Polony sequencing
Encyclopedia
Polony
Sequencing is an inexpensive but highly accurate multiplex sequencing technique that can be used to “read” millions of immobilized DNA sequences in parallel. This technique was first developed by Dr. George Church group in Harvard Medical School
. Unlike other sequencing technique, Polony sequencing technology is an open platform with freely downloadable, open source software and protocols. Also, the hardware of this technique can be easily set up with a commonly available epifluorescence microscope and a computer-controlled flowcell/ fluidics system. Polony sequencing is generally performed on paired-end Tags
library that each molecule of DNA template is of 135bp in length with two 17-18bp paired genomic tags separated and flanked by common sequences. The current read length of this technique is 26 bases per amplicon and 13 bases per tag, leaving a 4-5 bases gap in each tag.
Paired end-tag library construction
This protocol begins by randomly shearing the tested genomic DNA into a tight size distribution. The sheared DNA molecules are then subjected for the end repair and A-tailed treatment. The end repair treatment converts any damaged or incompatible protruding ends of DNA to 5’-phosphorylated and blunt-ended DNA, enabling immediate blunt-end ligation. While the A-tailing treatment adds an A to the 3’ end of the sheared DNA. DNA molecules with a length of 1kb are selected by loading on the 6% TBE PAGE gel. Next step, the DNA molecules are circularized with T-tailed 30bp long synthetic oligonucleotides (T30), which contains two outward-facing MmeI recognition sites, and the resulting circularized DNA undergoes rolling circle replication
. The amplified circularized DNA molecules are then digested with MmeI (type IIs restriction endonuclease) which will cuts at a distance from its recognition site, releasing the T30 fragment flanked by 17-18 bp tags (~70 bp in length). The paired-tag molecules need to be end-repaired prior to the ligation of ePCR (emulsion PCR) primer oligonucleotides (FDV2 and RDV2) to their both ends. The resulting 135 bp library molecules are size-selected and nick translated
. Lastly, amplify the 135bp paired end-tag library molecules with PCR to increase the amount of library material and eliminate extraneous ligation products in a single step. The resulted DNA template consists a 44bp FDV sequence, a 17-18 bp proximal tag, the T30 sequence, a 17-18 bp distal tag, and a 25 bp RDV sequence.
Template amplification
• Emulsion PCR
The Monosized, paramagnetic streptavidin
–coated beads are pre-loaded with dual biotin forward primer. Streptavidin
has a very strong affinity for biotin
, thus the forward primer will bind firmly on the surface of the beads. Next, an aqueous phase is prepared with the pre-loaded beads, PCR mixture, forward and reverse primers, and the paired end-tag library. This is mixed and vortexed with an oil phase to create the emulsion. Ideally, each droplet of water in the oil emulsion has one bead and one molecule of template DNA, permitting millions of non-interacting amplification within a milliliter-scale volume by performing PCR.
• Emulsion breaking
After amplification, the emulsion from preceding step is broken using isopropanol and detergent buffer (10mM Tris pH 7.5, 1mM EDTA pH 8.0, 100mM NaCl, 1% (v/v) Triton X‐100, 1% (w/v) SDS), following with a series of vortexing, centrifuging, and magnetic separation. The resulted solution is a suspension of empty, clonal and non-clonal beads, which arise from emulsion droplets that initially have zero, one or multiple DNA template molecules, respectively. The amplified bead could be enriched in the following step.
• Bead enrichment
The enrichment of amplified beads is achieved through hybridization to a larger, low density, non-magnetic polystyrene
beads that pre-loaded with a biotinylated capture oligonucleotides (DNA sequence that complementary to ePCR amplicon sequence). The mixture is then centrifuged to separate the amplified and capture beads complex from the unamplified beads. The amplified, capture bead complex has a lower density and thus will remain in the supernatant while the unamplified beads form a pellet. The supernatant is recovered and treated with NaOH which will break the complex. The paramagnetic amplified beads are separated from the non-magnetic capture beads by magnetic separation. This enrichment protocol is capable in enriching five times of amplified beads.
• Bead capping
The purpose of bead capping is to attach a “capping” oligonucleotide to the 3’ end of both unextended forward ePCR primers and the RDV segment of template DNA. The cap that being use is an amino group that prevents fluorescent probes from ligating to these ends and at the same time, helping the subsequent coupling of template DNA to the aminosilanated flow cell coverslip.
• Coverslip arraying
First, the coverslips are washed and aminosilane-treated, enabling the subsequent covalent coupling of template DNA on it and eliminating any fluorescent contamination. The amplified, enriched beads are mixed with acrylamide and poured into a shallow mold formed by a Teflon-masked microscope slide. Immediately, place the aminosilane-treated coverslip on top of the acrylamide gel and allow to polymerize for 45 minutes. Next, invert the slide/coverslip stack and remove the microscope slide from gel. The silane treated coverslips will bind covalently to the gel while the Teflon on the surface of microscope slide will enable the better removal of slide from the acrylamide gel. The coverslips then bonded to the flow cell body and any unattached beads will be removed.
