CSC Assignment1SequencingReview- 1109_Su N_NEXT_GENERATION_SEQUENCING.docx By Anonymous. Similarity Index

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1 Page 1 of 6 Document Viewer TurnitinUK Originality Report Processed on: 05-Dec-20 10:49 AM GMT ID: 13 Word Count: 1587 Submitted: 1 CSC Assignment1SequencingReview- 1109_Su N_NEXT_GENERATION_SEQUENCING.docx By Anonymous Similarity Index 55% Similarity by Source Internet Sources: 45% Publications: 35% Student Papers: 29% 16% match (Internet from 6/9/10) /Chung2009.pdf 15% match (Internet from 13/4/10) fileid=217 7% match (publications) Zhang, J.. "The impact of next-generation sequencing on genomics", Journal of Genetics and Genomics, % match (publications) Elena P. Ivanova. "Comparative Genomics of Pathogens", Infectious Disease Informatics, 2010

2 Page 2 of 6 4% match (Internet from 18/11/11) 2% match (Internet from 14/5/09) 1% match (publications) Xiaoguang Zhou. "The next-generation sequencing technology and application", Protein & Cell, 06/2010 1% match (student papers from 28/10/08) Submitted to The University of Manchester on % match (student papers from 05/12/11) Class: CSC8312 Assignment: Assignment1 Paper ID: % match (student papers from 14/01/11) Submitted to University of Abertay Dundee on % match (student papers from 06/12/10) Submitted to University of Newcastle upon Tyne on % match (student papers from 05/12/11) Class: CSC8312

3 Page 3 of 6 Assignment: Assignment1 Paper ID: < 1% match (Internet from 16/9/10) tid=2&pid= Introduction: Next Generation Sequencing Technology DNA sequencing technology has played a pivotal role in the advancement of molecular biology (Gilbert, 1980) (Xiaoguang Zhou et al,). The original sequencing methodology i.e. most commonly preferred Sanger s method which gave excellent accuracy and reasonable read length but very low throughput.after a series of technical innovations, the Sanger method has reached the capacity to read through bp. In order to sequence longer sections of DNA, a new approach called shotgun sequencing was developed during Human Genome Project (HGP). Next Generation Sequencing (NGS) techniques will provide radical insights and change the landscape of genomics.ngs reads DNA templates in a highly parallel manner to generate massive amounts of sequencing data but, the read length for each DNA template is very short ( bp) compared old methods. Therefore NGS technologies have increased the speed and throughput capacities of DNA sequencing and, as a result, dramatically reduced overall sequencing costs( Jun Zhanga et al, 2011). 2. Background: Student ID: ). The NGS technologies, represented by HiSeq 2000 from Illumina, SOLiD from Applied Biosystems, 454 from Roche and Heliscope single Molecule Sequencer technology from Helicos(Qin N et al, 2011) Methods: Illumina Solexa: In this process, one end of single DNA molecule is attached to a solid surface using an adapter; the molecules bend over and hybridize to complementary adapters.after the amplification step, a flow cell with more than 40 million clusters is produced, and each cluster contains approximately 1000 clonal copies of a single template molecule. The templates are sequenced in a massively parallel fashion using a DNA sequencing-by- synthesis approach that uses reversible terminators with removable fluorescent moieties and special DNA polymerases. In 2-3 days Illumina will generate 35-bp read. The arrival of the first practical massively parallel next generation sequencing (NGS) platform in 2005 marked the start of a revolution in genomic research. (Emma Meaburn and Reiner Schulz, 2011). Sanger sequencing is based on DNA polymerase- dependent synthesis of a complementary DNA strand in the presence of natural 2 deoxy nucleotides and 2, 3 di-deoxy nucleotides. (Olena Morozova and Marco A. Marra, Figure 1: Sequencing-by-synthesis with the Genome Analyzer (Illumina/Solexa): PCR clusters originating from a single fragment are sequenced by the addition of primers, fluorescently labelled reversible dntp terminators, and DNA polymerase. The fluorescent label is excited through a laser and the released signal is Next Generation

