# An introduction to Rbowtie

Michael Stadler, Dimos Gaidatzis and Anita Lerch

#### 26 April, 2022

#### Package

Rbowtie 1.36.0

# 1 Introduction

The Rbowtie package provides an R wrapper around the popular bowtie (Langmead et al. 2009) short read aligner and around SpliceMap (Au et al. 2010) a de novo splice junction discovery and alignment tool, which makes use of the bowtie software package.

The package is used by the QuasR (Gaidatzis et al. 2015) bioconductor package to _qu_antify and _a_nnotate _s_hort _r_eads. We recommend to use the QuasR package instead of using Rbowtie directly. The QuasR package provides a simpler interface than Rbowtie and covers the whole analysis workflow of typical ultra-high throughput sequencing experiments, starting from the raw sequence reads, over pre-processing and alignment, up to quantification.

# 2 Preliminaries

## 2.1 Citing Rbowtie

If you use Rbowtie (Hahne, Lerch, and Stadler 2012) in your work, you can cite it as follows:

    citation("Rbowtie")

    ##
    ## The Rbowtie package contains code from two separate software projects.
    ## If using bowtie only, it can be cited as Langmead et al. (2009). If
    ## using also SpliceMap, it can be cited in addition Au et al. (2010). The
    ## Rbowtie package can be cited using the third reference below:
    ##
    ##   Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and
    ##   memory-efficient alignment of short DNA sequences to the human
    ##   genome. Genome Biology 10(3):R25 (2009).
    ##
    ##   Au KF, Jiang H, Lin L, Xing Y, Wong WH. Detection of splice junctions
    ##   from paired-end RNA-seq data by SpliceMap. Nucleic Acids Research,
    ##   38(14):4570-8 (2010).
    ##
    ##   Hahne F, Lerch A, Stadler MB. Rbowtie: An R wrapper for bowtie and
    ##   SpliceMap short read aligners. (unpublished)
    ##
    ## This free open-source software implements academic research by the
    ## authors and co-workers. If you use it, please support the project by
    ## citing the appropriate journal articles.
    ## To see these entries in BibTeX format, use 'print(<citation>,
    ## bibtex=TRUE)', 'toBibtex(.)', or set
    ## 'options(citation.bibtex.max=999)'.

## 2.2 Installation

Rbowtie is a package for the R computing environment and it is assumed that you have already installed R. See the R project at (http://www.r-project.org). To install the latest version of Rbowtie, you will need to be using the latest version of R. Rbowtie is part of the Bioconductor project at (http://www.bioconductor.org). To get Rbowtie together with its dependencies you can use

    if (!require("BiocManager"))
        install.packages("BiocManager")
    BiocManager::install("Rbowtie")

## 2.3 Loading of Rbowtie

In order to run the code examples in this vignette, the Rbowtie library need to be loaded.

    library(Rbowtie)

## 2.4 How to get help

Most questions about Rbowtie will hopefully be answered by the documentation or references. If you’ve run into a question which isn’t addressed by the documentation, or you’ve found a conflict between the documentation and software itself, then there is an active support community which can offer help.

The authors of the package (maintainer: Michael Stadler michael.stadler@fmi.ch) always appreciate receiving reports of bugs in the package functions or in the documentation. The same goes for well-considered suggestions for improvements.

Any other questions or problems concerning Rbowtie should be posted to the Bioconductor support site (https://support.bioconductor.org). Users posting to the support site for the first time should read the helpful posting guide at (https://support.bioconductor.org/info/faq/). Note that each function in Rbowtie has it’s own help page, e.g. `help("bowtie")`. Posting etiquette requires that you read the relevant help page carefully before posting a problem to the site.

# 3 Example usage for individual Rbowtie functions

Please refer to the Rbowtie reference manual or the function documentation (e.g. using `?bowtie`) for a complete description of Rbowtie functions. The descriptions provided below are meant to give and overview over all functions and summarize the purpose of each one.

