A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI
© Shinozuka et al.; licensee BioMed Central. 2015
Received: 11 February 2015
Accepted: 30 March 2015
Published: 11 April 2015
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.
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.
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.
KeywordsSecond-generation sequencing technology Genotyping-by-sequencing 5-methylcytosine Restriction endonuclease DNA shearing
Massively parallel short-read sequencing technologies have become commonly used not only for de novo genome sequencing, but also for a wide range of biological purposes, such as resequencing and large-scale genotyping studies. Fragmentation at random nucleotide locations is an essential component of library construction for the various short-read sequencing instruments , through delivery of multiple read initiation points in template molecules. Sequence information may then be decoded through computational assembly of the short reads. Physical shearing is recommended by the manufacturers of all second-generation massively parallel DNA sequencing systems, due to the high reproducibility and randomness of fragmentation. However, the process is likely to require the use of dedicated instruments. The Nextera technology (Illumina, California, USA) and the NEBNext dsDNA Fragmentase kit (New England Biolabs, Massachusetts, USA) are alternative random DNA fragmentation methods which require only standard laboratory instruments [2-4]. The Nextera technology uses a transposon-transposase combination for random fragmentation of template DNA and attachment of transposon ends at the cleaved sites, permitting subsequent PCR amplification and sequencing. With the NEBNext dsDNA Fragmentase kit, double-stranded template DNA is fragmented in two sequential steps: nicks are enzymatically introduced into DNA, which is then cleaved at the nicked sites. These enzyme-based methods, however, require DNA sample preparation (buffer replacement and DNA concentration adjustment) for effective digestion, and the size of products is sensitive to both DNA sample quality and reaction duration, all of which require optimisation for each sample in order to achieve the desired outcome.
MspJI is a recently characterised modification-dependent endonuclease . The enzyme was isolated from Mycobacterium sp. JLS and recognises sites containing the sequence motif CNNR (R = G or A nucleotides) when the first base is a 5-methylcytosine (5mC) or 5-hydroxymethylcytosine, and cleaves DNA at N12/N16 bases distant from the modified cytosine on the 3′-side. Enzyme activity is enhanced by short double-stranded DNA that includes the MspJI recognition site (and so acts as an enzyme activator). Digestion of genomic DNA with the MspJI enzyme generates fragments 32-34 bp in length, containing mCpG or mCNG sites central to the fragment. Methylation status of the human genome has been analysed through sequencing of such fragments . Due to these unique features as a methylation-dependent restriction enzyme with adjacent non-specific cleavage activity, MspJI is expected to be highly useful for DNA modification and epigenomic studies .
FspEI and LpnPI are also recently characterised modification-dependent endonucleases, derived from Frankia sp. EAN1pec and Legionella pneumophila Philadelphia 1, respectively . The sequence recognition sites for FspEI and LpnPI are 5’-CC-3’ and 5’-CCDG-3’ (D = A, G or T nucleotides), respectively, when the second base is a 5mC or 5-hydroxymethylcytosine. Similar to MspJI, activity of the two enzymes is stimulated by short DNA templates containing the recognition site, and the enzymes produce DNA fragments with 5’- termini including cohesive ends. These two enzymes hence provide potential alternatives to MspJI as a tool for DNA fragmentation.
The present study describes a simple and inexpensive method for generation of semi-randomly fragmented DNA from amplicon templates. DNA amplicons with randomly-incorporated 5-methyl-2’-deoxycytidine-5’-triphosphate (5-methyl-dCTP) were synthesised with DNA polymerase, and then digested with the MspJI restriction enzyme. The size range of the MspJI-digested fragments was capable of control through optimisation of 5-methyl-dCTP concentration. A purification procedure is unnecessary for DNA digestion with MspJI, which permits high-throughput sequencing library preparation. Short DNA fragments were also generated from a range of templates with a whole genome amplification kit based on activity of the Φ29 DNA polymerase, using the same methodology. Illumina sequencing libraries with inserts of 200 or 550 bp in length were successfully prepared using the MspJI-digested DNA, and were processed on the Illumina MiSeq platform.
Results and discussion
DNA amplification with 5-methyl-dCTP and MspJI digestion
The Agro_gc50 sequence was also amplified with four types of DNA polymerase. The sequence was successfully amplified with all polymerases in the presence of 5-methyl-dCTP, and the amplicons were digested with MspJI. No significant differences in size range were observed, suggesting that a variety of DNA polymerases may be used for the proposed DNA fragmentation method (Additional file 2). A further characterisation of MspJI enzymatic activity indicated that components of the PCR solution do not significantly affect activity of the MspJI enzyme when diluted in the reaction mixture; the MspJI-mediated digestion of amplicons is completed within 4 hours; and the digestion result is independent of input DNA amount, when performed in an appropriate volume of reaction mixture (Additional file 3).
