title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI.
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.
authors.affiliation: Department of Economic Development, Jobs, Transport and Resources, Biosciences Research Division, AgriBio, Centre for AgriBioscience, 5 Ring Road, La Trobe University Research and Development Park, Bundoora, Victoria, 3083, Australia. hiroshi.shinozuka@ecodev.vic.gov.au.|Dairy Futures Cooperative Research Centre, Bundoora, Australia. hiroshi.shinozuka@ecodev.vic.gov.au.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shinozuka
authors.affiliation: Department of Economic Development, Jobs, Transport and Resources, Biosciences Research Division, AgriBio, Centre for AgriBioscience, 5 Ring Road, La Trobe University Research and Development Park, Bundoora, Victoria, 3083, Australia. noel.cogan@ecodev.vic.gov.au.|Dairy Futures Cooperative Research Centre, Bundoora, Australia. noel.cogan@ecodev.vic.gov.au.
authors.forename: Noel O I
authors.initials: NO
authors.lastname: Cogan
authors.affiliation: Department of Economic Development, Jobs, Transport and Resources, Biosciences Research Division, AgriBio, Centre for AgriBioscience, 5 Ring Road, La Trobe University Research and Development Park, Bundoora, Victoria, 3083, Australia. maiko.shinozuka@ecodev.vic.gov.au.|Dairy Futures Cooperative Research Centre, Bundoora, Australia. maiko.shinozuka@ecodev.vic.gov.au.
authors.forename: Maiko
authors.initials: M
authors.lastname: Shinozuka
authors.affiliation: Department of Economic Development, Jobs, Transport and Resources, Biosciences Research Division, AgriBio, Centre for AgriBioscience, 5 Ring Road, La Trobe University Research and Development Park, Bundoora, Victoria, 3083, Australia. alexis.marshall@ecodev.vic.gov.au.|Dairy Futures Cooperative Research Centre, Bundoora, Australia. alexis.marshall@ecodev.vic.gov.au.
authors.forename: Alexis
authors.initials: A
authors.lastname: Marshall
authors.affiliation: Department of Economic Development, Jobs, Transport and Resources, Biosciences Research Division, AgriBio, Centre for AgriBioscience, 5 Ring Road, La Trobe University Research and Development Park, Bundoora, Victoria, 3083, Australia. pippa.kay@ecodev.vic.gov.au.
authors.forename: Pippa
authors.initials: P
authors.lastname: Kay
authors.affiliation: Department of Economic Development, Jobs, Transport and Resources, Biosciences Research Division, AgriBio, Centre for AgriBioscience, 5 Ring Road, La Trobe University Research and Development Park, Bundoora, Victoria, 3083, Australia. yihan.lin@ecodev.vic.gov.au.|Dairy Futures Cooperative Research Centre, Bundoora, Australia. yihan.lin@ecodev.vic.gov.au.
authors.forename: Yi-Han
authors.initials: YH
authors.lastname: Lin
authors.affiliation: Department of Economic Development, Jobs, Transport and Resources, Biosciences Research Division, AgriBio, Centre for AgriBioscience, 5 Ring Road, La Trobe University Research and Development Park, Bundoora, Victoria, 3083, Australia. german.spangenberg@ecodev.vic.gov.au.|Dairy Futures Cooperative Research Centre, Bundoora, Australia. german.spangenberg@ecodev.vic.gov.au.|School of Applied Systems Biology, La Trobe University, Bundoora, Victoria, 3086, Australia. german.spangenberg@ecodev.vic.gov.au.
authors.forename: German C
authors.initials: GC
authors.lastname: Spangenberg
authors.affiliation: Department of Economic Development, Jobs, Transport and Resources, Biosciences Research Division, AgriBio, Centre for AgriBioscience, 5 Ring Road, La Trobe University Research and Development Park, Bundoora, Victoria, 3083, Australia. john.forster@ecodev.vic.gov.au.|Dairy Futures Cooperative Research Centre, Bundoora, Australia. john.forster@ecodev.vic.gov.au.|School of Applied Systems Biology, La Trobe University, Bundoora, Victoria, 3086, Australia. john.forster@ecodev.vic.gov.au.
authors.forename: John W
authors.initials: JW
authors.lastname: Forster
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics; D003845:Deoxycytosine Nucleotides; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D018744:DNA, Plant; D003845:Deoxycytosine Nucleotides; C034817:5-methyldeoxycytidine triphosphate; D004262:DNA Restriction Enzymes