## 3.1 Build the reference index with `bowtie_build`

To be able to align short reads to a genome, an index has to be build first using the function `bowtie_build`. Information about arguments can be found with the help of the `bowtie_build_usage` function or in the manual page `?bowtie_build`.

    bowtie_build_usage()

    ##  [1] "Usage: bowtie2-build-s [options]* <reference_in> <ebwt_outfile_base>"
    ##  [2] "    reference_in            comma-separated list of files with ref sequences"
    ##  [3] "    ebwt_outfile_base       write Ebwt data to files with this dir/basename"
    ##  [4] "Options:"
    ##  [5] "    -f                      reference files are Fasta (default)"
    ##  [6] "    -c                      reference sequences given on cmd line (as <seq_in>)"
    ##  [7] "    -a/--noauto             disable automatic -p/--bmax/--dcv memory-fitting"
    ##  [8] "    -p/--packed             use packed strings internally; slower, uses less mem"
    ##  [9] "    --bmax <int>            max bucket sz for blockwise suffix-array builder"
    ## [10] "    --bmaxdivn <int>        max bucket sz as divisor of ref len (default: 4)"
    ## [11] "    --dcv <int>             diff-cover period for blockwise (default: 1024)"
    ## [12] "    --nodc                  disable diff-cover (algorithm becomes quadratic)"
    ## [13] "    -r/--noref              don't build .3/.4.ebwt (packed reference) portion"
    ## [14] "    -3/--justref            just build .3/.4.ebwt (packed reference) portion"
    ## [15] "    -o/--offrate <int>      SA is sampled every 2^offRate BWT chars (default: 5)"
    ## [16] "    -t/--ftabchars <int>    # of chars consumed in initial lookup (default: 10)"
    ## [17] "    --threads <int>         # of threads"
    ## [18] "    --ntoa                  convert Ns in reference to As"
    ## [19] "    --seed <int>            seed for random number generator"
    ## [20] "    -q/--quiet              verbose output (for debugging)"
    ## [21] "    -h/--help               print detailed description of tool and its options"
    ## [22] "    --usage                 print this usage message"
    ## [23] "    --version               print version information and quit"

`refFiles` below is a vector with filenames of the reference sequence in `FASTA` format, and `indexDir` specifies an output directory for the index files that will be generated when calling `bowtie_build`:

    refFiles <- dir(system.file(package="Rbowtie", "samples", "refs"), full=TRUE)
    indexDir <- file.path(tempdir(), "refsIndex")

    tmp <- bowtie_build(references=refFiles, outdir=indexDir, prefix="index", force=TRUE)
    head(tmp)

    ## [1] "Settings:"
    ## [2] "  Output files: \"F:\\biocbuild\\bbs-3.15-bioc\\tmpdir\\RtmpCOi1Kp/refsIndex/index.*.ebwt\""
    ## [3] "  Line rate: 6 (line is 64 bytes)"
    ## [4] "  Lines per side: 1 (side is 64 bytes)"
    ## [5] "  Offset rate: 5 (one in 32)"
    ## [6] "  FTable chars: 10"

## 3.2 Create alignment with `bowtie`

Information about the arguments supported by the `bowtie` function can be obtained with the help of the `bowtie_usage` function or in the manual page `?bowtie`.

    bowtie_usage()

