Technique / Molecular Biology / DNA analysis techniques / DNA sequencing

Mab-seq protocol for DNA deep sequencing

Mab-seq protocol for DNA deep sequencing

DNA deep sequencing refers to coverage of particular base or number of reads during DNA sequencing for measurement of redundancy in genome sequencing. Mab-seq protocol is a new technique for deep sequencing using CHIP microarray.

Schmidt D, Stark R, Wilson MD, Brown GD, Odom DT 2008 Genome-Scale Validation of Deep-Sequencing Libraries. PLoS ONE 3(11): e3713

Chromatin immunoprecipitation (ChIP) is widely used to identify interactions between genomic DNA and proteins in eukaryotic cells [1]. Recent approaches have identified transcription factor binding or histone modifications by combining ChIP experiments with genomic-tiling or promoter-based microarrays (i.e. ChIP-chip) [2]–[9]. The subsequent maps of transcription factor binding, chromatin structure, and modifications established by ChIP-chip and similar assays have greatly broadened our understanding of mechanisms regulating transcription [2]–[9]. However, microarrays generally interrogate only genomic regions rich in unique sequences, and their designs typically start by eliminating half of a mammalian genome due to repeat regions.

The application of high-throughput (HTP) DNA sequencing to ChIP experiments (ChIP-seq) has overcome many microarray-related limitations in probe coverage and resolution [10]–[13]. ChIP-seq allows transcription factor and histone patterning of complex mammalian genomes to be identified at high resolution, and the interrogation of virtually an entire mammalian genome is now possible. As HTP sequencing technology can be difficult to access and expensive to use, a method to inspect sequencing library quality and genomic enrichment for ChIP-seq experiments in advance of deep sequencing would be of substantial utility to a wide number of researchers [14].

Here we report such a methodology (Microarrays-before-Sequencing, Mab-seq), which should also be of interest to investigators using chromatin immunoprecipitations who are working primarily with microarrays, but wish to bank material for later genome-wide interrogations. This method also offers advantages to investigators with access to HTP instruments (e.g. Illumina Genome Analyzer) who wish to test their libraries prior to sequencing without compromising either experimental approach by sample loss.

Last update 07-Dec-2008, Rating of 0 votes.