Read mapping with Bowtie2/Tophat2

The NGS reads of this project will be aligned against the reference genome sequence using Bowtie2/TopHat2 (Kim et al., 2013; Langmead et al., 2012). The parameter settings of the aligner are defined in the tophat.param file.

args <- systemArgs(sysma="param/tophat.param", mytargets="targets_trim.txt")
sysargs(args)[1] # Command-line parameters for first FASTQ file

Submission of alignment jobs to compute cluster, here using 72 CPU cores (18 qsub processes each with 4 CPU cores).

system("bowtie2-build ./data/tair10.fasta ./data/tair10.fasta")
resources <- list(walltime="20:00:00", nodes=paste0("1:ppn=", cores(args)), memory="10gb")
reg <- clusterRun(args, conffile=".BatchJobs.R", template="torque.tmpl", Njobs=18, runid="01", 

Alignment with Rsubread

The following example shows how one can use within the systemPipeR environment the R-based aligner Rsubread or other R-based functions that read from input files and write to output files.

args <- systemArgs(sysma="param/rsubread.param", mytargets="targets.txt")
buildindex(basename=reference(args), reference=reference(args)) # Build indexed reference genome
align(index=reference(args), readfile1=infile1(args), input_format="FASTQ",
      output_file=outfile1(args), output_format="SAM", nthreads=2)
for(i in seq(along=outfile1(args))) asBam(file=outfile1(args)[i], destination=gsub(".sam", "", outfile1(args)[i]), overwrite=TRUE, indexDestination=TRUE)
unlink(outfile1(args)); unlink(paste0(outfile1(args),".indel"))

Read mapping with HISAT2

args <- systemArgs(sysma="param/hisat2.param", mytargets="targets.txt")
# args <- systemArgs(sysma="param/hisat2.param", mytargets="targets_trim.txt")
sysargs(args)[1] # Command-line parameters for first FASTQ file
system("hisat2-build ./data/tair10.fasta ./data/tair10.fasta")

Check whether all BAM files have been created


Read and alignment stats

The following provides an overview of the number of reads in each sample and how many of them aligned to the reference.

args <- systemArgs(sysma="param/hisat2.param", mytargets="targets.txt")
read_statsDF <- alignStats(args=args) 
write.table(read_statsDF, "results/alignStats.xls", row.names=FALSE, quote=FALSE, sep="\t")
read.table(system.file("extdata", "alignStats.xls", package="systemPipeR"), header=TRUE)[1:4,]
##   FileName Nreads2x Nalign Perc_Aligned Nalign_Primary Perc_Aligned_Primary
## 1      M1A   192918 177961     92.24697         177961             92.24697
## 2      M1B   197484 159378     80.70426         159378             80.70426
## 3      A1A   189870 176055     92.72397         176055             92.72397
## 4      A1B   188854 147768     78.24457         147768             78.24457

The symLink2bam function creates symbolic links to view the BAM alignment files in a genome browser such as IGV. The corresponding URLs are written to a file with a path specified under urlfile in the results directory.

symLink2bam(sysargs=args, htmldir=c("~/.html/", "projects/tests/"),