Preparation of genome-wide DNA fragment libraries using bisulfite in polyacrylamide gel electrophoresis slices with formamide denaturation and quality control for massively parallel sequencing by oligonucleotide ligation and detection

Ranade, Swati; Chung, Christina B.; Zon, Gerald; Boyd, Victoria L.

doi:10.1016/j.ab.2009.04.020

Cited by 17 publications

(10 citation statements)

References 29 publications

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“…Therefore, the in solution method is preferred for larger DNA input (>2 ug starting material), while the in gel method is ideal for low amounts of starting material. As shown previously [15], 50 ng and even 5 ng can be bisulfite converted successfully in a gel with 15 cycles and 22 cycles of PCR amplification. This would correspond to about 500 ng or 50 ng of starting material, respectively, if losses are assumed to be the same as in the experiment described here.…”

Section: Resultssupporting

confidence: 63%

“…Two bisulfite-converted libraries of Escherichia coli ( E. coli ) DH10B were constructed, and the bisulfite conversion was performed in solution (bis-sol) or in a polyacrylamide gel (bis-gel) as previously described [15] (see Figure 1 for outline). Equal amounts of DNA (240 ng) were used for both bisulfite conversion methods to compare the efficiency of each method.…”

Section: Resultsmentioning

confidence: 99%

“…The enzymatic reaction was purified using the MinElute Reaction Cleanup Kit and the DNA recovered with two 20 ul buffer EB elutions, then quantified as described above. Adaptors used in this protocol deviated from the standard SOLiD fragment library protocol in that the top strand P1-A of the double-stranded P1 adaptor was synthesized using 5mC in place of C in order to protect the adaptor from modification during bisulfite conversion (for details and sequences see [15]) whereas double-stranded adaptor P2 was identical to the adaptor used in the standard protocol. The adaptors were ligated to the end-repaired DNA fragments in a 30∶1 molar ratio for 10 min at room temperature using the Quick Ligation Kit (New England BioLabs, Beverly, MA, USA) (1x quick ligase reaction buffer, 1 uL quick ligase/40 µL reaction volume).…”

Section: Methodsmentioning

confidence: 99%

“…During the adaptor ligation only the 5′-3′ strand of the adaptor ligated to the 5′P ends of the DNA fragments. After the DNA purification the 3′-5′ adaptor strands were filled in by nick-translation using a dNTP solution, containing 5m-dCTP instead of dCTP as previously described [15]. The nick translation reaction was carried out for 30 minutes at 16°C using DNA Polymerase I (New England BioLabs, Beverly, MA, USA) (1x NEB buffer 2, 2 mM 5mC-dNTP, 0.25 U/µl DNA Polymerase I).…”

Section: Methodsmentioning

confidence: 99%

“…The SOLiD sequencing reported here was performed with bacterial libraries that were prepared similar to our previous publication [15], but with some differences that were informative.…”

Section: Introductionmentioning

confidence: 99%

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Whole Methylome Analysis by Ultra-Deep Sequencing Using Two-Base Encoding

Chung¹,

Boyd²,

McKernan³

et al. 2010

PLoS ONE

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Methylation, the addition of methyl groups to cytosine (C), plays an important role in the regulation of gene expression in both normal and dysfunctional cells. During bisulfite conversion and subsequent PCR amplification, unmethylated Cs are converted into thymine (T), while methylated Cs will not be converted. Sequencing of this bisulfite-treated DNA permits the detection of methylation at specific sites. Through the introduction of next-generation sequencing technologies (NGS) simultaneous analysis of methylation motifs in multiple regions provides the opportunity for hypothesis-free study of the entire methylome. Here we present a whole methylome sequencing study that compares two different bisulfite conversion methods (in solution versus in gel), utilizing the high throughput of the SOLiD™ System. Advantages and disadvantages of the two different bisulfite conversion methods for constructing sequencing libraries are discussed. Furthermore, the application of the SOLiD™ bisulfite sequencing to larger and more complex genomes is shown with preliminary in silico created bisulfite converted reads.

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Section: Resultssupporting

confidence: 63%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The SOLiD sequencing reported here was performed with bacterial libraries that were prepared similar to our previous publication [15], but with some differences that were informative.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Whole Methylome Analysis by Ultra-Deep Sequencing Using Two-Base Encoding

Chung¹,

Boyd²,

McKernan³

et al. 2010

PLoS ONE

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Single Nucleotide Analysis of Cytosine Methylation by Whole‐Genome Shotgun Bisulfite Sequencing

Johnson

Mueller

Gamé

et al. 2012

CP Molecular Biology

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Unlike other methods to assess methylation, whole-genome shotgun bisulfite sequencing is used to generate quantitative genome-wide methylation profiles at single-nucleotide resolution. As described in this unit, this method allows for the quantitative measurement of methylation at each cytosine base by treatment of genomic DNA with sodium bisulfite followed by sequencing and alignment of the reads to a reference genome.

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High-Throughput Sequencing of the Methylome Using Two-Base Encoding

Chung

2012

Methods in Molecular Biology

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Methylation of Cytosine together with other epigenetic traits plays an important role in the development and regulation of both healthy and diseased cells. Changes in the methylation patterns have been shown to be associated with the development of cancer, growth, neurodevelopmental, and endocrine disorders (Laird PW, Nat Rev Genet 11:191-203, 2010; Tost J, Mol Biotechnol 44:71-81, 2010; Zuo T et al., Epigenomics 1:331-345, 2009). Thus, studying the methylation pattern can give important insights to the underlying causes of disease and development. A method for studying the methylome on a single base resolution is described, using bisulfite sequencing in combination with the high-throughput SOLiD(TM) sequencing technology.

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Preparation of genome-wide DNA fragment libraries using bisulfite in polyacrylamide gel electrophoresis slices with formamide denaturation and quality control for massively parallel sequencing by oligonucleotide ligation and detection

Cited by 17 publications

References 29 publications

Whole Methylome Analysis by Ultra-Deep Sequencing Using Two-Base Encoding

Whole Methylome Analysis by Ultra-Deep Sequencing Using Two-Base Encoding

Single Nucleotide Analysis of Cytosine Methylation by Whole‐Genome Shotgun Bisulfite Sequencing

High-Throughput Sequencing of the Methylome Using Two-Base Encoding

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