Category Archives: Uncategorized

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.

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 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.

1

212
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

12

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.

 

1

(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.

What Do You Know About Genomic Sequencing?

With the entry of the 21st century, Genomic sequencing has become one of the hottest research fields in biological world. The declining price of genomic sequencing has expanded the range of its application in the study of disease diagnosis and prediction. Despite of its importance, how much do you know about it?

genomics sequencing

What is genomic sequencing?

Genomic sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA. The advent of rapid genomic sequencing methods has greatly accelerated biological and medical research and discovery.

It is often compared to “decoding”, but a sequence is still very much in code. In a sense, a genome sequence is simply a very long string of letters in a mysterious language.

When you read a sentence, the meaning is not just in the sequence of the letters. It is also in the words those letters make and in the grammar of the language. Similarly, the human genome is more than just its sequence.

Imagine the genome as a book written without capitalization or punctuation, without breaks between words, sentences, or paragraphs, and with strings of nonsense letters scattered between and even within sentences.

Sequencing the genome doesn’t immediately lay open the genetic secrets of an entire species. Even with a rough draft of the human genome sequence in hand, much work remains to be done. Scientists still have to translate those strings of letters into an understanding of how the genome works: what the various genes that make up the genome do, how different genes are related, and how the various parts of the genome are coordinated. That is, they have to figure out what those letters of the genome sequence mean.

Why is genomic sequencing so important?

Sequencing the genome is an important step towards understanding it.

At the very least, the genome sequence will represent a valuable shortcut, helping scientists find genes much more easily and quickly. A genome sequence does contain some clues about where genes are, even though scientists are just learning to interpret these clues.

Scientists also hope that being able to study the entire genome sequence will help them understand how the genome as a whole works—how genes work together to direct the growth, development and maintenance of an entire organism.

Finally, genes account for less than 25 percent of the DNA in the genome, and so knowing the entire genome sequence will help scientists study the parts of the genome outside the genes. This includes the regulatory regions that control how genes are turned on an off, as well as long stretches of “nonsense” or “junk” DNA—so called because we don’t yet know what, if anything, it does.

images (2)

How do you sequence a genome?

The quick answer to this question is: in pieces. The whole genome can’t be sequenced all at once because available methods of DNA sequencing can only handle short stretches of DNA at a time.

So instead, scientists must break the genome into small pieces, sequence the pieces, and then reassemble them in the proper order to arrive at the sequence of the whole genome. Much of the work involved in sequencing lies in putting together this giant biological jigsaw puzzle.

There are two approaches to the task of cutting up the genome and putting it back together again. One strategy, known as the “clone-by-clone” approach, involves first breaking the genome up into relatively large chunks, called clones, about 150,000 base pairs (bp) long. Scientists use genome mapping techniques (discussed in further detail later) to figure out where in the genome each clone belongs. Next they cut each clone into smaller, overlapping pieces the right size for sequencing—about 500 BP each. Finally, they sequence the pieces and use the overlaps to reconstruct the sequence of the whole clone.

The other strategy, called “whole-genome shotgun” method, involves breaking the genome up into small pieces, sequencing the pieces, and reassembling the pieces into the full genome sequence.

More detailed genomic sequencing approaches will be introduced in the following posts.