1. A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI
- Author
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Alexis J. Marshall, Yi Han Lin, Pippa Kay, German Spangenberg, Hiroshi Shinozuka, Maiko Shinozuka, John W. Forster, and Noel O. I. Cogan
- Subjects
DNA, Bacterial ,DNA, Plant ,Genotyping Techniques ,DNA polymerase ,Arabidopsis ,Agrobacterium ,Computational biology ,DNA shearing ,DNA sequencing ,Recognition sequence ,Bacterial Proteins ,5-methylcytosine ,Genetics ,biology ,Methodology Article ,Second-generation sequencing technology ,High-Throughput Nucleotide Sequencing ,Nucleic acid amplification technique ,DNA Restriction Enzymes ,Sequence Analysis, DNA ,Amplicon ,Sequencing by ligation ,Restriction enzyme ,Deoxycytosine Nucleotides ,biology.protein ,Genotyping-by-sequencing ,Restriction endonuclease ,Nucleic Acid Amplification Techniques ,Biotechnology ,Reference genome - Abstract
Background Fragmentation at random nucleotide locations is an essential process for preparation of DNA libraries to be used on massively parallel short-read DNA sequencing platforms. Although instruments for physical shearing, such as the Covaris S2 focused-ultrasonicator system, and products for enzymatic shearing, such as the Nextera technology and NEBNext dsDNA Fragmentase kit, are commercially available, a simple and inexpensive method is desirable for high-throughput sequencing library preparation. MspJI is a recently characterised restriction enzyme which recognises the sequence motif CNNR (where R = G or A) when the first base is modified to 5-methylcytosine or 5-hydroxymethylcytosine. Results A semi-random enzymatic DNA amplicon fragmentation method was developed based on the unique cleavage properties of MspJI. In this method, random incorporation of 5-methyl-2’-deoxycytidine-5’-triphosphate is achieved through DNA amplification with DNA polymerase, followed by DNA digestion with MspJI. Due to the recognition sequence of the enzyme, DNA amplicons are fragmented in a relatively sequence-independent manner. The size range of the resulting fragments was capable of control through optimisation of 5-methyl-2’-deoxycytidine-5’-triphosphate concentration in the reaction mixture. A library suitable for sequencing using the Illumina MiSeq platform was prepared and processed using the proposed method. Alignment of generated short reads to a reference sequence demonstrated a relatively high level of random fragmentation. Conclusions The proposed method may be performed with standard laboratory equipment. Although the uniformity of coverage was slightly inferior to the Covaris physical shearing procedure, due to efficiencies of cost and labour, the method may be more suitable than existing approaches for implementation in large-scale sequencing activities, such as bacterial artificial chromosome (BAC)-based genome sequence assembly, pan-genomic studies and locus-targeted genotyping-by-sequencing. Electronic supplementary material The online version of this article (doi:10.1186/s12896-015-0139-7) contains supplementary material, which is available to authorized users.
- Published
- 2015