1、 microRNA transcription factor prediction analysis

In order to explore the relationship between upstream and downstream regulatory elements of the target miRNA sequence, extracting the target microRNA ‘s 4500bp upstream TSS sequence and downstream 500bp TSS sequence as transcription factor (TF) forecast data after finishing genome location. Then we complete transcription factor prediction analysis based on transcription factor database, and transcription factor binding site prediction program

 TSS is the transcription initiation site marked, phastCons is a conservative value of cross-species

2、 microRNA target gene prediction

2.1. MicroRNA target gene prediction of non-coding region of the project (recommended)

For non-coding region of the predicted target genes, we use multiple software to predict, respectively, reference miRGator software processing that can not correspond to the objectives of microRNA. We generally take the overlap of the results from several forecasting software part as the final result.

The overlapping of predicted results

2.2.microRNA coding region of the target gene prediction project

For the coding region of target genes predicted microRNA target extraction done after the coding region of multi-species sequence comparison. We predict target sites after finding the conservative section ,then we collate and do statistical analysis of the conservative sites. The forecast about  MicroRNA binding sites has been a lot of top-level article reports.

The references are as follows:

[1]Yvonne Tay. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature. 17 September 2008.

[2]Joshua J. Forman et al. A search for conserved sequences in coding regions reveals that the let-7 microRNA targets Dicer within its coding sequence. PNAS August 12, 2008.

[3]Anja M. Duursma. miR-148 targets human DNMT3b protein coding region. RNA. Mar 26, 2008.

Forecast Process:

(1) Extraction of coding sequence

(2) Multi-species comparison, seeking for the conservative sector

(3) Downloading miRanda and the TargetScan software for carrying on the target position spot forecast

(4) Reorganizing the conservative target position spot, and carries on the statistical analysis

3、Gene ontology analysis of target genes(may elect)

We carry on the biology function classification for the differential gene based on the gene annotation result of GO database,and screen significantly noticeable differences classes Based on statistical testing methods (P-value). Finally we carry on the output of result.

Tree chart:

Pie chart:

Bar chart:

4、Target gene pathway analysis

We establish signaling pathways and biological functions network, compare and integrate differential genes and signaling pathways, find out the relationship between genes ,carry out dynamic simulation path.And screen significantly noticeable differences in metabolic pathways based on statistical testing methods (P-value).It has a view of building a simulation of disease-causing genetic disease status of the access network, the purpose of genetic analysis for the target gene is finding out biological pathways and biochemical pathway related with diseases.

Take some pathway as the example:

What red mark surrenders to the state the gene, what blue color mark declines the gene

5、microRNA promoter methylation analysis

In order to explore the methylation effects on the target microRNA, we download microRNA target sequence of the promoter region, find potential microRNA target sites of methylation (CpG island) through correlation algorithm, and designed four pairs of primers: a pair of methylation , a pair of unmethylated primer to guide the experiment (MSP-PCR method) validation.

6、microRNA in different tissues, cells in the expression analysis

In order to investigate microRNA’s expression of different tissues,cloned and sequenced data was calculated as the relative expression levels of miRNA targets. The expression of miRNA is the copy number divided by the total number of organization miRNA and then multiplied by 1000, that copies per kilo.We analyze all microRNA expression.of the organization, cell lines, tumor.

microRNA distribution chart

Network construction

1、microRNA and the target gene network construction

We are building a network through integration between the microRNA and target gene regulation, microRNA target genes between the protein - protein interaction. This network is a overall direct-viewing of situation level between genes, and reflect gene regulation network’s stability. The high connection gene In network is called the hub gene. The Hub gene often plays the influential role for the network stability. Because hub geng will affect the majority of  all genes, it will be the gene’s regulation core, generally thought that its importance must be higher than the ordinary gene. Generally speaking, the majority of hub gene is the transcription factor, sometimes, is also possibly be the activating enzyme, like MAPK system.

microRNA and its target gene network.

 On the one hand the green expressed that microRNA to the target gene's regulative relations, purple all kinds on the other hand expresses between the microRNA target gene regulative relations.

2、microRNA and transcription factor regulatory network analysis

For some transcription factor, microRNA itself can be combined in the promoter region to regulate microRNA. And the microRNA may also be combined in transcription factor 3 'UTR in the same time.That in itself is a network. The way is to target gene prediction and promoter analysis of a comprehensive analysis..

 E2F and the mir-17-92 cluster of gene regulatory networks

3、microRNA interaction network

The method of NLP (Natural Language Processing) is used to analyse microRNA regulatory networks by literature abstracts from the PubMed database, and interaction of the microRNA, with doing GO analysis 、pathway nalysis.