DNA sequencing
The biochemistry of Polony sequencing mainly rely on the discriminatory capacities of polymerases and ligases. First, a series of anchor primers are flowed through the cells and hybridize to the synthetic oligonucleotide sequences at the immediate 3’ or 5’ end of the 17-18bp proximal or distal genomic DNA tags. Next, an enzymatic ligation reaction of the anchor primer to a population of degenerate nonamers that are labeled with fluorescent dyes is performed.
Differentially labeled nonamers:
5' Cy5‐NNNNNNNNT
5' Cy3‐NNNNNNNNA
5' TexasRed‐NNNNNNNNC
5' 6FAM‐NNNNNNNNG
The fluorophore-tagged nonamers selectively ligate onto the anchor primer, providing a fluorescent signal that indicates whether there is an A, C, G, or T at the query position on the genomic DNA tag. After four colour imaging, the anchor primer/nonamer complexes are stripped off and a new cycle is begun by replacing the anchor primer. A new mixture of the fluorescently tagged nonamers is introduced, for which the query position is shifted one base further into the genomic DNA tag.
5' Cy5‐NNNNNNNTN
5' Cy3‐NNNNNNNAN
5' TexasRed‐NNNNNNNCN
5' 6FAM‐NNNNNNNGN
Seven bases from the 5’ to 3’ direction and six bases from the 3’ end could be queried in this fashion. The ultimate result is a read length of 26 bases per run (13 bases from each of the paired tags) with a 4 to 5 bases gap in the middle of each tag.
(bacterial artificial chromosome) and bacterial genome resequencing, as well as SAGE (serial analysis of gene expression) tag and barcode sequencing. Furthermore, the polony sequencing technique is emphasized as an open system that shares everything including the software that have been developed, protocol and reagents.
However, although the raw data acquisition could be achieved as high as 786 gigabits but only 1 bit of information out of 10,000 bits collected is useful. Another challenge of this technique is the uniformity of the relative amplification of individual targets. The non-uniform amplification could lower the efficiency of sequencing and posted as the biggest obstacle in this technique.
G.007, based on polony sequencing technique. The current selling price of this machine is around US$ 170,000.
According to the calculation in year 2005, every kilobase of generated raw sequence was estimated to $0.11, while omitting the paired-end tag library construction cost, the cost of every kilobase of sequence could drops to $0.08.
technology which mainly developed by Dr. Rob Mitra. Together with a MD PhD student, Jay Shendure, Dr. Rob Mitra worked out ways to sequence in situ polonies using single-base extension which can achieved 5-6 bases reads. However, the existing polony sequencing technology was mainly developed by Jay Shendure and Greg Porreca. They have changed almost everything that was there in order to make this multiplex sequencing technology work.
Also, the highly parallel sequencing-by-ligation method of polony sequencing has contributed in forming the basis for ABI Solid Sequencing
and others.
Polony
Polony is a contraction of "polymerase colony," a small colony of DNA.Polonies are discrete clonal amplifications of a single DNA molecule, grown in a gel matrix. This approach greatly improves the signal-to-noise ratio. Polonies can be generated using several techniques that include solid-phase...
Sequencing is an inexpensive but highly accurate multiplex sequencing technique that can be used to “read” millions of immobilized DNA sequences in parallel. This technique was first developed by Dr. George Church group in Harvard Medical School
Harvard Medical School
Harvard Medical School is the graduate medical school of Harvard University. It is located in the Longwood Medical Area of the Mission Hill neighborhood of Boston, Massachusetts....
. Unlike other sequencing technique, Polony sequencing technology is an open platform with freely downloadable, open source software and protocols. Also, the hardware of this technique can be easily set up with a commonly available epifluorescence microscope and a computer-controlled flowcell/ fluidics system. Polony sequencing is generally performed on paired-end Tags
Paired-end Tags
Paired-end tags, also known as PET, refer to the short sequences at the 5’ and 3’ ends of the DNA fragment of interest, which can be a piece of genomic DNA or cDNA. These short sequences are called tags or signatures because, in theory, they should contain enough sequence information to be uniquely...
library that each molecule of DNA template is of 135bp in length with two 17-18bp paired genomic tags separated and flanked by common sequences. The current read length of this technique is 26 bases per amplicon and 13 bases per tag, leaving a 4-5 bases gap in each tag.
