In a particularly powerful advance to LM-PCR, ultrasonic shearing of the genomic DNA [39] is used instead of restriction enzyme digestion. Since the resulting shear sites are virtually random, this method has two major benefits. First, it abolishes the preferential amplification of integration sites that lie near a given restriction site. Second, each “sister” cell of the same infected clone (i.e., sharing the genomic integration site) has a shear site in its genome at a unique distance from the viral integration site. This makes it possible to count the sequenced sister cells of each clone.

High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed.

MethySite® DNA Global Differential Methylation (GDM)
Highlight:
Measure the methylation percentage of protein/transcriptional factor binding site at whole genome level 
High through put DDM check protein binding site methylation, The lower cost and higher throughput offered by NGS confers another advantage to researchers.
where DNA is methylated
The binding site methylation is one of important mechanism to control cell differentiation/development direction.

Methytome® DNA Differential Methylation (DDM)

 
Background
DNA methylation plays an important role in the control of gene activity. The mechanism either results from the direct effects of methylation on transcription factor binding, or be indirectly caused by repressor proteins that bind to methylated DNA.
A transcriptional factor have thousands binding site in gDNA.
RNA-seq only do RNA level 
Compare the methylated CCGG sequence among samples will show transcriptional factor binding difference between samples
High-throughput sequencing, also known as next-generation sequencing (NGS).
Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources.
 
Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed.

Principle
Hap-II is a restriction enzymes that recognize the unmethylated DNA sequence 5’-CCGG. HarborgenaseTM  (patent pending) is a novel ligase that link adapter and DNA restriction fragment, but not catalyses the ligations between fragments. Using HarborgenaseTM, Hap-II and a DNA adapter can make a fully tagged restriction fragment library, in which every end of DNA restriction fragments will be linked an adaptor and no fragment-fragment self ligation. All unmethylated 5’-CCGG sequence will be destroyed during the ligation reaction, but not methylated 5’-CCGG sequence. With the universal primer that matched adapter sequence, all the restriction fragments can be measured in NGS, in which the CCGG containing sequences left between libraries are caused by CCGG site methylation. The differences of CCGG containing sequence between libraries suggest that protein binding difference.

Application:
            DNA methylatome / profile / pattern
            Protein-DNA interaction / TF binding difference             Development / cell phenotype alterations  
If DNA binding site were methylated, the TF cannot bind DNA and will lose control on downstream gene even TF expressed in cell.
IF RNA expression level changed and the TF binding site methylation not changed, it suggest that the RNA expression change caused by other reason not the TF binding site methylation 
Compare with RNAseq/Chip assay: 

Procedure
1, make a Hpa-II library with adapter-A
2, make a regular NGS library (TA ligation) with Hpa II library and adapter-B
3, Do NGS
4, compare the differential methylation between samples with computer software.
5, Count CCGG site and adapter-A number with software to calculate Global methylation percentage (=unmethylated CCGG/methylated CCGG = Adapter-A seq number/ CCGG number in sequence)
 
Pitfall 1: NGS measured sequence too short to show the whole fragment sequence!
Possible Resolution: use genome sequence in gene bank!?
 
Pitfall 2: some Hap II sequence too long to be tagged
Possible Resolution: link one more adapter with TA ligation?! Or make another library with TA ligation after Hpa II digestion/library?
 
Pitfall 3: Protein-binding sequence in gDNA do not have CCGG site
Possible Resolution: No method to overcome it!
 
Pitfall 4: Global methylation calculation bias=same between samples
Pitfall 5: PCR application bias=same between samples
Pitfall 6: TA ligation bias=same between samples