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…

This paper was featured in the 12 October issue of Science, so really isn't from this week, nevertheless I thought it would be relevant given the previous post on library preparation.

Sequencing ancient DNA is a hugely challenging task. Not only is it very difficult to get any sort of yield of DNA from bones tens of thousands of years old, but the DNA itself is normally degraded to such an extent that conventional library preparation is highly inefficient. On top of this, there is the challenge to eliminate environmental contamination. To avoid much of this, the team, lead by Matthias Meyer at the Max Planck Institute for in Leipzig came up with a simple but efficient method to generate sequencing libraries from single stranded DNA. The basic steps in the library prep are:

DephosphorylateHeat denatureLigate single stranded biotinylated adaptorsImmobilize on streptavidin beadsGenerate the second strand with DNA polymeraseLigate a second adaptor by blunt end ligation

The main benefit…

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 …