BAM to BigWig without intermediary BedGraph

Question

I have a pipeline for generating a BigWig file from a BAM file:

BAM -> BedGraph -> BigWig

Which uses bedtools genomecov for the BAM -> BedGraph part and bedGraphToBigWig for the BedGraph -> BigWig part.

The use of bedGraphToBigWig to create the BigWig file requires a BedGraph file to reside on disk in uncompressed form as it performs seeks. This is problematic for large genomes and variable coverage BAM files when there are more step changes/lines in the BedGraph file. My BedGraph files are in the order of 50 Gbytes in size and all that IO for 10-20 BAM files seems unnecessary.

Are there any tools capable generate BigWig without having to use an uncompressed BedGraph file on disk? I'd like this conversion to hapen as quickly as possible.

I have tried the following tools, but they still create/use a BedGraph intermediary file:

• deepTools

Some Benchmarks, Ignoring IO

Here are some timings I get for creating a BigWig file from a BAM file using 3 different pipelines. All files reside on a tmpfs i.e. in memory.

BEDTools and Kent Utils

This is the approach taken by most.

time $(bedtools genomecov -bg -ibam test.bam -split -scale 1.0 > test.bedgraph \
  && bedGraphToBigWig test.bedgraph test.fasta.chrom.sizes kent.bw \
  && rm test.bedgraph)

real    1m20.015s
user    0m56.608s
sys     0m27.271s

SAMtools and Kent Utils

Replacing bedtools genomecov with samtools depth and a custom awk script (depth2bedgraph.awk) to output bedgraph format has a significant performance improvement:

time $(samtools depth -Q 1 --reference test.fasta test.bam \
  | mawk -f depth2bedgraph.awk \
  > test.bedgraph \
  && bedGraphToBigWig test.bedgraph test.fasta.chrom.sizes kent.bw \
  && rm test.bedgraph)

real    0m28.765s
user    0m44.999s
sys     0m1.166s

Although it is has less features, we used mawk here as it's faster than gawk (we don't need those extra features here).

Parallelising with xargs

If you want to have a BigWig file per chromosome, you can easily parallelise this across chromosome/reference sequences. We can use xargs to run 5 parallel BAM->BedGraph->BigWig pipelines, each using the tmpfs mounted /dev/shm for the intermediary BedGraph files.

cut -f1 test.fasta.chrom.sizes \
  | xargs -I{} -P 5  bash -c 'mkdir /dev/shm/${1} \
  && samtools depth -Q 1 --reference test.fasta -r "${1}" test.bam \
  | mawk -f scripts/depth2bedgraph.awk \
  > "/dev/shm/${1}/test.bam.bedgraph" \
  && mkdir "./${1}" \
  && bedGraphToBigWig \
    "/dev/shm/${1}/test.bam.bedgraph" \
    test.fasta.chrom.sizes \
    "./${1}/test.bam.bw" \
  && rm "/dev/shm/${1}/test.bam.bedgraph"' -- {}

deepTools

Let's see how deepTools performs.

time bamCoverage --numberOfProcessors max \
  --minMappingQuality 1 \
  --bam test.bam --binSize 1 --skipNonCoveredRegions \
  --outFileName deeptools.bw

real    0m40.077s
user    3m56.032s
sys     0m9.276s

Answer 1

This can be done in R very easily from an indexed .bam file.

Given single-end file for sample1.

library(GenomicAlignments)
library(rtracklayer)

## read in BAM file (use readGAlignmentPairs for paired-end files)
gr <- readGAlignments('sample1.bam')

## convert to coverages
gr.cov <- coverage(gr)

## export as bigWig
export.bw(gr.cov,'sample1.bigwig')

Be aware that this method doesn't include normalization steps (such as normalizing to total coverage). Most of these additional steps can be added if necessary.

Answer 2

Just for reference, there is a not very in depth, but first-hand reasoning (Jim Kent) given here about why bedGraphToBigWig does not support streaming http://genome.soe.ucsc.narkive.com/2S1Z3VpG/bedgraphtobigwig-reading-in-from-stdin

Edit: as noted wigToBigWig allows bedgraph streaming input via stdin but takes (possibly much) more memory than bedGraphToBigWig itself does

cat file.bedgraph | wigToBigWig stdin chrom.sizes output.bigwig

Note: You can also use wiggletools bin 10 for example to bin rows and downsample

Answer 3

The QuasR package in R has a useful function called qExportWig.

To use it you first need to create a sample sheet and a qProject object - this can be done starting with bam files or fastq files (in which case it also performs alignments). You can then create wig or bigWig files without writing a bedGraph file to disk.

An example would look something like this:

library(QuasR)

# Create qProject object using qAlign
# Sample file should contain tab-separated file names (BAM or fastq) and sample names
myproj <- qAlign("samples.txt", genome=mygenome, paired="no")

# Export all samples in project as bigWig, scaling to mean number of alignments by default
qExportWig(myproj, binsize=50, createBigWig=T)

It has a lot of flexible options, including scaling or log-transforming counts, shifting reads and changing the bin size.