    ##  [1] "Usage: "
    ##  [2] "bowtie-align-s [options]* -x <ebwt> {-1 <m1> -2 <m2> | --12 <r> | --interleaved <i> | <s>} [<hit>]"
    ##  [3] ""
    ##  [4] "  <ebwt>  Index filename prefix (minus trailing .X.ebwt)."
    ##  [5] "  <m1>    Comma-separated list of files containing upstream mates (or the"
    ##  [6] "          sequences themselves, if -c is set) paired with mates in <m2>"
    ##  [7] "  <m2>    Comma-separated list of files containing downstream mates (or the"
    ##  [8] "          sequences themselves if -c is set) paired with mates in <m1>"
    ##  [9] "  <r>     Comma-separated list of files containing Crossbow-style reads.  Can be"
    ## [10] "          a mixture of paired and unpaired.  Specify \"-\" for stdin."
    ## [11] "  <i>     Files with interleaved paired-end FASTQ reads."
    ## [12] "  <s>     Comma-separated list of files containing unpaired reads, or the"
    ## [13] "          sequences themselves, if -c is set.  Specify \"-\" for stdin."
    ## [14] "  <hit>   File to write hits to (default: stdout)"
    ## [15] "Input:"
    ## [16] "  -q                 query input files are FASTQ .fq/.fastq (default)"
    ## [17] "  -f                 query input files are (multi-)FASTA .fa/.mfa"
    ## [18] "  -F k:<int>,i:<int> query input files are continuous FASTA where reads"
    ## [19] "                     are substrings (k-mers) extracted from a FASTA file <s>"
    ## [20] "                     and aligned at offsets 1, 1+i, 1+2i ... end of reference"
    ## [21] "  -r                 query input files are raw one-sequence-per-line"
    ## [22] "  -c                 query sequences given on cmd line (as <mates>, <singles>)"
    ## [23] "  -Q/--quals <file>  QV file(s) corresponding to CSFASTA inputs; use with -f -C"
    ## [24] "  --Q1/--Q2 <file>   same as -Q, but for mate files 1 and 2 respectively"
    ## [25] "  -s/--skip <int>    skip the first <int> reads/pairs in the input"
    ## [26] "  -u/--qupto <int>   stop after first <int> reads/pairs (excl. skipped reads)"
    ## [27] "  -5/--trim5 <int>   trim <int> bases from 5' (left) end of reads"
    ## [28] "  -3/--trim3 <int>   trim <int> bases from 3' (right) end of reads"
    ## [29] "  --phred33-quals    input quals are Phred+33 (default)"
    ## [30] "  --phred64-quals    input quals are Phred+64 (same as --solexa1.3-quals)"
    ## [31] "  --solexa-quals     input quals are from GA Pipeline ver. < 1.3"
    ## [32] "  --solexa1.3-quals  input quals are from GA Pipeline ver. >= 1.3"
    ## [33] "  --integer-quals    qualities are given as space-separated integers (not ASCII)"
    ## [34] "Alignment:"
    ## [35] "  -v <int>           report end-to-end hits w/ <=v mismatches; ignore qualities"
    ## [36] "    or"
    ## [37] "  -n/--seedmms <int> max mismatches in seed (can be 0-3, default: -n 2)"
    ## [38] "  -e/--maqerr <int>  max sum of mismatch quals across alignment for -n (def: 70)"
    ## [39] "  -l/--seedlen <int> seed length for -n (default: 28)"
    ## [40] "  --nomaqround       disable Maq-like quality rounding for -n (nearest 10 <= 30)"
    ## [41] "  -I/--minins <int>  minimum insert size for paired-end alignment (default: 0)"
    ## [42] "  -X/--maxins <int>  maximum insert size for paired-end alignment (default: 250)"
    ## [43] "  --fr/--rf/--ff     -1, -2 mates align fw/rev, rev/fw, fw/fw (default: --fr)"
    ## [44] "  --nofw/--norc      do not align to forward/reverse-complement reference strand"
    ## [45] "  --maxbts <int>     max # backtracks for -n 2/3 (default: 125, 800 for --best)"
    ## [46] "  --pairtries <int>  max # attempts to find mate for anchor hit (default: 100)"
    ## [47] "  -y/--tryhard       try hard to find valid alignments, at the expense of speed"
    ## [48] "  --chunkmbs <int>   max megabytes of RAM for best-first search frames (def: 64)"
    ## [49] " --reads-per-batch   # of reads to read from input file at once (default: 16)"
    ## [50] "Reporting:"
    ## [51] "  -k <int>           report up to <int> good alignments per read (default: 1)"
    ## [52] "  -a/--all           report all alignments per read (much slower than low -k)"
    ## [53] "  -m <int>           suppress all alignments if > <int> exist (def: no limit)"
    ## [54] "  -M <int>           like -m, but reports 1 random hit (MAPQ=0); requires --best"
    ## [55] "  --best             hits guaranteed best stratum; ties broken by quality"
    ## [56] "  --strata           hits in sub-optimal strata aren't reported (requires --best)"
    ## [57] "Output:"
    ## [58] "  -t/--time          print wall-clock time taken by search phases"
    ## [59] "  -B/--offbase <int> leftmost ref offset = <int> in bowtie output (default: 0)"
    ## [60] "  --quiet            print nothing but the alignments"
    ## [61] "  --refidx           refer to ref. seqs by 0-based index rather than name"
    ## [62] "  --al <fname>       write aligned reads/pairs to file(s) <fname>"
    ## [63] "  --un <fname>       write unaligned reads/pairs to file(s) <fname>"
    ## [64] "  --no-unal          suppress SAM records for unaligned reads"
    ## [65] "  --max <fname>      write reads/pairs over -m limit to file(s) <fname>"
    ## [66] "  --suppress <cols>  suppresses given columns (comma-delim'ed) in default output"
    ## [67] "  --fullref          write entire ref name (default: only up to 1st space)"
    ## [68] "SAM:"
    ## [69] "  -S/--sam           write hits in SAM format"
    ## [70] "  --mapq <int>       default mapping quality (MAPQ) to print for SAM alignments"
    ## [71] "  --sam-nohead       supppress header lines (starting with @) for SAM output"
    ## [72] "  --sam-nosq         supppress @SQ header lines for SAM output"
    ## [73] "  --sam-RG <text>    add <text> (usually \"lab=value\") to @RG line of SAM header"
    ## [74] "Performance:"
    ## [75] "  -o/--offrate <int> override offrate of index; must be >= index's offrate"
    ## [76] "  -p/--threads <int> number of alignment threads to launch (default: 1)"
    ## [77] "Other:"
    ## [78] "  --seed <int>       seed for random number generator"
    ## [79] "  --verbose          verbose output (for debugging)"
    ## [80] "  --version          print version information and quit"
    ## [81] "  -h/--help          print this usage message"

