How mappable is a specific repeat?

If you've ever wondered how mappable a specific repeat is, here's a quick post on creating a plot showing the mappability of a repetitive element along its consensus sequence. Specifically, the consensus sequence of a repeat was taken and sub-sequences were created by a sliding window approach (i.e. moving along the sequence) at 1 bp intervals and these sub-sequences were mapped to hg19.

I will use bwa for the mapping, so firstly download and compile bwa:

wget http://sourceforge.net/projects/bio-bwa/files/latest/bwa-0.7.7.tar.bz2
tar -xjf bwa-0.7.7.tar.bz2
cd bwa-0.7.7
make

#index hg19
bwa index hg19.fa

Continue reading

ENCODE RNA polymerase II ChIP-seq

Chromatin immunoprecipitation sequencing (ChIP-seq) is a high throughput method for investigating protein-DNA interactions and aims to determine whether specific proteins are interacting with specific genomic loci. The workflow consists of crosslinking DNA and protein together, usually via the use of formaldehyde, which induces protein-DNA and protein-protein crosslinks. Importantly, these crosslinks are reversible by incubation at 70°C. Next the crosslinked DNA-protein complexes are sheared into roughly 500 bp fragments, usually by sonication. At this point we have "sheared DNA" and "sheared DNA crosslinked with proteins". Now comes the immunoprecipitation step, which is a technique that precipitates a protein antigen out of solution using an antibody that recognises that particular antigen. The crosslinking would result in many DNA-protein interactions and we use immunoprecipitation to pull down the protein of interest with the DNA region it was interacting with. After immunoprecipitation, the formaldehyde crosslinks are reversed by heating and the DNA strands are purified and sequenced. There's a nice graphic depicting this workflow at the Wikipedia article for ChIP-seq.

Continue reading