The 454 DNA sequencing?

454 sequencing was first propelled by Life Science in 2005 with the beginning of first next-generation DNA sequencing which had made a great innovation in the field of DNA sequencing.

Introduction

454 strategy is the method to arrange DNA proportionate up to 1 billion bases in a solitary day.

454 DNA sequencing

Compared to the other methods, 454 is less expensive and speedier used to the arrangement.

In any cases, without its shortcomings, the stage is nothing. To recognize the quantity of the bases in indistinguishable bases, it experiences issues, like AAAA.

The work flow of 454 sequencing

To start the procedure, a specimen of double-stranded DNA is needed.

The DNA separates into 400-600 base pieces through cutting the DNA at a particular focuses by utilizing the confinement compounds.

Short groupings of DNA are joined to the DNA parts, which is called connectors.

In the blend, minor resin dots are added.

On the heads, the DNA groupings are corresponding to arrangements on the connectors, which permit the DNA part can tie to the dots straightforwardly in a perfect section.

At the point, the DNA pieces append to the DNA on the dots to join the the double-strand together, getting the single-stranded DNA.

After that, on each dot, the DNA parts are replicated various times with polymerase chain response. This step can makes huge number of indistinguishable duplicates of the DNA sequencing.

To evacuate any one that has either neglected or connected any DNA which contains more than kind of DNA section, the globules are sifted.

At that point, all the remaining globules are placed into the wells. And the enzyme dots contain the DNA polymerase for the purpose of the sequencing response.

On the globules, the polymerase catalyst and groundwork also join to the DNA section.

Nucleotide bases are added to the wells at once.

At the point when every base is consolidated into the DNA. Camera will record it that the light is given out.

By plotting this example of light power on a diagram, the succession of the first bit of DNA can be decoded.

Research Methods and Main Progress in Recent Years of Comparative genomics

There are two research methods for Comparative genomics, namely comparative mapping and comparative bioinformatics. Comparative mapping is to do physical or genetic mapping of related species with the common genetic markers (molecular marker, gene cDNA cloning and genomic clones), comparing the different species distribution of those markers in the genome, suggesting synteny and collinearity on chromosome or chromosome segment, in order to analyze accurately genome structure and genome evolution of different species. Genome comparative mapping study prompts the genome homology and difference of the same species, which has great inspiration in the study of the origin and evolution in different species. Thus, comparative mapping has become an important part of plant genomics research in recent years.

There are two main methods for plant comparative physical mapping. One is the analysis of the homology sequence by analyzing the similarity to study the molecular phylogenetic process and interpretation sequence in different species; the other one is the comparison in situ hybridization localization, which is due to the true position, fragments of syntenic blocks on the chromosomes of different species. So the comparison in situ hybridization physical mapping has become an increasingly important research field of comparative mapping in plants.

The research significance of comparative mapping:

According to the highly conservative characteristics of different kinds of genome genes and their arrangements, you can study and explore their evolutionary clues.

The interpretation of the genome sequence. That is, through the comparison of homology to speculate the function of unknown genes.

Promote gene mapping. Due to similarity in different biological homologous genes, genome genetic information can be obtained by smaller studies related genomes, and the small genome of plant species is conducive to the straight homologous gene(ortholongus gene) for cloning.

Concept of proteome

Proteome refers to all protein components expressed by a gene, a cell or tissue expressed. Proteomics is the study of specific protein groups in different time and space, so as to reveal and explain the basic rules of life activities.

Why Is So Different in Analysis Results Provided by the Gene Sequencing Companies?