In the example below, `readsFiles` is the name of a file containing short reads to be aligned with `bowtie`, and `samFiles` specifies the name of the output file with the generated alignments.

    readsFiles <- system.file(package="Rbowtie", "samples", "reads", "reads.fastq")
    samFiles <- file.path(tempdir(), "alignments.sam")

    bowtie(sequences=readsFiles,
           index=file.path(indexDir, "index"),
           outfile=samFiles, sam=TRUE,
           best=TRUE, force=TRUE)
    strtrim(readLines(samFiles), 65)

    ## [1] "@HD\tVN:1.0\tSO:unsorted"
    ## [2] "@SQ\tSN:chr1\tLN:100000"
    ## [3] "@SQ\tSN:chr2\tLN:100000"
    ## [4] "@SQ\tSN:chr3\tLN:100000"
    ## [5] "@PG\tID:Bowtie\tVN:1.3.0\tCL:\"F:\\biocbuild\\bbs-3.15-bioc\\tmpdir\\RtmpMt3"
    ## [6] "HWUSI-EAS1513_0012:6:48:5769:946#0/1\t0\tchr1\t819\t255\t101M\t*\t0\t0\tTGGAGTTCATG"
    ## [7] "HWUSI-EAS1513_0012:6:48:6908:952#0/1\t0\tchr2\t1133\t255\t101M\t*\t0\t0\tAACATAGTGA"
    ## [8] "HWUSI-EAS1513_0012:6:48:8070:953#0/1\t0\tchr1\t7543\t255\t101M\t*\t0\t0\tGTCTGTCTAG"

## 3.3 Create spliced alignment with `SpliceMap`

While `bowtie` only generates ungapped alignments, the `SpliceMap` function can be used to generate spliced alignments. `SpliceMap` is itself using `bowtie`. To use it, it is necessary to create an index of the reference sequence as described in ??. `SpliceMap` parameters are specified in the form of a named list, which follows closely the configure file format of the original `SpliceMap` program(Au et al. 2010). Be aware that `SpliceMap` can only be used for reads that are at least 50bp long.

    readsFiles <- system.file(package="Rbowtie", "samples", "reads", "reads.fastq")
    refDir <- system.file(package="Rbowtie", "samples", "refs", "chr1.fa")
    indexDir <- file.path(tempdir(), "refsIndex")
    samFiles <- file.path(tempdir(), "splicedAlignments.sam")

    cfg <- list(genome_dir=refDir,
                reads_list1=readsFiles,
                read_format="FASTQ",
                quality_format="phred-33",
                outfile=samFiles,
                temp_path=tempdir(),
                max_intron=400000,
                min_intron=20000,
                max_multi_hit=10,
                seed_mismatch=1,
                read_mismatch=2,
                num_chromosome_together=2,
                bowtie_base_dir=file.path(indexDir, "index"),
                num_threads=4,
                try_hard="yes",
                selectSingleHit=TRUE)
    res <- SpliceMap(cfg)

