 bytes of the alignment read data. It contains constant-length data including chromosome refID and the left-most genomic coordinates of the alignment. It also contains metadata including cigar size and sequence length.

#### Usage

    uint32_t block_size();  // Size of the alignment data (in bytes)
    int32_t refID();        // refID of chromosome of this alignment
    int32_t pos();          // leftmost 0-based genome coordinate of this alignment
    uint8_t l_read_name();  // length of read name, including terminating '\0' null
    uint8_t mapq();         // mapping quality
    uint16_t bin();         // BAI index bin
    uint16_t n_cigar_op();  // number of cigar operations
    uint32_t flag();        // Bitwise flags
    uint32_t l_seq();       // Sequence length
    int32_t next_refID();   // refID of next segment
    int32_t next_pos();     // pos of next segment
    int32_t tlen();         // template length

#### Parameters

None

#### Return value

See comments in the “Usage” section above. Also, it is helpful to refer to SAMv1.pdf - section 4.2 for further details regarding the BAM format.

#### Examples

    pbam1_t read = inbam.supplyRead(i);

    Rcpp::Rcout << "The alignment block size is " << read.block_size() << '\n';
    Rcpp::Rcout << "The alignment refID is " << read.refID() << '\n';
    Rcpp::Rcout << "The alignment left-most coordinate is " << read.pos() << '\n';
    Rcpp::Rcout << "The read name length is "
      << unsigned(read.l_read_name()) << '\n';
    Rcpp::Rcout << "The alignment mapping quality is "
      << unsigned(read.mapq()) << '\n';
    Rcpp::Rcout << "The BAI index bin is " << read.bin() << '\n';
    Rcpp::Rcout << "The number of cigar ops is " << read.n_cigar_op() << '\n';
    Rcpp::Rcout << "The flag binary value is " << read.flag() << '\n';
    Rcpp::Rcout << "The sequence length is " << read.l_seq() << '\n';
    Rcpp::Rcout << "The next alignment refID is " << read.next_refID() << '\n';
    Rcpp::Rcout << "The next alignment pos " << read.next_pos() << '\n';
    Rcpp::Rcout << "The alignment template length is " << read.tlen() << '\n';

## (4f) Getters of Cigar Data

The cigar is data which specifies the nature of the alignment.

#### Usage

    // Direct uint32_t pointer to raw data
    uint32_t * cigar();

    // number of cigar operations; includes compatibility for long-read data
    uint32_t cigar_size();

    // Fills a string with the SAM-based cigar string
    // - Returns cigar_size() if success
    // - Returns 0 if failed to validate
    int cigar(std::string & dest);

    // Returns the cigar operation / value (as char / uint32_t)
    //   given the position of the cigar operation
    char cigar_op(const uint16_t pos);
    uint32_t cigar_val(const uint16_t pos);

    // Returns the cigar operations and values (as char / uint32_t) as a vector
    int cigar_ops_and_vals(std::vector<char> & ops, std::vector<uint32_t> & vals);

#### Parameters

- `std::string & dest` The reference to a string to contain the cigar string
- `const uint16_t pos` The index of the cigar operation (`0 <= pos < cigar_size()`)
- `std::vector<char> & ops` The reference to a `char` vector to contain the cigar operation
- `std::vector<uint32_t> & vals` The reference to a `uint32_t` vector to contain the number of nucleotides to apply the cigar operation

#### Return value

`uint32_t * cigar()` returns a pointer to the data buffer at which the cigar data begins.

`uint32_t cigar_size()` returns the number of cigar operations. The BAM format is limited at 65535 operations which is insufficient for some long-read applications. Recently, the “CG” tag has been implemented that allows storage of alignment cigar data beyond this limit. ompBAM allows for this by first checking whether the “CG” tag exists. If it does, `cigar_size()` returns the length of this tag. If it does not, `cigar_size()` returns `n_cigar_op` which is the 16-bit storage of the cigar length.

`int cigar()` takes as reference a string and fills it with a string (in SAM format) of the entire cigar string.