Whole genome amplification (WGA) was performed using the QIAGEN REPLI-g mini kit in the presence of 5-methyl-dCTP (10 to 20 μM) with genomic DNA samples from Arabidopsis thaliana (L.) Heynh. ecotype Columbia (Arab gDNA), a field pea (Pisum sativum L. subsp. sativum var. arvense (L.) Poir.) genotype (PsgDNA), a perennial ryegrass (Lolium perenne L.) genotype (LpgDNA), a bovine (Bos taurus L.) genotype (BtgDNA), Agro gDNA, and a DNA sample from soil harvested in South Australia (Soil DNA) as templates. Amplified product was visualised on an agarose gel, revealing no significant differences in DNA amplification due to variation of 5-methyl-dCTP concentration. The amplified DNA was digested with MspJI (Figure 1c). Similar size distribution patterns were detected across varying 5-methyl-dCTP concentrations: a majority of DNA fragments from the 20 μM 5-methyl-dCTP-containing solutions was shorter than 250 bp, and a proportion of DNA fragments from 10 μM 5-methyl-dCTP-containing solutions was close to 1,000 bp, or larger, in size.
Massively parallel sequencing of MspJI-digested templates
Result of Agrobacterium and Arabidopsis genome resequencing using the two different DNA fragmentation methods
Number of reads
Ratio of coverage (%)
Average coverage depth (time)
Potential application 1: sequencing of BAC clones
Although high-throughput DNA sequencing technologies have delivered a cost-efficient whole-genome shotgun sequencing method for those species with large genome sizes, information from BAC-based genomic libraries is valuable for effective DNA sequence assembly . Sequence information from BAC-ends is commonly used for de novo assembly of large genomes [11,12]. The BAC-end sequencing procedure, however, requires a large investment, as it depends on the Sanger sequencing method . A simple sequencing method for BAC clones using high-throughput sequencing technologies is described here.
Resequencing results of the BAC clones
100,000 read subset
Aligned reads (%)
Ratio of coverage (%)
Average coverage depth (time)
Ratio of coverage (%)
Average coverage depth (time)
Potential application 2: whole genome amplification and sequencing of bacterial and fungal genomes
High-throughput DNA sequencing technologies also provide an efficient method for pan-genome studies, especially for bacterial and fungal species [14,15]. Due to high levels of genomic diversity, a substantial number of bacteria or fungal strains must, however, be sequenced in order to define both core- and pan-genome constituents, and so a high-throughput library preparation method is required. Previously, direct WGA from fungal tissues was suggested as an efficient DNA sample preparation method . A combination of the WGA and MspJI-based DNA fragmentation methods may permit high-throughput library processing.
Resequencing results of the ryegrass endophyte genome
Ratio of coverage (%)
Average coverage depth (time)
Max coverage depth (time)
Potential application 3: sequencing of PCR amplicons
Massively parallel sequencing technologies have permitted whole genome re-sequencing in a cost-effective manner . Subsequently, genome-wide association studies (GWASs) have identified DNA polymorphisms that are correlated with trait-specific variation . The numbers of relevant DNA polymorphisms identified through GWASs have, however, been relatively small [17,18]. Identification of trait locus variation-related DNA polymorphisms could hence be usefully followed by conversion into specific PCR-based markers, permitting locus-targeted genotyping over larger numbers of individuals [19,20].
The present study has reported a novel method for DNA fragmentation using the MspJI enzyme, which has been exemplified for a range of template types. A DNA sample preparation procedure, such as buffer replacement and DNA concentration adjustment, is not essential for MspJI digestion, which permits a simple DNA library preparation procedure from amplicons. A modified method involving combined use with other modification-dependent restriction enzymes may improve the random nature of the fragmentation. The size range of the resulting fragments was capable of control through adjustment of the 5-methyl-dCTP concentration in the amplification reaction solution, providing various fragment ranges from <100 bp to >2 kb. The method may hence be applicable for recombinant DNA purposes other than second-generation massively parallel short read sequencing technologies. Development of a computational methodology may improve sequencing efficiency with this method, through optimisation of 5-methyl-dCTP concentration and prediction of coverage for each nucleotide.