    ## [SpliceMap] splitting reads into 25mers...done
    ## [SpliceMap] aligning 25mers...done
    ## [SpliceMap] sorting 25mer-alignments...done
    ## [SpliceMap] indexing 25mer-alignments...done
    ## [SpliceMap] finding spliced alignments for 1 chromosomes using 2 parallel processes...done
    ## [SpliceMap] extracting unmapped reads (single read mode)...done
    ## [SpliceMap] combining spliced alignments...done
    ## [SpliceMap] reordering final output...done

    res

    ## [1] "F:\\biocbuild\\bbs-3.15-bioc\\tmpdir\\RtmpCOi1Kp/splicedAlignments.sam"

    strtrim(readLines(samFiles), 65)

    ## [1] "@HD\tVN:1.0\tSO:coordinate"
    ## [2] "@SQ\tSN:chr1\tLN:100000"
    ## [3] "@PG\tID:SpliceMap\tVN:3.3.5.2 (55)"
    ## [4] "HWUSI-EAS1513_0012:6:48:5769:946#0\t0\tchr1\t819\t255\t101M\t*\t0\t0\tTGGAGTTCATGTG"
    ## [5] "HWUSI-EAS1513_0012:6:48:6908:952#0\t4\t*\t0\t0\t*\t*\t0\t0\tAACATAGTGAAGAAACCTCATAG"
    ## [6] "HWUSI-EAS1513_0012:6:48:8070:953#0\t0\tchr1\t7543\t255\t101M\t*\t0\t0\tGTCTGTCTAGTG"
    ## [7] "HWUSI-EAS1513_0012:6:48:9942:949#0\t4\t*\t0\t0\t*\t*\t0\t0\tCGGTTCCTGTATCCTTAATAAGT"

# 4 Session information

The output in this vignette was produced under:

    sessionInfo()

    ## R version 4.2.0 RC (2022-04-19 r82224 ucrt)
    ## Platform: x86_64-w64-mingw32/x64 (64-bit)
    ## Running under: Windows Server x64 (build 20348)
    ##
    ## Matrix products: default
    ##
    ## locale:
    ## [1] LC_COLLATE=C
    ## [2] LC_CTYPE=English_United States.utf8
    ## [3] LC_MONETARY=English_United States.utf8
    ## [4] LC_NUMERIC=C
    ## [5] LC_TIME=English_United States.utf8
    ##
    ## attached base packages:
    ## [1] stats     graphics  grDevices utils     datasets  methods   base
    ##
    ## other attached packages:
    ## [1] Rbowtie_1.36.0   BiocStyle_2.24.0
    ##
    ## loaded via a namespace (and not attached):
    ##  [1] bookdown_0.26       digest_0.6.29       R6_2.5.1
    ##  [4] jsonlite_1.8.0      magrittr_2.0.3      evaluate_0.15
    ##  [7] stringi_1.7.6       rlang_1.0.2         cli_3.3.0
    ## [10] jquerylib_0.1.4     bslib_0.3.1         rmarkdown_2.14
    ## [13] tools_4.2.0         stringr_1.4.0       parallel_4.2.0
    ## [16] xfun_0.30           yaml_2.3.5          fastmap_1.1.0
    ## [19] compiler_4.2.0      BiocManager_1.30.17 htmltools_0.5.2
    ## [22] knitr_1.38          sass_0.4.1

# References

Au, K. F., H. Jiang, L. Lin, Y. Xing, and W. H. Wong. 2010. “Detection of Splice Junctions from Paired-End Rna-Seq Data by Splicemap.” Nucleic Acids Research 38 (14): 4570–8.

Gaidatzis, D., A. Lerch, F. Hahne, and M. B. Stadler. 2015. “QuasR: Quantify and Annotate Short Reads in R.” Bioinformatics 31 (7): 1130–2. https://doi.org/10.1093/bioinformatics/btu781.

Hahne, F., A. Lerch, and M. B. Stadler. 2012. “Rbowtie: A R Wrapper for Bowtie and SpliceMap Short Read Aligners.”

Langmead, B., C. Trapnell, M. Pop, and S. L. Salzberg. 2009. “Ultrafast and Memory-Efficient Alignment of Short Dna Sequences to the Human Genome.” Genome Biology 10 (3): R25.