Workflow
The protocol of Polony sequencing can be break into three main parts which are the paired end-tag library construction, template amplification and DNA sequencing.Paired end-tag library construction
This protocol begins by randomly shearing the tested genomic DNA into a tight size distribution. The sheared DNA molecules are then subjected for the end repair and A-tailed treatment. The end repair treatment converts any damaged or incompatible protruding ends of DNA to 5’-phosphorylated and blunt-ended DNA, enabling immediate blunt-end ligation. While the A-tailing treatment adds an A to the 3’ end of the sheared DNA. DNA molecules with a length of 1kb are selected by loading on the 6% TBE PAGE gel. Next step, the DNA molecules are circularized with T-tailed 30bp long synthetic oligonucleotides (T30), which contains two outward-facing MmeI recognition sites, and the resulting circularized DNA undergoes rolling circle replication
Rolling circle replication
Rolling circle replication describes a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids...
. The amplified circularized DNA molecules are then digested with MmeI (type IIs restriction endonuclease) which will cuts at a distance from its recognition site, releasing the T30 fragment flanked by 17-18 bp tags (~70 bp in length). The paired-tag molecules need to be end-repaired prior to the ligation of ePCR (emulsion PCR) primer oligonucleotides (FDV2 and RDV2) to their both ends. The resulting 135 bp library molecules are size-selected and nick translated
Nick translation
Nick translation was developed in 1977 by Rigby and Paul Berg. It is a tagging technique in molecular biology in which DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labeled analogues, creating a tagged DNA sequence which can be used as a probe in...
. Lastly, amplify the 135bp paired end-tag library molecules with PCR to increase the amount of library material and eliminate extraneous ligation products in a single step. The resulted DNA template consists a 44bp FDV sequence, a 17-18 bp proximal tag, the T30 sequence, a 17-18 bp distal tag, and a 25 bp RDV sequence.
Template amplification
• Emulsion PCR
The Monosized, paramagnetic streptavidin
Streptavidin
Streptavidin is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin . With a dissociation constant on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest...
–coated beads are pre-loaded with dual biotin forward primer. Streptavidin
Streptavidin
Streptavidin is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin . With a dissociation constant on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest...
has a very strong affinity for biotin
Biotin
Biotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
, thus the forward primer will bind firmly on the surface of the beads. Next, an aqueous phase is prepared with the pre-loaded beads, PCR mixture, forward and reverse primers, and the paired end-tag library. This is mixed and vortexed with an oil phase to create the emulsion. Ideally, each droplet of water in the oil emulsion has one bead and one molecule of template DNA, permitting millions of non-interacting amplification within a milliliter-scale volume by performing PCR.
• Emulsion breaking
After amplification, the emulsion from preceding step is broken using isopropanol and detergent buffer (10mM Tris pH 7.5, 1mM EDTA pH 8.0, 100mM NaCl, 1% (v/v) Triton X‐100, 1% (w/v) SDS), following with a series of vortexing, centrifuging, and magnetic separation. The resulted solution is a suspension of empty, clonal and non-clonal beads, which arise from emulsion droplets that initially have zero, one or multiple DNA template molecules, respectively. The amplified bead could be enriched in the following step.
• Bead enrichment
The enrichment of amplified beads is achieved through hybridization to a larger, low density, non-magnetic polystyrene
Polystyrene
Polystyrene ) also known as Thermocole, abbreviated following ISO Standard PS, is an aromatic polymer made from the monomer styrene, a liquid hydrocarbon that is manufactured from petroleum by the chemical industry...
beads that pre-loaded with a biotinylated capture oligonucleotides (DNA sequence that complementary to ePCR amplicon sequence). The mixture is then centrifuged to separate the amplified and capture beads complex from the unamplified beads. The amplified, capture bead complex has a lower density and thus will remain in the supernatant while the unamplified beads form a pellet. The supernatant is recovered and treated with NaOH which will break the complex. The paramagnetic amplified beads are separated from the non-magnetic capture beads by magnetic separation. This enrichment protocol is capable in enriching five times of amplified beads.
• Bead capping
The purpose of bead capping is to attach a “capping” oligonucleotide to the 3’ end of both unextended forward ePCR primers and the RDV segment of template DNA. The cap that being use is an amino group that prevents fluorescent probes from ligating to these ends and at the same time, helping the subsequent coupling of template DNA to the aminosilanated flow cell coverslip.
• Coverslip arraying
First, the coverslips are washed and aminosilane-treated, enabling the subsequent covalent coupling of template DNA on it and eliminating any fluorescent contamination. The amplified, enriched beads are mixed with acrylamide and poured into a shallow mold formed by a Teflon-masked microscope slide. Immediately, place the aminosilane-treated coverslip on top of the acrylamide gel and allow to polymerize for 45 minutes. Next, invert the slide/coverslip stack and remove the microscope slide from gel. The silane treated coverslips will bind covalently to the gel while the Teflon on the surface of microscope slide will enable the better removal of slide from the acrylamide gel. The coverslips then bonded to the flow cell body and any unattached beads will be removed.
