Genome Spot

Bismark is currently the de facto standard for primary analysis of high throughput bisulfite sequencing data. Bismark can align the reads to the genome and perform methylation calling. In this post, I'll go through Illumina whole genome bisulfite sequence (WGBS) alignment and methylation calling using Bismark. First I want to mention that this post is just a summary, not meant to be a user manual or thorough troubleshooting guide. Fortunately, Bismark has some of the best documentation for any bioinformatics suite and is mandatory reading. The Bismark crew are very proactive with responding to user queries on various forums as well.

First step in getting Bismark to work is to index the genome, in this case with Bowtie2:

bismark_genome_preparation --bowtie2 /pathto/refgenome/

Conventionally, multiplexed libraries will be sequenced over a number of lanes. Resist concatenating or merging the smaller fastq files for each patient/sample until after the alignment, as the concatenated fil…

Gene regulation is a really complicated thing. We have covalent marks to DNA, histones and transcription factors. Chromatin remodeling and long range enhancer interactions. Enhancer elements located in introns of genes hundreds of kilobases away from the gene they're controlling. Transcriptional control from microRNA networks and now there is an emerging model for the function of some of the thousands of long non-coding RNAs which are just now being uncovered with high resolution (directional) transcriptome analysis.

Many of you which studied molecular biology at Uni would (should) remember the model for how X chromosome inactivation is achieved. The mechanism centers around XIST, one of the first non-coding RNA genes identified. Expression of XIST from the inactive X chromosome essentially wraps it up at the same time that repressive epigenetic marks are established through its interaction with the Polycomb Repressive Complex 2 (PRC2). Sounds simple enough, but the model also inv…

There have been a number of high profile profile articles in recent times discussing the function of TET proteins, mostly in the conversion of methylated cytosine (5mC) into hydroxymethylated cytosine (5hmC), the 5th base. Hydroxymethylcytosine is much rarer than methylcytosine and is thought to be an intermediate towards demethylation of cytosine, a mechanism which remains incompletely resolved. A paper last year showed that TET proteins also convert 5hmc to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), termed the 6th and 7th bases.


OGT on the other hand is a fairly unique protein because it is the only known known O-GlcNAc transferase in mammals. What is GlcNAc you say? It stands for N-acetylglucosamine, a hexosamine. There has been a series of papers (here, here, here) discussing OGT as a nutrient sensor, transferring GlcNAc during period of surplus nutrient supply. GlcNAc can be transferred to the same amino acids as phosphorylation, so there is a suggested crosstalk betwee…

Nearly every baby born in Australia since 1970 has had a few drops of blood taken and stored on a so-called Guthrie card, and this practise is widely adopted in the developed world. As DNA analysis technologies become ever more sensitive and economical, these cards will become ever more important in diagnosis of genetic disease and also in identifying genetic and epigenetic variations which contribute to complex disease. The paper I showcase today from Beyan et al, describes the development of genome-wide assays for DNA methylation using methylation microarrays and methylcytosine immunoprecipitation followed by Illumina sequencing (MeDIP-Seq). Authors find differential methylation regions which are stable from birth to 3 years of age.

The methodology is fairly novel, but the conclusions are a bit vague and it would have been best to apply Guthrie card analysis for a specific disease. It would be really neat if they analysed material from discordant twins for a complex disease i.e; juv…