Kira Peikoff, 28 year old expressed concern about her future health because of family disease. She hoped to find some tips and suggestions from gene sequencing. But she found that three different gene sequencing companies gave different analysis results. Since the analysis results of different companies are different, what is the significance of such sequencing?Sequencing Companies

In the human body and almost all the living body (except for some RNA viruses and prion), each cell has a complete set of genomic DNA, such as a complete blueprint + construction manual. From the beginning of the fertilized egg, the body will choose different chapters from this manual to set up different functions of the cells, and let them perform the corresponding functions. Each one of these manuals is slightly different (most of them are the aforementioned SNP). These differences define race, skin, hair, eye color and other traits, but also defines the sensitivity of the disease. Three companies have represented the genetic health consulting industry, trying to find some SNPs associated disease, to detect their state, then calculate a probability, and finally hand over in the detected hand.

What kind of SNP locus is really associated with disease? How much relationship does it have ?

The former problem basically relies on large-scale association analysis. In fact, it is a statistical concept.

Actually only a few single gene diseases (such as a type of congenital deafness) have resolute and decisive conclusions. Height, weight, hypertension, diabetes and cancer are caused by hundreds of intertwined genes, coupled with the impact of the environment factor accumulation, coupled with the time, and show the differences. So now in the human genetics, in fact, everyone is trying to increase the statistics of the population, find race and background conditions consistent with the crowd, improve the effectiveness of their study of statistics and probability as far as possible. Even so, the conclusions between different groups of researchers are different. Because they are less likely to be shared by a group of people who are selected, the conclusion is seldom that to be repeatedly verified by other groups.

The next-generation sequencing technology—beyond Sanger sequencing

With the development of science, the traditional Sanger sequencing has not fully meet the needs of research, organism model for genome sequencing and some non model organisms genome sequencing both need a new sequencing with lower cost, higher flux and fast rate. The Next-generation sequencing comes into being.

The core idea of the next-generation sequencing technology is Sequencing by Synthesis, that is, DNA sequencing is identified by capturing the end tags composed newly. DNA FLX Illumina/Solexa, Genome Analyzer system and Biosystems SOLID Roche/454 by capturing the end of the new synthesis. The existing technology platforms include Roche/454 FLX、Illumina/Solexa Genome Analyze and Applied Biosystems SOLID system. These three technologies have their own advantages. 454 FLX sequencing is longer, and its read length can reach 400bp; Solexa sequencing is the most popular, because not only the machine price is lower than the other two, and running costs are also low. Besides, in the same data volume, the cost is only 1/10 of 454 sequencing; SOLID sequencing’s accuracy is high, the accuracy of the original base data is more than 99.94%, and the accuracy of 15X coverage can reach 99.999%, which is currently the highest sequencing technology among the next-generation technology.sanger sequencing

Solexa sequencing’s read length can reach 75bp. The size is much shorter than the traditional Sanger sequencing. But the advantage of Solexa sequencing is able to get massive data, and the price is low. By the same amount of data, Solexa sequencing is much cheaper than other sequencing. The length of 75bp is definitely not suitable for direct analysis. The reads of sequencing need to be mosaic before the actual use, which requires a strong biological information (bioinformation) analysis ability as a support.

Compared with the traditional sequencing technologies, the error rate of Solexa sequencing is relatively high, and the sequencing errors prone to distribute in the base of the reads. How to distinguish between the sequencing error and real DNA polymorphism is also a big problem.

The Next Step of Human genome project

The scientific community has completed the human genome map and medical genetic map. It is currently launching Cancer Genome Project.

5 years ago, when the first to genes, the public have been curious to ask: “what is the gene?” “What is the human genome project for what?” With the completion of human genome project sequencing work, “Where will human genome research go?” “When will the human genome project really benefit human health?” become hot topics of public concern.

Completing the human genome map and medical genetic map

Now the human genome project has finished a specific white genome map which can not represent the genome of yellow, black and other races. Before October last year, the human genome project completed a medical genetic map. The genetic map tried to choose more than 380 people with white, yellow, Mongoloid and Negroid races in the world, comparing their structure differences in the process of human evolution. Scientists hoped that they can be inspired by comparing the differences in the structure of the human race on the way of life and the clinical drug use.

