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In silico analysis identifies novel restriction enzyme combinations that expand reduced representation bisulfite sequencing CpG coverage.

ABSTRACT: Epigenetics is the study of gene expression changes that are not caused by changes in the deoxyribonucleic acid (DNA) sequence. DNA methylation is an epigenetic mark occurring in C-phosphate-G sites (CpGs) that leads to local or regional gene expression changes. Reduced-representation bisulfite sequencing (RRBS) is a technique that is used to ascertain the DNA methylation of millions of CpGs at single-nucleotide resolution. The genomic coverage of RRBS is given by the restriction enzyme combination used during the library preparation and the throughput capacity of the next-generation sequencer, which is used to read the generated libraries. The four-nucleotide cutters, MspI and Taq?I, are restriction enzymes commonly used in RRBS that, when combined, achieve ~12% genomic coverage. The increase in throughput of next-generation sequencers allows for novel combinations of restriction enzymes that provide higher CpG coverage.We performed a near-neighbor analysis of the four nucleotide sequences most frequently found within 50 nt of all genomic CpGs. This resulted in the identification of seven methylation-insensitive restriction enzymes (AluI, BfaI, HaeIII, HpyCH4V, MluCI, MseI, and MspI) that shared similar restriction conditions suitable for RRBS library preparation. We report that the use of two or three enzyme combinations increases the theoretical epigenome coverage to almost half of the human genome.We provide the enzyme combinations that are more likely to increase the CpG coverage in human, rat, and mouse genomes.

SUBMITTER: Martinez-Arguelles DB

PROVIDER: S-EPMC4141122 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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In silico analysis identifies novel restriction enzyme combinations that expand reduced representation bisulfite sequencing CpG coverage.

Martinez-Arguelles Daniel B DB Lee Sunghoon S Papadopoulos Vassilios V

BMC research notes 20140815

<h4>Background</h4>Epigenetics is the study of gene expression changes that are not caused by changes in the deoxyribonucleic acid (DNA) sequence. DNA methylation is an epigenetic mark occurring in C-phosphate-G sites (CpGs) that leads to local or regional gene expression changes. Reduced-representation bisulfite sequencing (RRBS) is a technique that is used to ascertain the DNA methylation of millions of CpGs at single-nucleotide resolution. The genomic coverage of RRBS is given by the restrict ...[more]

PMID: 25127888

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Project description:Microbial community profiles have been associated with a variety of traits, including methane emissions in livestock. These profiles can be difficult and expensive to obtain for thousands of samples (e.g. for accurate association of microbial profiles with traits), therefore the objective of this work was to develop a low-cost, high-throughput approach to capture the diversity of the rumen microbiome. Restriction enzyme reduced representation sequencing (RE-RRS) using ApeKI or PstI, and two bioinformatic pipelines (reference-based and reference-free) were compared to bacterial 16S rRNA gene sequencing using repeated samples collected two weeks apart from 118 sheep that were phenotypically extreme (60 high and 58 low) for methane emitted per kg dry matter intake (n = 236). DNA was extracted from freeze-dried rumen samples using a phenol chloroform and bead-beating protocol prior to RE-RRS. The resulting sequences were used to investigate the repeatability of the rumen microbial community profiles, the effect of laboratory and analytical method, and the relationship with methane production. The results suggested that the best method was PstI RE-RRS analyzed with the reference-free approach, which accounted for 53.3±5.9% of reads, and had repeatabilities of 0.49±0.07 and 0.50±0.07 for the first two principal components (PC1 and PC2), phenotypic correlations with methane yield of 0.43±0.06 and 0.46±0.06 for PC1 and PC2, and explained 41±8% of the variation in methane yield. These results were significantly better than for bacterial 16S rRNA gene sequencing of the same samples (p<0.05) except for the correlation between PC2 and methane yield. A Sensitivity study suggested approximately 2000 samples could be sequenced in a single lane on an Illumina HiSeq 2500, meaning the current work using 118 samples/lane and future proposed 384 samples/lane are well within that threshold. With minor adaptations, our approach could be used to obtain microbial profiles from other metagenomic samples.

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In silico analysis identifies novel restriction enzyme combinations that expand reduced representation bisulfite sequencing CpG coverage.

Publications

In silico analysis identifies novel restriction enzyme combinations that expand reduced representation bisulfite sequencing CpG coverage.

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