Second Generation DNA-Sequencing - Polonator (References (SANGER…
Second Generation DNA-Sequencing -
:star: Beads bind to aminosaline coating of coverslip.
:star: Polymerization of acrylamide gel.
:star: Beads from ePCR mixed with acrylamide and poured into a teflon-masked microscope slide.
:star: Coverslips washed and treated with
to eliminate fluorescent contamination and allow covalent coupling of template DNA and beads.
:star: Seperate bead population by centrifugation.
:star: Anneal capture beads to emulsion PCR beads
:star: Bind capture oligonucleotides to capture beads.
:star: Bind forward PCR primer to
and perform emulsion PCR reaction. Beads are then recovered from the emulsion (Porreca
:star: Takes place within water droplet embedded within an oil solution.
:star: amplify the 135bp paired end-tag library molecules
Amplify Circular DNA
:star: Releases T30 fragment
:star: Newly generated circularized DNA digested by restriction enzyme
:STAR: Rolling circle replication
DNA repair II
:star: Results in a 135bp library molecules.
:star: Primers, FDV2 and RDV2 added on each ends
:star: Ligate genomic DNA in presence of adapter oligonucleotides, T30 (Porreca
DNA repair I
:star: DNA fragment also undergo
which adds an A to the 3' end of the sheared DNA.
:star: To make DNA ends blunt-ended with a phosphate group attached at the 5' which allows ligation of adapter oligonucleotides.
is performed to fix any damaged or incompatible edges.
:star: Genomic DNA sheared to desired size (Porreca
:star: After imaging, the array of annealed primer-fluorescent probe complex is chemically stripped and anchor primer is replaced. Fluorescently-tagged nonamers is introduced to sequence the adjacent base (Liu
:star: Anchor primers has four components that are labelled with one of four fluorophores with the help of T4 DNA, which corresponds to base type at the query position. During ligation, T4 DNA ligase is sensitive to mismatches on 3'-side of the gap which improves the accuracy of sequencing. (Liu
:star: Decodes base by single-base probe in nonanucleotides or nonamers. Fluorescent-tagged nonamers degenerated by selectively ligation to a series of anchor primers (Liu
:star: Employs sequencing-by-ligation approach using a randomly arrayed, bead-based, emulsion PCR to amplify DNA fragments for parallel sequencing (Zhang
Langmead, B. (2012). Introduction to second-generation sequencing (powewrpoint slides). Retrieved from University of Maryland, CMSC858B: Computational Systems Biology and Functional Genomics (Spring 2012)
Chen, F., Dong, M., Ge, M., Zhu, L., Ren, L., Liu, G., & Mu, R. (2013). The History and Advances of Reversible Terminators Used in New Generations of Sequencing Technology.
Genomics, Proteomics & Bioinformatics, 11
Schatz, M. C., Delcher, D. L., & Salzberg, S. L. (2018). Assembly of large genomes using second-generation sequencing.
Heather, J. M., & Chain, B. (2016). The sequence of sequencers: The history of sequencing DNA. Genomics, 107(1), 1-8
Bayés, M., Heath, S., Gut, I. G. (2011). Applications of second generation sequencing technologies in complex disorders. In J. F. Cryan, & A. Reif, (eds)
Behavioral neurogenetics. current topics in behavioral neurosciences
(pp 321-343). Berlin: Heidelberg: Springer
H. L., J. G., S. Y., M. N., S. M., S. G., . . . Schatz, M. C. (2016, April 13). Third-generation sequencing and the future of genomics. Retrieved May 9, 2018, from
SANGER SEQUENCING: GATC Biotech. (2018). Retrieved from
Porreca, G. J., Shendure, J., & Church, G. M. (2006). Polony DNA sequencing.
Current Protocols in Molecular Biology
Metzker, M.L. (2010). Sequencing technologies — the next generation. Nature Reviews Genetics, 11, 31-46.
Liu, L., Li, Y. H., Li, S. L., Yu, N., He, Y. M., Pong, R., . . . Law, M. (2012). Comparison of Next-Generation Sequencing systems.
Journal of Biomedicine and Biotechnology, 2012
Zhang, J., Chiodini, R., Badr, A., & Zhang, G. (2011). The impact of next-generation sequencing on genomics.
Journal of Genetics Genomics, 38
Janitz., M. (2008). Next generation genome sequencing: Toward personalized medicine. Berlin: Wiley-Blackwell.
