High-throughput bisulfite sequencing in mammalian genomes - PubMed (original) (raw)
High-throughput bisulfite sequencing in mammalian genomes
Zachary D Smith et al. Methods. 2009 Jul.
Abstract
DNA methylation is a critical epigenetic mark that is essential for mammalian development and aberrant in many diseases including cancer. Over the past decade multiple methods have been developed and applied to characterize its genome-wide distribution. Of these, reduced representation bisulfite sequencing (RRBS) generates nucleotide resolution DNA methylation bisulfite sequencing libraries that enrich for CpG-dense regions by methylation-insensitive restriction digestion. Here we provide an extensive, optimized protocol for generating RRBS libraries and discuss the power of this strategy for methylome profiling. We include information on sequence analysis and the relative coverage over genomic regions of interest for a representative mouse MspI generated RRBS library. Contemporary sequencing and array-based technologies are compared against sample throughput and coverage, highlighting the variety of options available to investigate methylation on the genome-scale.
Figures
Figure 1. Reduced Representation Bisulfite Sequencing
- General outline of RRBS library construction protocol including all key steps
- Overview of RRBS read alignment
- Sequencing coverage at selected regions of interest within the mouse genome as generated from a representative V6.5 mouse Embryonic Stem (ES) cell library.
*CpG Islands are as described in Ref. and extend beyond 700 bp in length.
Figure 2. _Msp_I Generated RRBS Libraries from Mouse Genomic DNA
- Overlap of sequenced/aligned CpGs with specific regions of interest in the mouse genome for typical low (40–120bp) and high (120–220bp) fragment libraries (single CpGs can simultaneous overlap with more than one region class)
- Size distribution of in silico digested fragments compared to experimentally generated RRBS libraries (low and high are combined). Only unique sequences are aligned. Asterisks (*) denote redundant microsatellite fragments.
- Diagnostic gel of high (H) and low (L) fragment libraries generated from V6.5 mouse Embryonic Stem (ES) cells. Asterisks (*) correspond to microsatellite markers as predicted in silico.
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