`cigar_op()` and `cigar_val()` returns the cigar operation and length, respectively, given the index of cigar operation `pos` (where `0 <= pos < cigar_size()`)

`cigar_ops_and_vals()` takes two vectors by reference (`ops` and `vals`) and fills these with the cigar operations and lengths, respectively, of the entire cigar.

Refer to SAMv1.pdf - section 1.4 for further details regarding the cigar string (in SAM format).

#### Examples

    pbam1_t read = inbam.supplyRead(i);

    // Stores raw pointer to the data containing the cigar
    uint32_t * cigar_buffer = read.cigar();

    uint32_t cigar_len = read.cigar_size();
    Rcpp::Rcout << "The cigar has " << cigar_len << " operations\n";

    std::string cigar_string;
    read.cigar(cigar_string);
    Rcpp::Rcout << "The cigar string is " << cigar_string << '\n';

    Rcpp::Rcout << "The cigar operation at index 0 is "
        << read.cigar_op(0)
        << " and the length to apply this is "
        << std::to_string(read.cigar_val(0)) << '\n';

    std::vector<char> ops;
    std::vector<uint32_t> vals;
    read.cigar_ops_and_vals(ops, vals);

    Rcpp::Rcout << "The cigar operation at index 0 is "
        << ops.at(0)
        << " and the length to apply this is "
        << std::to_string(vals.at(0)) << '\n';

## (4g) Getters of Alignment Sequence and Quality score

Reads consist of a sequence of nucleotides. FASTQ files also contain per- nucleotide quality scores representing the level of statistical significance of each sequenced nucleotide. These values are often stored in BAM files after alignment.

#### Usage

    uint8_t * seq();
    char * qual();

    int seq(std::string & dest);
    int qual(std::vector<uint8_t> & dest);

#### Parameters

- `std::string & dest` A string reference to contain the sequence.
- `std::vector<uint8_t> & dest` A 8-bit unsigned integer vector to contain the list of quality scores

#### Return value

`uint8_t * seq()` and `char * qual()` returns the pointer to the raw data containing the sequence (in `uint8_t`) and quality scores (in `char`). Advanced users will have to convert the 4-bit sequence to nucleotides as described in the SAM documentation: SAMv1.pdf.

It is more convenient to use the latter functions. `int seq(std::string & dest)` takes a string reference as parameter and fills this reference with the sequence of the read. It returns the length of the read. `qual(std::vector<uint8_t> & dest)` takes a `uint8_t` vector by reference and fills this with per-nucleotide quality scores for the sequence. It returns the length of the read.

#### Details

Quality score can take values between [0,93] (without ASCII conversion). Users wishing to convert these to ASCII must add +33 to these scores before ASCII conversion. Alignments without quality scores will have all QUAL scores set at 255 (0xFF).

It is helpful to refer to SAMv1.pdf - section 4.2.3 for further details regarding SEQ and QUAL encoding.

#### Examples

    pbam1_t read = inbam.supplyRead(i);

    std::string sequence;
    std::vector<uint8_t> qual_scores;

    read.seq(sequence);
    read.qual(qual_scores);

    Rcpp::Rcout << "The sequence is " << sequence << '\n';

    if(qual_scores.at(0) != 255) {
      // After checking quality scores are not missing, convert to printable ASCII
      for(unsigned int k = 0; k < qual_scores.size(); k++) {
        qual_scores.at(k) += 33;
      }
      Rcpp::Rcout << "The Phred-scale per-nucleotide score ASCII is "
        << std::string(qual_scores.begin(), qual_scores.end()) << '\n';
    }

## (4h) Tag Getters

Tags are auxiliary information about each alignment.