Chances of false-positive Single Nucleotide Polymorphisms (SNP) detection rates
Inadequate coverage of the genome
Too much of raw data are produced and only a small proportion of raw data are useful
The non-uniform amplification could lower the efficiency of sequencing
Has shortest NGS read lengths
Users are required to maintain and quality control reagents
Able to decode the base by single-base probe in nanonucleotides (nanomers)
Establishing the basis of other sequencing chemistries,
including SOLiD sequencing
Very flexible technique with variable applications, protocols and reagents
Open-source nature to adapt alternative NGS chemistries
Least expensive platform in combining a high-performance instrument
Data Analysis of Polonator
Nucleotides position that is different will be write in a file.
Data will be mapped on reference genome and position consistent positions of nucleotide are recognised. (Janitz, 2008)
These files are processed to produce sequences for whole genome.
DNA sequencing for each base is generated as file.
Quality score is calculated for each base
The fluorescence intensity in four channels is read.
Match every bead on entire array with exactly the same bead for every position.
Images are aligned
Large amount of raw data are produced in form of 4 image
History & Background
By 2005, these early attempts had been overhauled to develop the existing polony sequencing technology.
Polony sequencing is a development of the polony technology from the late 1990s and 2000s.
Polony sequencing technique was first developed by Dr,George Church and his group at Havard Medical School.
Different sequencing platforms came to market and data produced by these new technologies mushroomed exponentially.
Therefore, second-generation sequencing technology appeared.
Dideoxy sequencing method that developed by Frederick Sanger er al. 1997 is high cost and low throughput inherited within the method had limited its application (Chen et al. 2013).
Differences between Sanger Sequencing, Second Generation Sequencing and Third Generation Sequencing
Third Generation Sequencing
-Oxford Nanopore Technologies Sequencing Platform
-Illumina Tru-Seq Synthetic Long Read Technology
-Single molecule real time (SMRT)
-Pacific bioscience (pacbio)
Have been used as:
-produce highly accurate de nove and highly contiguous reconstruction of plant animal genomes enabling new insight into evolution and sequence diversity
-create detailed map of structural variations and phasing variants across large region of human chromosomes
-the new technology have been used to fill many gaps in human reference genome and bring many important to medical such as the human leukocyte 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 methyltransferases to be discover and for the role of methylation in pathogens to be better studied.
-By combining third generation sequencing and mapping technologies it is possible to form super-contigs (“scaffolds”) can span entirely chromosome arms.
-Produce more uniform coverage of the genome
-Improved analysis of genome structure thus enable improve “split-read” analyses so that insertion, deletion, translocation and other can be more readily recognized.
-Single molecule which generate over 10000bp read or map over 100000bp molecules
-Mapping technology create a renaissance in high quality genome sequence
-Long range DNA sequencing
Second Generation Sequencing
Sequence throughput history
HiSeq 2000 sequencing 25 billion bp per day (2010)
GA IIx sequencing 5 billion bp per day (2009)
GA II sequncing 1.6 billion bp per day (2008)
Profiling epigenetic modifications
Transcriptome sequencing and identification of infectious agents.
Uses luminescent method for measuring pyrophosphate synthesis
Then used as substrate for luciferase which produce light propotional to the amount of pyrophosphate
Consist of two-enzyme process where ATP sulfurylase is used to convert pyrophosphate into ATP
Did not infer nucleotide identity using radio- or fluroscently-labelled dNTPs or oligonucleotides before visualising in electrophoresis
Generating millions of relatively short reads from amplified single DNA fragments using iterative cycles of nucleotide extensions
Short reads with higher assembly quality yield, approximate 100bp
Able to sequence a human genome in a few weeks
Generate unprecedented amounts of sequence data very rapidly and at low costs
Sanger sequencing is preferable over Next Generation Sequencing for:-
:white_flower:: Sequencing of single genes
:white_flower:: Cost-efficient sequencing of single samples
:white_flower:: Verification sequencing for site-directed mutagenesis or the presence of cloned inserts
:white_flower: In some cases, less error-prone than Next Generation Sequencig
Sanger DNA sequencing is widely used for analysis purposes like:-
:white_flower:Targeting smaller genomic regions during a larger range of samples
:white_flower:Sequencing of variable regions
:white_flower:Genotyping of microsatellite markers
:white_flower:Identifying single disease-causing genetic variants
The method has been extensively accustomed advance the sector of practical and comparative genomics, evolutionary genetics and complicated disease analysis
The process relies on the detection of labeled chain-terminating nucleotides that are incorporated by a DNA polymerase throughout the replication of a template.