Converting BAM files to BigWig format

One of the reasons to convert bam to BigWig is to get a normalized coverage in viewers such as IGB.

Tools needed

samtools (if your bam file is not sorted by position), bedtools and ucsc's bedGraphToBigWig in the ucsc command line utilities

Procedure

samtools sort input.bam input.sorted

mysql --user=genome --host=genome-mysql.cse.ucsc.edu -A -e "select chrom, size from hg19.chromInfo" > hg19.genome

bedtools genomecov -ibam input.sorted.bam -bg hg19.genome > input.sorted.bedGraph

bedGraphToBigWig input.sorted.bedGraph hg19.genome input.sorted.bigwig

Unfortunately we can't pipe genomecoverage directly into bedGraphToBigWig because bedGraphToBigWig does a few passes over the file (for improved speed).

Other things to consider:
1) Scaling : setting a scale factor (within bedtools) would normalize your bedgraph/bigwig files (the default=1, no scaling). I generally scale all files to say, 10 million reads (get total reads from eg. samtools flagstat output) so that they can be consistently used throughout different comparisons.
2) -split option should be used for RNA-seq to properly represent spliced reads.
3) -strand option in theory would give you strand-specific files (meaning you'll have a +ve and -ve set) but for my paired-end directional reads, it seems to only work for the first in read pair. I've left out this option until I figure out a solution.