Launching the cancer genome project

The international human genome project revealed the cancer genome project united by Britain and the United States has started 2 months ago in the United States. As the most direct and specific application of human genome project in medical practice, its goal is to apply the existing knowledge, tools and methods to the research of cancer genome, and to find out the variation of DNA in the DNA sequence level.

Cancer genome project in the United States is an innovative research program, with a total investment of $100,000,000. The current study focuses on brain cancer, lung cancer and ovarian cancer, which are three highest cancers in USA.

Although the cancer genome project has not yet formed like the human genome project, it has a clear plan that which countries will participate and what tasks each country will undertake. By 2020, personalized treatment will become possible.

The Analysis of Gene Sequencing Industries in 2015

The year is a challenging year for sequencing industry. The improvement of technology makes it gradually plays a more important role in all kinds of research, especially the medical research.
Sequencing technology is Under the Spotlight
For three reasons people pay more attention to gene sequencing. The first and most important reason is the receding technical costs which exceed the Moore’s law. The 2008 is a historic year for sequencing. The appearance and spread of next generation sequencing(NGS) low the cost of sequencing and quickly made it go beyond the prediction according to the Moore’s law. The cost of human whole genome sequencing has been down to $1000, and it will continuously decline with the development of new sequencing technology.

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Another one is the improvement of big data analysis tool. The sequencing is only the first step. To know the meaning of sequencing data, we need comprehensive analysis. The analysis tool makes rapid analysis of a huge amount possible.
The last one is the support from government and the promotion of medical needs. In February FDA approved a test made by 23andMe. This means the growing needs of medical application of sequencing.
The Challenge and Chance of Sequencing Industry
Overview

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According to the budget of Illumina, the market scale of gene sequencing will reach $20 billion: $12 billion for oncology; $5 billion for life science(which include the life science tools, complex diseases, agricultural genetic research, and metagenomics);$2 billion for reproductive and genetic health;$1 billion for other application.
As you can see in the chart, the market of sequencing is divided into three parts. The market of supplier almost shared by monopolies:Illumina, Life Tech( purchased by Thermo Fisher in 2014), Roche and Pacific Biosciences. According to the market research of Genome Web in 2013, the illumina share the biggest cake– seventy percent of market share of supplier.
The service providers are facing the biggest challenge. Most of them are small and middle business. It’s hard to share a profit with big companies. To survival in this industry, they need improve their service of data analysis. The market for data analysis will divide into two parts: for company with the capacity of scientific research they will do this job by themselves; for small business and individual research, they will find a third company to do this work. Due to the increase of different needs of service, companies which provide data analysis service will increase. This part is the most challenge part in sequencing industry, but it also provides the biggest chance for people who want to find a position in this industry.
Reference:
FDA permits marketing of first direct-to-consumer genetic carrier test for Bloom syndrome

Genomic sequencing–Next Generation Sequencing

The finish of Human Genome Project is a milestone for genome research. With ten-year development, the technology of genomic sequencing has changed a lot, which greatly improved the efficiency of sequencing. Last week researchers announced their result of UK10K project. Since it was proposed by David Cameron in 2014, this big project has analyzed the whole genome of nearly ten thousand people for only one year. Next generation sequencing plays an important role here.

Next generation sequencing, also called deep sequencing or high throughput sequencing, is a sequencing technology to meet the need of all kinds of research. The main characteristic of next generation sequencing is sequencing by synthesis. It has a higher throughput, and is more rapid but cost less than Sanger sequencing.
Roche 454
The coming of Genome Sequencer 20 System opened the gate of next generation sequencing. This is a high throughput genome sequencing system based on pyrosequencing technology which was introduced by 454 company. Through the purchase, Roche owned this technology and upgraded it. This is the 454 GS FLX sequencer.