Three-dimensional structure prediction

Using correlation techniques reported by Nature and PNAS, we developed the microRNA three-dimensional structure’s forecast platform, this platform can carry on the precise forecast aiming at the microRNA three-dimensional structure . It already passed through in microRNA inhibitor screening, the microRNA target gene exact search and so on many domains,it is also already succeeded for many units about microRNA research, the projects progress smoothly.

Applications 1: microRNA small molecule inhibitors

Objective: To construct a three-class structure of microRNA and find their drug targets; then predict miroRNA inhibitor small molecule compounds based on three pairs of small molecule compounds structure prediction.MicroRNA has been carried out on a number of small molecule medicines related to forecasts, the results are verified in the experiments.

Chart of symbols 1: Some microRNA three dimensional structure drawing

 Chart of symbols 2: Some microRNA member docking union position spot chart

Application 2: microRNA target gene locus precise positioning
Conventional microRNA target gene identification method is that finding the genes can be matched from TargetScan, PicTar and other target genes database, but such a large number of genes found in the other hand are difficult,and if you know a targeted gene how to find the combination of its microRNA accurately? it has been more difficult to resolve the problem. In this regard, we use a combination of microRNA target gene with the free energy and species such conservative standards, it starts from the secondary and tertiary structure, and exacts matches to each other mciroRNA and target gene. Now, Huashan Hospital, Chinese Academy of Sciences and other health units have done relevant analysis, their experimental validation results are gratifying.

Other analysis

SVM model for screening of target genes (may select)

Research objectives is for identifying all the genes involved in the research ,building SVM model base these genes, and re-use this model to screen the target gene.

Solexa

Solexa sequencing Description:

     Solexa this approach can be a reaction by adding four kinds of nucleotides with labled sing synthetic dye while sequencing (SBS-sequencing by synthesis), it may reduce the secondary structure mismatches caused by the absence of a section of the region. And it is high-throughput, high precision, with simple and easy to operate the automation platform and powerful characteristics required less sample.This reaction can simultaneously detect the 100 million nucleotide fragments, so a single chip or a few the chip (only 1% of the conventional method) will finfish the whole genome sequencing cost of very little. 
    With DNA sequencing, expression profiling (DNA sequencing, expression profiling), as well as microRNA analysis platform-Solexa Genome Analysis System. One after another, making this method be applied in more fields. 
    Illumina's Solexa Sequencing technology to provide customers with a powerful next-generation sequencing methods, for the present genetic analysis and functional genomics and other popular areas of research applications. 

Solexa's applications:

1, sequencing and re-sequencing

2, expression profiling analysis

3, Identification and quantitative analysis of small RNA

4, directional repeat regions of the genome sequence - determination and the discovery of new genetic polymorphism, such as the SNP

5, genomic methylation analysis

6, nucleic acid and protein interactions and positioning research, such as ChIP-Seq Studies

Small RNA sequencing

Small RNA is a large class of regulatory molecules, found in almost all of the organisms. Small RNA, including: miRNA, ncRNA, siRNA, snoRNA, piRNA, rasiRNA, and so on. Small RNA through a variety of pathways, including mRNA degradation, translation inhibition, heterochromatin formation and DNA removal, to control the growth and development of organisms and disease. Small RNA transcriptome sequencing is the identification and quantification of small RNA analysis, new methods and a powerful tool

1、 can be any species of small RNA sequencing - no precursors based on sequence information or structural information of the two probes.

2、genome-wide - study of samples of any size, known or unknown to all small RNA

3、Digital transcript expression analysis ，an absolute quantitative expression levels across five orders of magnitude of transcript abundance.

4、detection of rare transcripts and transcript variants, such as single nucleotide mutation (SNP).

Experiment

sample preparation:

Provide the concentration of ≥ 1μg/μL, the total ≥ 12μg of total RNA, into two sub-installed, in which a tube of less than 10μg, for sequencing; another tube 2 ~ 3μg, for the 2100 detection;

Total RNA detected after passing through 2100, by gel electrophoresis to isolate 18-30bp length smallRNA (of specific length can be customized according to customer demand), and then the following steps:

Sample preparation process at least 4 days:

- sequencing

Through high-throughput sequencing for each sample can be at least 5 million reads numbers;

Bioinformatic analysis of sequencing results, see the microRNA sequence analysis services

the mRNA sequence experiment serves

This method first cuts 21bp TAG fragment from 3’end of enzyme to obtain a section of (specificity to mark this gene) for each mRNA; Then through the high flux sequence, obtains the massive TAG sequence, different TAG sequence's quantity has represented the corresponding gene expression quantity; Through the biological information sciences analysis, Obtain representative TAG gene, the gene expression level, as well as the sample information and so on gene expression difference.