DNA sequencing
The biochemistry of Polony sequencing mainly rely on the discriminatory capacities of polymerases and ligases. First, a series of anchor primers are flowed through the cells and hybridize to the synthetic oligonucleotide sequences at the immediate 3’ or 5’ end of the 17-18bp proximal or distal genomic DNA tags. Next, an enzymatic ligation reaction of the anchor primer to a population of degenerate nonamers that are labeled with fluorescent dyes is performed.
Differentially labeled nonamers:
5' Cy5‐NNNNNNNNT
5' Cy3‐NNNNNNNNA
5' TexasRed‐NNNNNNNNC
5' 6FAM‐NNNNNNNNG
The fluorophore-tagged nonamers selectively ligate onto the anchor primer, providing a fluorescent signal that indicates whether there is an A, C, G, or T at the query position on the genomic DNA tag. After four colour imaging, the anchor primer/nonamer complexes are stripped off and a new cycle is begun by replacing the anchor primer. A new mixture of the fluorescently tagged nonamers is introduced, for which the query position is shifted one base further into the genomic DNA tag.
5' Cy5‐NNNNNNNTN
5' Cy3‐NNNNNNNAN
5' TexasRed‐NNNNNNNCN
5' 6FAM‐NNNNNNNGN
Seven bases from the 5’ to 3’ direction and six bases from the 3’ end could be queried in this fashion. The ultimate result is a read length of 26 bases per run (13 bases from each of the paired tags) with a 4 to 5 bases gap in the middle of each tag.
Analysis and software
The polony sequencing generates millions of 26 reads per run and this information needed to be normalized and converted to sequence. These can be done by the software that has been developed by Church Lab. All of the software is free and could be downloaded from the website. http://arep.med.harvard.edu/Polonator/Strength and Weaknesses
Polony sequencing allows for a high throughput and high consensus accuracies of DNA sequencing based on a commonly available, inexpensive instrument. Also, it is a very flexible technique that enables variable application including BACBAC
- Arts and entertainment :* Batman: Arkham City, a 2011 video game* Battersea Arts Centre, London, England, United Kingdom* Benedicta Arts Center, St...
(bacterial artificial chromosome) and bacterial genome resequencing, as well as SAGE (serial analysis of gene expression) tag and barcode sequencing. Furthermore, the polony sequencing technique is emphasized as an open system that shares everything including the software that have been developed, protocol and reagents.
However, although the raw data acquisition could be achieved as high as 786 gigabits but only 1 bit of information out of 10,000 bits collected is useful. Another challenge of this technique is the uniformity of the relative amplification of individual targets. The non-uniform amplification could lower the efficiency of sequencing and posted as the biggest obstacle in this technique.
Cost
The sequencing instrument used in this technique could be set up by the commonly available fluorescence microscope and a computer controlled flowcell. According to the calculation in the year of 2005, the set up of a complete set of instrument will cost around US$ 130,000. However, the cost could be further lower to US$ 100,000 in the near future. A biotech company, Dover, is in collaboration with the Church Laboratory of Harvard Medical School to produce an automated sequencing machine, PolonatorPolonator
The Polonator is an automated, second-generation genome sequencing machine that is based on polony sequencing technology developed by George Church's group at Harvard Medical School. It will be used for the PGP , and is available for sale at a price well below that of other second generation...
G.007, based on polony sequencing technique. The current selling price of this machine is around US$ 170,000.
According to the calculation in year 2005, every kilobase of generated raw sequence was estimated to $0.11, while omitting the paired-end tag library construction cost, the cost of every kilobase of sequence could drops to $0.08.
History
The polony sequencing is a “distant relative” of the classical polonyPolony
Polony is a contraction of "polymerase colony," a small colony of DNA.Polonies are discrete clonal amplifications of a single DNA molecule, grown in a gel matrix. This approach greatly improves the signal-to-noise ratio. Polonies can be generated using several techniques that include solid-phase...
technology which mainly developed by Dr. Rob Mitra. Together with a MD PhD student, Jay Shendure, Dr. Rob Mitra worked out ways to sequence in situ polonies using single-base extension which can achieved 5-6 bases reads. However, the existing polony sequencing technology was mainly developed by Jay Shendure and Greg Porreca. They have changed almost everything that was there in order to make this multiplex sequencing technology work.
Also, the highly parallel sequencing-by-ligation method of polony sequencing has contributed in forming the basis for ABI Solid Sequencing
ABI Solid Sequencing
SOLiD is a next-generation sequencing technology developed by Life Technologies and has been commercially available since 2008. These next generation technologies generate hundreds of millions to billions of small sequence reads at one time...
and others.