Roche 454

(Roche 454 GS FLX sequencer)

The biggest advantage of Roche 454 is its reads. It can remain a high accuracy over 400bp reads. That’s the reason that it can be widely used in de novo sequencing, genome analysis and transcriptome analysis.
Solexa
In 2006, Solexa company also launched its own NGS system, Genome Analyzer. For its advantage of cost, compatibility and operability, Solexa has become the most widely used technology in sequencing. From GA to Illumina Hiseq 2000/2500, the Solexa technology also changed a lot. Now it only takes ten days for the completion of three human whole genome sequences. The application of Solexa contains de novo sequencing, re-sequencing, small RNA sequencing, lncRNA sequencing, transcriptomics sequencing and etc.

 

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(Illumina Hiseq 2000)

SOLID

As one of the magnates in sequencing industry, ABI is the monopolistic company in Sanger sequencing. However, ABI did not grab the chance of NGS. Until 2007, ABI purchased a research company, Agencourt, and launched its SOLID sequencing platform. The whole name is Sequencing by Oligo Ligation Detection. Compared to Roche 454 and Solexa, SOLID has the highest accuracy and flexibility. It can be used to the whole genome mapping analysis, and more RNA research.

Controversies Lead by Genome Sequencing for New Born Babies

For 51 years, newborn babies have gotten a heel-prick test in which their blood is screened for dozens of congenital diseases. Routine newborn baby screening has basically eliminated the risk of death or irreversible brain damage that some of these disorders can pose if they are not identified right away. Even though, it has brought about and is bringing great controversies in this field.

 

1Last December, Mercy Children’s Hospital of Kansa released its results of genome sequencing for sick new born babies on Science Translational Medicine. In this research, they practiced whole genome sequencing or exome sequencing for children with severe neurological developmental disorders from 100 families, among which there are families that have been seeking for diagnostic methods for their children for years. According to the data, 45% of the families have experienced genome sequencing. Moreover, 73% of the babies with congenital disease have accepted this new testing approach.

 

Doctor Stephen F. Kingsmore, the leader of this project hopes that the whole genome sequencing project could be carried on to around 14% of the newborns observed in ICU among the total 4 million per year.

 

Researchers from the USA are also investigating families’ attitudes towards genome sequencing. Last December, they made a research on parents of 514 healthy babies born within the past 48 hours in Brigham and Women’s Hospital, which aimed to get a general knowledge of how much do these parents know about their babies’ genome information, risks of getting genetic diseases and the meaning of genome information. When been asked whether they would be willing to attend the project of whole genome sequencing for newborn babies, results showed that 83% said yes and were happy to be in this project.

 

Moreover, Donald Chaplin of Acton, the farther a 17 months old baby, was also interested in it. As a pharmacist, he worried that the gene data might have the risk of been misused, he still wants to learn more about the gene information of his son. Jamaican engineer Nicholas Catella, however, said that he wouldn’t care much about the result unless it shows that his babies have great risks of getting severe diseases. Mr. Catella has two children, one is three old and the other 16months. “Although gene sequencing is able to reveal the risks of getting chronic diseases like Alzheimer’s, we still have no good invention methods at present”, said Mr. Catella.

 

We hope, one day when this new testing skill develops greatly, that the gene sequencing data since the birth of babies can accompany throughout all their life, and that gene sequencing can lead the growing process of newborn babies.

Genome Sequencing—Direct Sequencing of PCR Products

Nowadays, the direct sequencing of PCR products has already played a significant role in molecular biology and genomics research. Such sequencing is widely applied to the detection of gene mutation, diagnosis of genetic diseases, and polymorphism research of single nucleotide. Compared with traditional clone sequencing, direct sequencing of PCR products conducts sequencing towards the amplified DNA directly, which eliminates time-consuming cloning procedures and avoids the traditional repetitive operations like extraction of template. In this way, the correct DNA sequence information can be received from a small number of original samples. Direct sequencing of PCR products are equipped with the following advantages: fast, convenient, simple, and stable.