#### Usage

    // Provides an index of tags available to the alignment
    int AvailTags(std::vector<std::string> & tags);

    // Provides metadata about the specific tag
    char Tag_Type(const std::string tag);
    char Tag_Subtype(const std::string tag);
    uint32_t Tag_Size(const std::string tag);
    char Tag_Type_SAM(const std::string tag);

    // Returns raw char pointer to the beginning of the info stored by the tag
    // - For advanced users only
    char * p_tagVal(const std::string tag);

    // Returns values of fixed length
    // - For tags of type AcCsSiIf
    // Returns '\0' or 0 if tag does not exist or if the type is inappropriate
    // for the given tag
    char tagVal_A(const std::string tag);       // tags of type 'A'
    int8_t tagVal_c(const std::string tag);     // tags of type 'c'
    uint8_t tagVal_C(const std::string tag);    // tags of type 'C'
    int16_t tagVal_s(const std::string tag);    // tags of type 's'
    uint16_t tagVal_S(const std::string tag);   // tags of type 'S'
    int32_t tagVal_i(const std::string tag);    // tags of type 'i'
    uint32_t tagVal_I(const std::string tag);   // tags of type 'I'
    float tagVal_f(const std::string tag);      // tags of type 'f'

    // Fills given string reference by given Z-type tag
    // Returns tag length of string if success, -1 if fail
    int tagVal_Z(const std::string tag, std::string & dest);  // tags of type 'Z'

    // Returns a B-tag by reference to its respective type
    // Returns tag length if success, -1 if fail
    int tagVal_B(const std::string tag, std::vector<int8_t> & dest);     // 'B, c'
    int tagVal_B(const std::string tag, std::vector<uint8_t> & dest);    // 'B, C'
    int tagVal_B(const std::string tag, std::vector<int16_t> & dest);    // 'B, s'
    int tagVal_B(const std::string tag, std::vector<uint16_t> & dest);   // 'B, S'
    int tagVal_B(const std::string tag, std::vector<int32_t> & dest);    // 'B, i'
    int tagVal_B(const std::string tag, std::vector<uint32_t> & dest);   // 'B, I'
    int tagVal_B(const std::string tag, std::vector<float> & dest);      // 'B, f'

#### Parameters

- `std::vector<std::string> & tags` A reference to a string vector in which to store a list of available tags for the read.
- `const std::string tag` A string containing the two-character tag to query
- `std::string & dest` A string reference to store the given string in Z-type tags.
- `std::vector<T> & dest` A reference to a vector of type <T> in which to store the data contained in ‘B’-type tags.

#### Return value

- `AvailTags()` returns the number of tags in the read. It also fills the given string vector with a list of available tags that can be queried.

- `Tag_Type()`, `Tag_Subtype()` and `Tag_Size()` returns the type, subtype and tag length of the given `tag`. `Tag_Type_SAM()` returns the type as displayed in SAM format. See details for further information

- `p_tagVal()` returns the raw pointer to the beginning of the data contained within the given tag. That is, the pointer is 3 bytes downstream to the start of the tag label in all tags except tags of type ‘B’, where it is 8 bytes downstream of the tag label. See SAMv1.pdf for more details.

- `tagVal_{A/c/C/s/S/i/I/f}()` returns the 1-length value of the given tag type stored in the given `tag`.

- `tagVal_Z()` takes by reference a string `dest` in which to store the string contained in Z-type tags. It returns the length of the tag if success, or `-1` if fail.

- `tagval_B()` takes by reference a vector `dest` of the appropriate type, in which to store the vector of values associated with B-type tags. It returns the length of the tag if success, or `-1` if fails.

#### Details

Tags of type A/c/C/s/S/i/I/f are of length 1. Tags of type Z are of the length of its stored string (including the terminating ‘\0’ byte). Tags of type B are of the length of its vector.

Tag types designate the data type of its stored value. These are ‘A’ `char`, ‘c’ `int8_t`, ‘C’ `uint8_t`, ‘s’ `int16_t`, ‘S’ `uint16_t`, ‘i’ `int32_t`, ‘I’ `uint32_t`, and ‘f’ `float`.

Subtypes only apply to tags of type B. They can be of type c/C/s/S/i/I/f which refers to the data type of its stored value (see above).

SAM types are slightly different. Tags of BAM type c/C/s/S/i/I are displayed in SAM format as type ‘i’. Other types remain as they are.

Note that tags of type ‘H’ are not supported in ompBAM.

For more details, refer to SAMv1.pdf, section 4.2.4 for more information about how tags are stored in BAM format.

