Second Generation DNA-Sequencing - Polonator
Second Generation DNA-Sequencing -
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Data Analysis of Polonator
Large amount of raw data are produced in form of 4 image
Images are aligned
Match every bead on entire array with exactly the same bead for every position.
The fluorescence intensity in four channels is read.
Quality score is calculated for each base
DNA sequencing for each base is generated as file.
These files are processed to produce sequences for whole genome.
Data will be mapped on reference genome and position consistent positions of nucleotide are recognised. (Janitz, 2008)
Nucleotides position that is different will be write in a file.
:star: Employs sequencing-by-ligation approach using a randomly arrayed, bead-based, emulsion PCR to amplify DNA fragments for parallel sequencing (Zhang
: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: 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: 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: Genomic DNA sheared to desired size (Porreca
DNA repair I
is performed to fix any damaged or incompatible edges.
:star: To make DNA ends blunt-ended with a phosphate group attached at the 5' which allows ligation of adapter oligonucleotides.
:star: DNA fragment also undergo
which adds an A to the 3' end of the sheared DNA.
:star: Ligate genomic DNA in presence of adapter oligonucleotides, T30 (Porreca
DNA repair II
:star: Primers, FDV2 and RDV2 added on each ends
:star: Results in a 135bp library molecules.
Amplify Circular DNA
:STAR: Rolling circle replication
:star: Newly generated circularized DNA digested by restriction enzyme
:star: Releases T30 fragment
:star: amplify the 135bp paired end-tag library molecules
:star: Takes place within water droplet embedded within an oil solution.
:star: Bind forward PCR primer to
and perform emulsion PCR reaction. Beads are then recovered from the emulsion (Porreca
:star: Bind capture oligonucleotides to capture beads.
:star: Anneal capture beads to emulsion PCR beads
:star: Seperate bead population by centrifugation.
:star: Coverslips washed and treated with
to eliminate fluorescent contamination and allow covalent coupling of template DNA and beads.
:star: Beads from ePCR mixed with acrylamide and poured into a teflon-masked microscope slide.
:star: Polymerization of acrylamide gel.
:star: Beads bind to aminosaline coating of coverslip.
History & Background
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).
Therefore, second-generation sequencing technology appeared.
Different sequencing platforms came to market and data produced by these new technologies mushroomed exponentially.
Polony sequencing technique was first developed by Dr,George Church and his group at Havard Medical School.
Polony sequencing is a development of the polony technology from the late 1990s and 2000s.
By 2005, these early attempts had been overhauled to develop the existing polony sequencing technology.
Users are required to maintain and quality control reagents
Has shortest NGS read lengths
The non-uniform amplification could lower the efficiency of sequencing
Too much of raw data are produced and only a small proportion of raw data are useful
Inadequate coverage of the genome
Chances of false-positive Single Nucleotide Polymorphisms (SNP) detection rates
Least expensive platform in combining a high-performance instrument
Open-source nature to adapt alternative NGS chemistries
Very flexible technique with variable applications, protocols and reagents
Establishing the basis of other sequencing chemistries,
including SOLiD sequencing
Able to decode the base by single-base probe in nanonucleotides (nanomers)
Differences between Sanger Sequencing, Second Generation Sequencing and Third Generation Sequencing
The process relies on the detection of labeled chain-terminating nucleotides that are incorporated by a DNA polymerase throughout the replication of a template.
The method has been extensively accustomed advance the sector of practical and comparative genomics, evolutionary genetics and complicated disease analysis
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
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
Second Generation Sequencing
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, approximate 100bp
Generating millions of relatively short reads from amplified single DNA fragments using iterative cycles of nucleotide extensions
Did not infer nucleotide identity using radio- or fluroscently-labelled dNTPs or oligonucleotides before visualising in electrophoresis
Uses luminescent method for measuring pyrophosphate synthesis
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 pyrophosphate
Transcriptome sequencing and identification of infectious agents.
Profiling epigenetic modifications
Sequence throughput history
GA II sequncing 1.6 billion bp per day (2008)
GA IIx sequencing 5 billion bp per day (2009)
HiSeq 2000 sequencing 25 billion bp per day (2010)
Third Generation Sequencing
-Long range DNA sequencing
-Mapping technology create a renaissance in high quality genome sequence
-Single molecule which generate over 10000bp read or map over 100000bp molecules
-Improved analysis of genome structure 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:
-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.
Also widely used to study transcriptomes, recognizing thousand novel of isoform and gene fusion
-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).
-create detailed map of structural variations and phasing variants across large region of human chromosomes
-produce highly accurate de nove and highly contiguous reconstruction of plant animal genomes enabling new insight into evolution and sequence diversity
-Pacific bioscience (pacbio)
-Single molecule real time (SMRT)
-Illumina Tru-Seq Synthetic Long Read Technology
-Oxford Nanopore Technologies Sequencing Platform