Please enable JavaScript.
Coggle requires JavaScript to display documents.
Second Generation DNA-Sequencing Technique - Illumina Genome Analyzer…
Second Generation DNA-Sequencing Technique - Illumina Genome Analyzer
Sanger Sequencing
.
process of selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication
targeted sequencing technique that uses oligonucleotide primers to seek out specific DNA region
retains an essential place in clinical genomics, evolutionary genetics and complicated disease analysis
Preferable over next generation sequencing for:
target smaller genomic regions during a larger range of samples
sequencing of variable regions.
genotyping of microsatellite markers
identify single disease-causing genetic variants
sequencing of single genes
cost-efficient sequencing of single samples
less error-prone than Next Generation Sequencing (in some cases).
Used for analysis purposes like:
to determine the sequences of many relatively small fragments of human DNA
used for the sequencing of individual pieces of DNA (fragments used in DNA cloning or PCR)
Third Generation Sequencing
Benefit
Long range DNA sequencing
Mapping technology DNA sequencing
Single molecule which geneate over 10000bp read or map over 1000000bp molecules
Improved analysis of genome stucture thus enable improve "split-read" analyses so that insertion, deletion, translocation and other can be more readily recognized
Produce more uniform coverage of the genome
By combining third generation sequencing and mapping technologies it is possible to form super- contigs ("scaffolds') can span entirely chromosome arms.
Have been used as
produce highly accurate dont move and hightly contiguous reconstruction of plant animal genomes enabling new insight into evolution and sequence diversity.
create detailed map of structure variation and phasing variants across large region of human chromosomes
the new technology have been used to fill many gaps in human reference genome bring many important to medical such as the human leukaocyte antigen (HLA)
Also widely used to study transcriptomes, recognizing thousand novel of isoform and gene fusion.
some allow direct measurement of epigenetic modification from single molecules, allowing for many new methytransferases to be discover and for the role of methylation from single molecules, allowing for many new methytransferases to be discover and for the role ofmethylation in pathagens to be better studied.
Example
Pacific bioscience (pacbio)
Single molecule real time (SMRT)
Illumina Tru- Seq Synthetic Long Read Technology
Oxford Nanopore Technologies Sequencing Platform
Detail about Technique-Illumina
Genome Analyzer.
For genetic analysis and functional genomics
To sequence DNA in large scale (>100 sequence cycles)
Design to generate data quickly and reduce time (>350 million reads per run)
Ready to use kit
Designed to fit in any lab
Techniques of Illumina Genome Analyzer System
Sequencing Process
Sample preparation (6 hours)
Genomic DNA were cut
Adapter ligation and denaturation
Cluster Generation (5 hours)
Samples applied to flow cell
Bridge amplification was performed
Flow cell clamped into Cluster Station
Sequencing by Synthesis (2-9 days)
Sequencing performed on forward strand
Regenerate reverse strand
Flow cell and pre-filled reagents placed on Genome Analyzer
Sequencing Paired Ends
Sequence strand is stripped off
Regenerate clusters and cleave forward strand > sequence reverse strand
Module was added for automated reagent
Second read prepared and sequenced while flow cell remains on Genome Analyzer
Data Analysis
Data transfer to automated analysis pipeline
Automated base calling
Paired-end alignment
Polymorphism detection
SNP detection & error masking
Disadvantages
Shorter read length sequencer
Different biases
Accuracy of sequence reads and quality values not yet well understood
Current IT infrastructure cannot handle the file sizes
Advantages
It is generated by bridge amplification on a glass surface that increase the density of DNA
It use reversible terminator chemistry to sequence up to 100million clonal DNA cluster
Each sequence is from a unique DNA molecule
Paired ends sequencing is possible
Second- Generation Sequencing
Principle
It helps in the faster diagnosis
helps in the identification of disease-causing mutations
Cheaper and faster alternative sequencing which can now sequence whole small genome in a day
millions of fragments of DNA from a single sample are accurately sequenced using iterative cycles of nucleotide extension
enables sequencing of large stretch of DNA base pairs, producing hundreds of gigabases of data in a single sequential run.
Did not infer nucleotide identify using radio- or fluroscently- labelled dNTPs or oligonucleotides
Uses luminescent mehtod for measuring pyrophospahte synthess
Consist of two- enzyme process where ATP sulfurylase is used to convert pyrophosphate into ATP
Then used as substrate for luciferase which produce light propotional to the amount of pyrophospate
Benefit
Generate unprecedented amounts of sequence data very rapidly and at low costs
Able to sequence a human genome in a few weeks
Short reads with higher assembly quality yield
Application
Whole- genome sequencing
Target resequencing
Transcriptome sequencing and identication of infectious agents
History/background of the technique
Illumina began offering single nucleotide polymorphism (SNP) genotyping services in 2001 and launched its first system, the Illumina BeadLab, in 2002, using GoldenGate Genotyping technology
In June 2009, Illumina announced the launch of their own Personal Full Genome Sequencing Service at a depth of 30X for $48,000 per genome, and a year later dropped the price to $19,500
This is still too expensive for true commercialization but the price will most likely decrease substantially over the next few years as they realize economies of scale and given the competition with other companies such as Complete Genomics and Knome. As of May 2011, Illumina reduced the price to $4,000
Illumina was founded in April 1998 by David Walt, Ph.D., Larry Bock, John Stuelpnagel, D.V.M., Anthony Czarnik, Ph.D., and Mark Chee, Ph.D
Illumina acquired Epicentre Biotechnologies, based in Madison, Wisconsin, on January 11, 2011
On January 25, 2012, Hoffmann-La Roche made an unsolicited bid to buy Illumina for $44.50 per share or about $5.7 billion. Roche tried other tactics, including raising its offer (to $51.00, for about $6.8 billion)
In 2014, the company announced a multi-million dollar product, HiSeqX Ten, that it forecast would provide large-scale whole-genome sequencing for $1,000/genome. The company claimed that forty such machines would be able to sequence more genomes in one year than had been produced by all other sequencers to date
Group Members
Niana Mun anak John (63516)
Suraya binti Shahidan (62663)
Vaenessa anak Noni @ Boni (63851)
Ernadia Elsie binti Lawrence Jaya (63189)
April Annastashia anak Daniel Subuh (63109)
Alvira Natasha anak Chundi (63077)
Fionna Urei Avit (63218)9
Anis binti Abdullah (63102)
Ahmad Irsyaduddin bin Lokhman (63031)
Mohammad Mirza Zulhilmi bin Mohd. Salleh (63361)