Dear All,
I would like to have the bigwig or BAM output files after aligning my Chip-Seq data with the reference genome and then use these files for peak calling. Could you please advice me about the tools and links to accomplish the above tasks. I tried on Galaxy where it gives only BAM output but the visualization is better with Wig files in UCSC browser.
Also, I couldn't find a way how to run ELAND alignment algorithm on my data but succeeded in running Bowtie which gives SAM output.
The organism is Mouse.
Many Thanks for your help.
cheers,
Aravind
Get a WIG file directly from a sorted BAM file using SAMtools (from http://www.ecseq.com/NGSsnippets.html)
samtools mpileup -BQ0 run.sorted.bam | perl -pe '($c, $start, undef, $depth) = split;if ($c ne $lastC || $start != $lastStart+1) {print "fixedStep chrom=$c start=$start step=1 span=1\n";}$_ = $depth."\n";($lastC, $lastStart) = ($c, $start);' | gzip -c > run.wig.gz
Do you have a Wig file? If you use GALAXY, you can use MACS peak calling to generate a Wig file that can then be converted to BigWig for display on UCSC.
Hi Lawrence....i don't have a Wig file....i'm looking for a tool or software which can generate Wig file out of alignment with reference genome.
As I said, if you use GALAXY, one of the output options for MACS is a Wig file. Use that program to perform peak detection.
Get a WIG file directly from a sorted BAM file using SAMtools (from http://www.ecseq.com/NGSsnippets.html)
samtools mpileup -BQ0 run.sorted.bam | perl -pe '($c, $start, undef, $depth) = split;if ($c ne $lastC || $start != $lastStart+1) {print "fixedStep chrom=$c start=$start step=1 span=1\n";}$_ = $depth."\n";($lastC, $lastStart) = ($c, $start);' | gzip -c > run.wig.gz
And to create a bigWig file out of your wig file, you can use wigToBigWig (from http://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/)
MACS (http://liulab.dfci.harvard.edu/MACS/) allows you both, get a wig file and detect enriched regions.It can also be used together with peak splitter (http://www.ebi.ac.uk/research/bertone/software) to isolate discrete peaks in enriched regions.
Dear Aravind
If you use Bowtie you can convert the SAM output format by using samtools.
In a terminal :
samtools view -Shb input.sam > output.bam
Once you have a bam format you can convert it in bigwig or bedgraph format. you will find the informations in the links below.
https://www.biostars.org/p/64495/
https://www.biostars.org/p/2699/
cheers,
F
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