4 Page 4 of 6 Sequencing Technology recorded by a camera. After removing the fluorescent label and the terminal group another round of fluorescent labelled dntps and DNA polymerase are added to investigate the next base in the sequence. (Source: biochem218.stanford.edu/projects 2009/Chung 2009.pdf) SEQUENCING: The 454 technology, the first NGS technology released to the market, avoids the cloning requirement by using highly efficient and accurate in vitro DNA amplification method known as emulsion PCR. In emulsion PCR, individual DNA fragment-carrying streptavidin beads, are captured into separate emulsion droplets. The droplets act as individual amplification reactors, producing 10 7 clonal copies of a unique DNA template per bead. Each template that contains the bead is transferred into a well of a picotiter plate and the clonally related templates are analyzed using a pyrosequencing reaction. The current 454 platform marketed by Roche Applied Science is capable of generating Mb of sequence in bp reads in a 4-h run. Student ID: ABI /SOLiD: Massively parallel sequencing by hybridization ligation, implemented in the supported oligonucleotide ligation and detection system from Applied Biosystems, has recently become available. Construction of sequences for analysis on the SOLiD instrument begins with an emulsion PCR single-molecule amplification step. The amplification products are transferred onto a glass surface where sequencing occurs by sequential rounds of hybridization and ligation with 16 dinucleotide combinations labelled by 4 different fluorescent dyes. Using the 4 -dye encoding scheme, each position is effectively probed twice, and the identity of the nucleotide is determined by analysing the colour that results from 2 successive ligation reactions. The newly released SOLiD instrument is capable of producing 1 3 Gb of sequence data in 35bp reads per an 8 - day run. (OlenaMorozova, and Marco A. Marra, 2008) Figure 2: Sequencing-by-synthesis with the GS FLX 454 Life Sciences): During the incorporation of a nucleotide though polymerase activity, PPi is released and converted into ATP. Upon contact with the enzyme luciferase, light is released, which is recorded by a camera. (Source: biochem218.stanford.edu/projects 2009/Chun g2009.pdf) Figure 3. Sequencing-by-ligation with the SOLiD System (Applied Biosystems): A sequencing primer is annealed to the DNA fragment, followed by the hybridization and ligation of one of 16 possible di-base probes. Through excitation of the fluorescent label of the probe, one of four colors is recorded by a camera. After cleaving of the label and last three bases of the 8-base long probe, a new round of hybridization and ligation occurs, thus interrogating every fifth di-base. At the end all probes get removed and a new primer gets annealed to the sequence, Next Generation Sequencing Technology starting a new sequencing round. This process is repeated for five primers total, leading to the interrogation of each base twice. (source: /Chung 2009.pdf) 2.2. Application of the next generation sequencing: Next generation sequencing has a wide range of applications in the field of biology as genomics, identification of Disease-Causing Mutations in Autosomal Dominant Retinitis Pigmentosa (adrp), RNA sequencing, in clinical Diagnostics for Targeted Resequencing for Hypertrophic Cardiomyopathy, sequencing of Cancer transcriptomes, and modern genomics Identification of Disease-Causing Mutations in

