Genome Spot

Library preparation for next generation sequencing is becoming easier with the quality of kits and protocols improving substantially in the past few years. With the price of NGS decreasing, we are finding that our throughput is increasing, both in terms of the number of experiments as well as the average size of these experiments. With this in mind, the ability to accurately pool barcoded libraries in equimolar ratios (also called "balancing") is even more critical. Accurate quantification is thus vital. There are several ways to quantify library DNA: Qubit fluorometer. Gives very accurate concentrations in nanogram per microlitre, but agnostic of fragment size distribution.Bioanalyzer. Gives accurate readings of fragment size, but is only fairly accurate in terms of concentrations of each fragmentQuantitative PCR. Most accurate, but expensive and most time consuming.NanoDrop UV-Spec. Easiest method but least accurate. Not recommended. So I'll lead you through my favouri…

It is often said that RNA-seq is overtaking microarray as the gold-standard for transcriptome wide gene expression analysis, but what most people don't understand is, that this "gold standard" is far from standard. There are an ever increasing number of methods available to apply high throughput sequencing to gene expression analysis. Thus "RNA-Seq" is actually very generic term describing a range of techniques which aim to use sequencing to profile transcripts. Let's go through some of the more common applications  and work our way to the more niche applications.

mRNA-Seq This method aims to profile the pool of transcripts which encode for proteins. The idea is to enrich the RNA mixture for mRNAs by depleting the concentration of rRNAs and other abundant ncRNAs. Usually, this is achieved by polyA enrichment using hybridization to magnetic beads decorated with oligo dT strands. After the enrichment the resulting RNA is fragmented by heat exposure in the pre…

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…

Library preparation is a process in which we modify DNA into a form that it is compatible for high throughput sequencing, and is becoming a key molecular biology technique. While there are an amazing variety of different library preparation methods available, I thought I'd start the the blog with a description of the classic method:
- Shearing/fragmentation
- End Repair
- DNA clean-up
- A tailing
- Adaptor ligation
- Size selection
- Amplification
- Quality control

Shearing/Fragmentation
The DNA needs to be in a size range that is compatible with the sequencing platform. The most commonly used sequencing platforms require DNA construct in the range of 300-500 bp, although this depends on the specific platform and the application. Fragmentation can be done by mechanical disruption through sonication like we do in our lab, but can also be done with a nebuliser or with enzymatic fragmentation. Our thoughts are that sonication/nebuliser has a lesser degree of sequence specificity bias …