DNA sample preparation
DNA samples were prepared using the QIAGEN DNeasy kit (QIAGEN, Hilden, Germany) (Arab gDNA, PsgDNA and LpgDNA) and the Gentra PUREGENE® DNA Purification Kit (QIAGEN) (BtgDNA), BioRad AquaPure Genomic DNA Kit (Bio-Rad Laboratories, CA, USA) (Agro gDNA). The Soil DNA sample was extracted with the MoBio Powersoil kit (MoBio, CA, USA), following a modified protocol . DNA concentrations were adjusted to 5-30 ng/μl in the TE buffer using the NanoDrop system (Thermo Fisher Scientific, MA, USA).
DNA amplification with 5-methyl-dCTP
Locus-specific primers were prepared for amplification of the Agro_gc40, Agro_gc50 and Agro_gc60, BtKIT1-10 and BtKIT27-37 sequences (Additional file 1), and PCR was performed with Phusion Hot Start DNA polymerase (Thermo Fisher Scientific) and The Expand Long Range dNTPack (Roche Applied Science, Penzberg, Germany), following the manufacturer’s protocol. 5-methyl-dCTP (TriLink, CA, USA) was added to the PCR mixture at final concentrations of from 0 to 60 μM. WGA was performed using the REPLI-g mini kit (QIAGEN). Following the manufacturer’s protocol, 2.5 μl DNA (12.5-75 ng) was denatured with the D1 solution for three minutes, and then neutralised with the N1 solution. The amplification was performed in the reaction mixture with the presence of from 0 to 100 μM 5-methyl-dCTP (final concentrations) at 30°C for 16 hours. After incubation, the DNA polymerase was heat-inactivated, and the products were diluted with the same amount of water.
Restriction enzyme digestion
The amplified DNA (5 μl) was digested with 3 U of the MspJI, FspEI or LpnPI restriction enzyme (NEB) following manufacturer’s protocol. After incubation at 37°C for 4-16 hours, the enzyme was heat-inactivated at 70°C for 20 minutes.
Illumina sequencing library construction
Sequencing libraries were constructed with the TruSeq DNA Sample Preparation kit (Illumina) or NEBNext® DNA Library Prep Master Mix Set for Illumina® (NEB) with modifications. End-filling and adenine-tailing reactions for the MspJI-digested REPLI-g products were performed with the A-Tailing Mix or Klenow Fragment (3’ → 5’ exo-). For the PCR amplicons with the Expand Long Range dNTPack, the heat-inactivation procedure of the restriction enzyme also permitted end-filling of the restriction enzyme-digested fragments in the presence of the activated heat-resistant DNA polymerase and dNTPs. Blunt-ended DNA was purified with the AMPure XP bead kit (Life Technologies, CA, USA) and was used for the adenine-tailing reaction of the NEB kit. The ‘Purify Ligation Products’ process based on agarose gel electrophoresis (Illumina kit) was not performed. Following ligation of the DNA adapter index, the ligated DNA was purified with the AMPure XP bead kit and enriched through PCR. The size range of enriched DNA fragments was determined with the Agilent 2100 Bioanalyzer and Agilent DNA 1000 Kit (Agilent Technologies, CA, USA). The sequencing library was quantified with the KAPA Library Quantification Kit (Kapa Biosystems, MA, USA), following the manufacturer’s protocol.
Following the standard procedure, sequencing libraries from the Agro gDNA and Arab gDNA templates were prepared with the Illumina and NEB library prep kits, respectively. For these libraries, genomic DNA was fragmented with the S2 focused-ultrasonicator system (Covaris, MA, USA) following the manufacturer’s protocol.
Sequencing library preparation from BAC-containing clone glycerol stocks
Arabidopsis BAC clones (MIXK3, F20D21 and F20B17) were amplified with the QIAGEN REPLI-g mini kit. Glycerol stock (4 μl) of a BAC-containing E. coli clone was mixed with the Buffer D1 (4 μl) and incubated on ice for 5 minutes. The Buffer N1 (8 μl) was added into the sample and mix by stirring with a tip. The sample was incubated at room temperature for 3 minutes. The reaction mixture, consists of 5.8 μl REPLI-g Reaction Buffer, 0.2 μl REPLI-g Mini DNA Polymerase and 0.4 μl 5-methyl-dCTP (750 μM), was added into 3.4 μl denatured sample, and the reaction mixture was incubated at 30°C for 16 hours. The amplicons (5 μl) were digested with MspJI and the end-filling reaction was performed with Klenow Fragment (3’ → 5’ exo-). Sequencing adopter ligation was performed with T4 ligase (NEB) and ligated DNA was cleaned with AMPure XP bead solution (x0.8) to exclude short DNA. DNA fragments were subsequently enriched through PCR with the phusion DNA polymerase Kit. Small fragments (<500 bp), in which fraction E. coli genome-derived fragments were highly prevalent, were removed through size-selection with AMPure XP bead solution (x0.6). The sequencing library was characterised with the Agilent 2100 Bioanalyzer, Agilent DNA 1000 Kit, and the Qubit® Fluorometer (Life Technologies), following the manufacturer′s protocols.