5 Page 5 of 6 Autosomal Dominant Retinitis Pigmentosa (adrp) Using Next- Generation DNA Sequencing: The low-cost, high-throughput attributes make it particularly attractive and advantageous for use in highly heterogeneous monogenic diseases such as retinitis pigmentosa where the potential disease-causing genes and mutations are high, and many are still unknown. RP is highly heterogeneous in patient populations and has multiple patterns of inheritance. Next-generation sequencing was used to identify mutations in pairs of affected individuals from 21 families with autosomal dominant RP, which was selected from cohort of families without mutations in common RP genes amplicons which targeted 249,267 unique bases of 46 candidate genes were sequenced with the 454GS FLX Titanium and GAIIx Illumina Solexa platforms. The result showed an average sequence depth of 70 and 125 for the 454GS FLX and Student ID: GAIIx platforms, respectively. More than 9000 sequence variants were identified and analysed, 112 of these were selected as likely candidates and tested for segregation with di-deoxy capillary electrophoresis sequencing of additional family members and control subjects and 5 disease-causing mutations (24%) were identified in the 21 families. Thus this project demonstrates that next-generation sequencing is an effective approach for detecting novel, rare mutations causing heterogeneous monogenic disorders such as RP. (Sara J. Bowne et al, 2011). 3. Discussion: Next-generation sequencing technologies have found a broad applicability in functional genomics research.compared to Sanger sequencing, advantages of the nextgeneration technologies mentioned so far, including 454, Illumina Solexa and ABiSOLiD, alleviate the need for in vivo cloning by clonal amplification using either emulsion PCR (454/Roche and ABI/SOLiD) or bridge amplification on solid surface (Illumina/Solexa). 3.1 Challenges of next generation technologies to conventional sequencing technologies: The new technologies produce up to 30 GB of data through massively parallel sequencing within a few days and for a fraction of the cost compared to conventional sequencing methods. (Christina Bormann Chung, 2009). NGS has the potential and the ability to accelerate the biological and biomedical research, by enabling the comprehensive analysis of genomes, transcriptomes and interactomes to become inexpensive and Next Generation Sequencing Technology widespread, rather than requiring significant production-scale efforts compared to conventional technologies. The new techniques require specific training, skills a software (Jun Zhang et al, 2011). Thus NGS technologies are posing challenges to conventional sequencing technologies in terms of time, costs, read length, utility in application, labour requirement, results, software and sequence quality and various other research aspects. (Jay Shendure1 & Hanlee Ji, 2008) 4. CONCLUSIONS: Next generation sequencing are very vast in number, its applications in biology has revolutionised genomic research. It has numerous advantages and few disadvantages. There are many other unknown sequencing techniques which still are being researched. Some of the extensively used applications include Illumina Solexa, ABiSOLiD, 454 Pyrosequencing and Heliscope single Molecule Sequencer. The specific application mentioned above includes the use of NGS technology to identify the disease causing Mutations in Autosomal Dominant Retinitis Pigmentosa (adrp). Sanger s method was the most preferred method that was used, from the introduction of many sequencing techniques, until the existence of NGS, but it is still used in experiments to obtain certain results. When

6 Page 6 of 6 a comparative study between the NGS and conventional bioinformatics techniques is done the NGS is more time and cost effective. The very recent techniques developed in the later stages have posed a number of challenges in the next generation sequencing methods. REFERENCES: Student ID: Christina Bormann Chung, (16 march 2009). Mapping Short Sequencing Reads: Challenges & Solutions. Emma Meaburn and Reiner Schulz, (18 October 2011). Seminars in Cell & Developmental Biology. Next generation sequencing in epigenetics: Insights and challenges. Jun Zhanga et al, (20 March 2011). Journal of Genetics and Genomics. The impact of next generation sequencing on genomics, 38(3), Jay Shendure1 & Hanlee Ji, (2008). Nature Biotechnology. Next-generation DNA sequencing. 26, Olena Morozova, and Marco A. Marra, (November 2008). Genomics. Applications of next-generation sequencing technologies in functional genomics. 92(5), Qin N et al, (4 april 2011). Next- generation sequencing technologies and the application in microbiology, 51(4), PMID: (Link: ) Sara J. Bowne et al, (4 april 2011). Invest Ophthalmol Vis Sci. Identification of Disease-Causing Mutations in Autosomal Dominant Retinitis Pigmentosa (adrp) Using Next-Generation DNA Sequencing, 52(1), Xiaoguang Zhou et al, Protein & Cell, The nextgeneration sequencing technology and application, 1(6),

7 From: SuXXXX Sent: 12 December 20XX 18:06 To: Dr XXXX Subject: RE: assessment irregularity CSC8313 Dear Dr XXXX I am sorry for the problem and did not intend. This is first work for the masters. I am taiwan national and not sure how to do english work. I tried to write the work from the papers and do right. But I did not do right. I am sorry and full of remorse. i worried about disgrace. Forgive poor student. SuXXXX Notes: This was the student s first assignment. The student is international and has not previously studied in the UK. The assignment is for CSC8313 Bioinformatics (a 20 credit module) the assignment counts for 10% of the module mark.