Technical advantages:

The advantages of using high-throughput sequencing to study the expression pattern is very clear, as follows: 
1. Digital signals: 
2. High repeatability: 
3. High sensitivity: 
4. Genome-wide analysis, cost-effective: 
5. High-throughput sequencing: expression tags. 
6. No need to repeat the experiment. 
7. The same time the discovery of new transcripts, gene expression regulation of regional and other groups. 
8. Complete in-depth bioinformatics analysis support, but also help to make important scientific discoveries, made high-quality articles. 

Technical protocol:

1、 sample preparation:

Provides the density ≥500ng/μL, total quantity ≥6ug total RNA, loads separately two, 4~5 μg, use in the sequence; Another 1~2 μg use in 2100 examinations, OD260/280 is 1.8~2.2 total RNA samples;

2、sample preparation:

It is similar with SAGE technology, the method which cuts through the specificity enzyme 3 ' the terminal obtains a section of 21bp specificity fragment from each mRNA, uses for to mark this gene, is called TAG, uses in the sequence in the TAG fragment both sides connection the attachment directing the thing;

3、hands-on sequence:

Through high-throughput sequencing for each sample can be at least 2.5 million TAG sequence;

The result of sequencing analysis of biological information please see the mRNA sequence analysis services

ChIP-seq sequencing service

A new generation of high-throughput sequencing technology for epigenetic studies provide a new set of research ideas, through chromatin immunoprecipitation (ChIP) to obtain a large-scale sequencing of DNA fragments can obtain high-resolution maps within the framework of whole-genome DNA methylation, various histone modifications, transcription factors. High-quality, high-throughput, low-cost data throughput, laid the technical foundation of studying epigenetics. 
ChIP-SEQ is the second ChIP-Chip, the protein / nucleic acid interaction study of another technological breakthrough. Next-generation sequencing-based technology, ChIP-SEQ, the researchers access to millions of sequence tags, and can concern the DNA protein binding site on the precise positioning into the genome. 
Application areas: DNA methylation, histone modification, transcription factors and so on. 

Technical advantages:
• High reliability: ChIP-Chip lower than the background level and high signal to noise ratio to ensure high reliability of the experimental results
• High throughput: a lane is almost covered all the binding domain of transcription factors in whole genome by the data generated
Low Cost: a single read sequencing and the cost of analysis is only 1 / 100 of traditional sequencing

ChIP-seq experiment and analysis

Sample preparation: 
1, please provide a concentration of ≥ 10 ng / ul, total ≥ 200 ng, OD260/280 1.8 ~ 2.2 of the DNA samples.
2, if the amount of single DNA after ChIP is not enough, it is recommended 2 to 3 times ChIP of DNA together. 
3, please provide the detection of DNA interrupted gel map, DNA gel electrophoresis after the request to interrupt the main belt in 200-500bp range. 
4, please obtain DNA for the ChIP primers were designed for QPCR validation and quantitative, to provide test sites test report. Attached to positive and negative controls. 
5, for sending samples samples, please fill out to send a single sample 
Sample transportation requirements: 
1, sample requests placed in 1.5 ml tube, pipe indicate the sample name, concentration and preparation time, the use of Parafilm sealing nozzle. Before all samples in the transport tubes fixed at 50 ml centrifuge tube with lid, and then 50 ml tubes placed in sealed bags. 
2, in order to prevent the adhesion of low-concentration samples in the centrifuge wall, use non-stick tube transport DNA. 
3, it is recommended to use ice packs transport, post and try to use a faster way to reduce the transport process in the possibility of sample degradation. 
4, will fill in the ChIP-Seq Sample Information Form with the sample transport. 
5, immediately after delivery to ensure timely receive the sample.

Bacterial whole-genome experiment service

At present there is rapid progress in bacterial genome research, it has published 684 real bacterial genomes and 51 archaeal genome sequences. Application of new technologies to enable more laboratories can afford the cost of bacterial genome sequencing, many bacterial genome sequencing work has been underway. Bacterial genome research will be a faster pace, and competitive. Our company technology has the introduction of 10M following bacterial genome sequencing services based on Solexa sequencing.

The bacterium genome team changes is quick, is big, the re-sequence may discover its variation part again:

Reads by Solexa a run generated: 1000 million;
Single End Reads alkaloids base: 10 ^ 7 * 40 = 400M;
Paired End Reads alkaloids base: 10 ^ 7 * 80 = 800M;
The size of bacterial genomes: 2 ~ 4M;
Coverage generally up to a few hundred times;
The new 20-25X coverage can be spliced;

Technical protocol

Solexa-based high-throughput genome sequencing technology enables greatly reduce the cost, Solexa Paired-End technology greatly reduces the difficulty of the genome assembly. Therefore, based on Solexa's technology to reduce the bacterial genome sequencing costs a lot to the traditional sanger sequencing technology 1 / 5 to 1 / 10, and time is significantly reduced.