sequencing-PCRThen, below are the test reagents.
1. Amplified double-stranded DNA template of PCR
2. DNA primers with the length of 20 nucleotides
3. DNA polymerase
4. Sequencing gel
5. 0.1mol/L DDT
6. α-32P-DATP
7. DNTP/DDNTP mixture (80μmol/L/8μmol/L)
8. DNTP (DCTP, DGTP, DTTP-0.75μmol/L for each item)
9. Sequencing reaction buffer: 40mmol/L Tris-HCl (pH7.5), 20mmol/L MgCl2, 50mmol/L NaCl
10. Stop buffer: 95% (formamide), 20mmol / L EDTA, 0.05% (bromophenol blue), 0.05% (xylene nitrile)

Test procedure:
1. Add 2.5μl of DNTP / DDNTP mixture into four microcentrifuge tubes (amount is for each microcentrifuge tube). The mixture should be incubated at 37ºC for 5 min to alternate.
2. Add 1pmol amplified double-stranded DNA of PCR, 10pmol sequencing primer, 2μl 5×sequencing buffer, and double-distilled water into an empty microcentrifuge tube with a total volume of 10μl, then it should be heated at 96ºC for 8 min and ice cooled for 1 min (centrifugal 10s at 4ºC in 10000g).
3. Add 2μl prechilled marked mixture (DCTP, DGTP, DTTP-0.75μmol/L for each item), 5μCi α-32P-DATP, 1μl 0.1mol / L DDT, 2U sequenase, and water to the total amount of 15μl. Place it on the ice for 2 min after mixing it and label newly synthesized DNA strand.
4. Add 3.5μl marked reactive mixture into the four tubes in the first step (for each tube). It should be incubated at 37ºC for 5 min. Then each tube should be added 4μl termination.
5. Samples should be thermal denaturation for 5 min at 80ºC’ water, next 2μl should be added to the sequencing gel for each lane, and then these fragments are separated by electrophoresis.

Notes:
1.PCR products should have a certain length (>200bp), because the accuracy of the electrophoretic peak figure around 20-30bp is low.
2.If the amplified specificity were high, it could be purified through phenol directly: chloroform extraction and ethanol precipitation.
3.The design principles of sequencing primer is similar to the primer design of PCR which could take about 20 nucleotides as primers. After the purification, it can be used as a sequencing primer.

To be brief, the direct sequencing of PCR products should be payed more attention for its various benefits such as its accuracy in sequencing. Whereas, during the test procedure, the conductors should be cautious about the products as well. All in all, such technology will embrace a brighter future!

The Era of Antibody Sequencing

Antibody sequencing is a technique focuses on the determination of the amino acid sequence of an antibody, as well as which conformation the antibody adopts and the extent to which it is complexed with any non-peptide molecules. Discovering the structures and functions of antibodies in living organisms is an important tool for understanding cellular processes, and allows drugs that target specific metabolic pathways to be invented more easily.

The Rise of New Antibody Sequencing

With the development of biotechnology, antibody specialists have developed a novel strategy to determine the complete sequence of an antibody with unparalleled speed and accuracy. The technique combines all the advantages of existing methods is called “Database Assisted Shotgun Sequencing” (DASS).

antibody sequencing

Antibody sequencing has now turned into a routine measurement that typically can be completed within 10 working days for IgGs. The method, on teh other hand, is applicable for all antibody formats, namely IgMs, fluorochrome conjugates, immobilized antibodies and mixtures. Creative Biolabs, an emerging qualified service provider in this field, showed their technique of antibody sequencing.

In the first step, the antibody is fragmented to peptides by a special technique, which creates up to 5000 different peptides per chain. This set of peptides will be analyzed by high end mass spectrometers to generate extensive sequence information. MS/MS spectra are then de Novo sequenced by the latest algorithms and matched against a database with related sequences. Special “in house” data mining tools allow Creative Biolabs to extract the sequence information from ten thousands of MS/MS spectra within hours.

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