Also, refer to SAMtags.pdf for a list of the commonly-used BAM/SAM tags annotated with their SAM-types and the type of information they store.

#### Examples

    // The code below iterates through all the tags in the read and prints them in
    // SAM format. B-type tag data is not displayed for brevity of this example.

    pbam1_t read = inbam.supplyRead(i);

    std::vector<std::string> tags;
    read.AvailTags(tags);

    for(unsigned int j = 0; j < tags.size(); j++) {
      std::string Z_val;
      Rcpp::Rcout << tags.at(j) << ":" << read.Tag_Type_SAM(tags.at(j)) << ":";
      switch(read.Tag_Type(tags.at(j))) {
        case 'A':
          Rcpp::Rcout << read.tagVal_A(tags.at(j)) << '\t'; break;
        case 'c':
          Rcpp::Rcout << std::to_string(read.tagVal_c(tags.at(j))) << '\t'; break;
        case 'C':
          Rcpp::Rcout << std::to_string(read.tagVal_C(tags.at(j))) << '\t'; break;
        case 's':
          Rcpp::Rcout << std::to_string(read.tagVal_s(tags.at(j))) << '\t'; break;
        case 'S':
          Rcpp::Rcout << std::to_string(read.tagVal_S(tags.at(j))) << '\t'; break;
        case 'i':
          Rcpp::Rcout << std::to_string(read.tagVal_i(tags.at(j))) << '\t'; break;
        case 'I':
          Rcpp::Rcout << std::to_string(read.tagVal_I(tags.at(j))) << '\t'; break;
        case 'f':
          Rcpp::Rcout << std::to_string(read.tagVal_f(tags.at(j))) << '\t'; break;
        case 'Z':
          read.tagVal_Z(tags.at(j), Z_val);
          Rcpp::Rcout << Z_val << '\t'; break;
        case 'B':
          // write code to store B-type tag in vector of appropriate type
          Rcpp::Rcout << '\t'; break;
      }
    }
    Rcpp::Rcout << '\n';    // Line break

# (5) SessionInfo

    sessionInfo()
    #> R version 4.3.0 RC (2023-04-13 r84269)
    #> Platform: x86_64-pc-linux-gnu (64-bit)
    #> Running under: Ubuntu 22.04.2 LTS
    #>
    #> Matrix products: default
    #> BLAS:   /home/biocbuild/bbs-3.17-bioc/R/lib/libRblas.so
    #> LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0
    #>
    #> 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
    #>
    #> time zone: America/New_York
    #> tzcode source: system (glibc)
    #>
    #> attached base packages:
    #> [1] stats     graphics  grDevices utils     datasets  methods   base
    #>
    #> other attached packages:
    #> [1] MyPkg_0.0.0.9000 ompBAM_1.4.0
    #>
    #> loaded via a namespace (and not attached):
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    #>  [5] digest_0.6.31     magrittr_2.0.3    evaluate_0.20     pkgload_1.3.2
    #>  [9] fastmap_1.1.1     rprojroot_2.0.3   jsonlite_1.8.4    processx_3.8.1
    #> [13] sessioninfo_1.2.2 pkgbuild_1.4.0    brio_1.1.3        urlchecker_1.0.1
    #> [17] ps_1.7.5          promises_1.2.0.1  purrr_1.0.1       fansi_1.0.4
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    #> [33] memoise_2.0.1     httpuv_1.6.9      vctrs_0.6.2       R6_2.5.1
    #> [37] mime_0.12         lifecycle_1.0.3   zlibbioc_1.46.0   stringr_1.5.0
    #> [41] htmlwidgets_1.6.2 fs_1.6.2          usethis_2.1.6     miniUI_0.1.1.1
    #> [45] pkgconfig_2.0.3   desc_1.4.2        callr_3.7.3       pillar_1.9.0
    #> [49] bslib_0.4.2       later_1.3.0       profvis_0.3.7     glue_1.6.2
    #> [53] Rcpp_1.0.10       xfun_0.39         tibble_3.2.1      rstudioapi_0.14
    #> [57] knitr_1.42        xtable_1.8-4      htmltools_0.5.5   rmarkdown_2.21
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