Whole genome amplification and sequencing library preparation from perennial ryegrass-derived endophyte mycelium
Ryegrass endophyte genomic DNA was amplified with the QIAGEN REPLI-g mini kit. A section (2-3 mm2) of endophyte mycelium was placed into 6 μl PBS solution. The Buffer D2 (7 μl) was added into the sample and incubated on ice for 10 minutes, following mixing by a vortex. The Stop Solution was, then, added and mixed by a vortex. The reaction mixture, consisting of 29 μl REPLI-g Reaction Buffer, 1 μl REPLI-g Mini DNA Polymerase and 1 μl 5-methyl-dCTP (750 μM), was added into 9 μl of denatured sample, and the sample was incubated at 30°C for 16 hours. The amplicons (5 μl) were digested with MspJI and the sequencing library preparation was performed following the BAC clone sequencing protocol. The PCR-enriched DNA was cleaned with AMPure XP bead solution (x0.8). The sequencing library was characterised with the Agilent 2100 Bioanalyzer and Qubit® Fluorometer.
Sequencing library preparation from PCR amplicon
Locus-specific primers for the LpAP1, LpCO, LpCRY1, LpFLD, LpFT, LpLHY, LpPHYC, LpTOC1 and LpVrn5 sequences, and the MyFi™ DNA Polymerase kit (BIOLINE), which contains DNA polymerase that lacks 3′ → 5′ exonuclease activities, were used for PCR amplification (Additional file 1). The DNA samples of the p150/112 F1 mapping population were used as DNA templates. In the PCR solution, 8 μM 5-methyl-dCTP was included . The PCR products were pooled for MspJI digestion (37°C for 4 hours) and MspJI was inactivated through incubation at 70°C for 20 mins. The heat-inactivation procedure also permitted end-filling and adenine-tailing of the MspJI-digested fragments in the presence of the activated heat-resistant DNA polymerase and dNTPs. Sequencing adapter ligation was performed with T4 ligase and ligated DNA was cleaned with AMPure XP bead solution (x0.8) to remove short DNA. DNA fragments were, then, enriched through PCR and the product was cleaned with AMPure XP bead solution (x0.8). The sequencing library was characterised with the 2200 TapeStation system (Agilent) and Qubit® Fluorometer.
Massively parallel sequencing and read assembly
The Illumina MiSeq sequencing platform was used to generate sequence output for sequencing libraries with the Illumina MiSeq Reagent Kit v2 or v3. Reads were attributed by the use of sample-specific DNA bar codes. The generated sequence reads were then checked for quality and integrity using a custom PERL script. Any reads with more than 3 consecutive Ns or more than 3 nucleotides with PHRED score ≤ 20 or a median PHRED score < 20 or a read length <50 nucleotides were trimmed or removed. The specific DNA sequence reads were then reference-aligned to the respective amplicon, Agrobacterium C58 (NCBI accession numbers: AE007869 and AE007870)  or Arabidopsis Columbia sequence  (http://www.arabidopsis.org/index.jsp). Reference alignments were performed using the BWA software package and then converted to a sorted BAM file using the SAMtools software package (http://samtools.sourceforge.net/). The Seqtk software package was used for generation of a subset sequence data (https://github.com/lh3/seqtk). Alignment of the sequencing reads to the reference sequences was visualised using the Tablet software .
Availability of supporting datasets
The data sets supporting the results of this article are included within the article and its additional files.
This work was supported by funding from the Victorian Department of Economic Development, Jobs, Transport and Resources and the Dairy Futures Cooperative Research Centre (DFCRC). The authors would like to acknowledge Drs. Amanda Chamberlain, Tony Gendall, Helen Hayden Jatinder Kaur and Sukhjiwan Kaur, Ms. Joanne Rachel Ernest, Mr. Brett Mason and Mrs. Shimna Sudheesh for provision of the DNA samples, and plant and fungal materials. We would also like to acknowledge Mr. Sami Hakim for assistance during the sequencing process.
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