Polony Sequencing: Principle, Steps, Uses, Diagram microbiologystudy

Polony sequencing is an open-source DNA sequencing technology that involves clonal amplification of DNA fragments into polymerase colonies called polonies.

It was developed by Dr. George Church and his team at Harvard Medical School. This method of sequencing is known for its cost-effectiveness and ability to produce highly accurate short reads which is ideal for applications like resequencing genomes and studying genetic variations.

This technique bridges the gap between traditional Sanger sequencing and modern high-throughput technologies by offering a low-cost yet accurate approach suitable for large-scale projects. With its high sensitivity, affordability, and open-source accessibility, polony sequencing has influenced the development of many modern high-throughput DNA sequencing methods.

An advanced variation of polony sequencing is the multiplex polony sequencing which builds on the foundations of classical polony sequencing. It is capable of handling more DNA templates simultaneously. This evolution improved the throughput and accuracy of the sequencing method. 

Principle of Polony Sequencing

Polony sequencing works on the principle of amplifying and analyzing DNA within a solid matrix or on beads to allow accurate and simultaneous sequencing of millions of fragments. This method involves creating tiny clusters of identical DNA molecules called polonies which are immobilized in a matrix and serve as templates for sequencing. Each polony represents a single DNA template, ensuring the amplified sequences remain distinct. This prevents mixing or contamination between DNA fragments which helps maintain high accuracy. Sequencing is then performed on these polonies using fluorescently labeled nucleotides to identify each base in the DNA. 

The process begins with constructing a paired-tag library from genomic DNA followed by clonal amplification on microbeads using emulsion PCR (ePCR). This amplification produces discrete polymerase colonies that serve as the basis for sequencing. The amplified beads are then enriched. The enriched beads are embedded in a polyacrylamide matrix, creating a two-dimensional array that allows simultaneous sequencing of millions of DNA templates. 

What are Polonies?

• Polonies, or polymerase colonies, are clusters of DNA molecules amplified from a single nucleic acid template. It is conceptually similar to bacterial colonies on an agar plate. 
• Polonies are composed of identical DNA copies derived from a single template molecule just like each bacterial colony originates from a single bacterium and contains identical cells. 
• These are amplified within an acrylamide gel matrix, providing distinct advantages over traditional in vivo cloning.

Process/Steps of Polony Sequencing

1. DNA Isolation and Library Construction

• Constructing a sequencing library for polony sequencing starts with isolating and preparing genomic DNA.
• Isolated genomic DNA is randomly broken into fragments of a specific size. These fragments undergo end repair to convert damaged ends to blunt-ended DNA which allows blunt-end ligation. They also undergo A-tailing which adds an adenine (A) nucleotide to the 3’ end of the DNA.
• The DNA fragments are then circularized by ligating them to synthetic oligonucleotide sequences containing specific enzyme recognition sites and sequencing primer sites. 
• The circularized DNA is amplified using rolling circle replication and digested into paired-end tags using a restriction enzyme. 
• Sequencing primers are ligated to the fragments and the library is loaded onto beads for amplification and sequencing.

2. Template Amplification using Emulsion PCR

• The DNA library is amplified using emulsion PCR which isolates individual DNA molecules within tiny droplets of water in oil. Streptavidin-coated beads with biotinylated forward primers are used for emulsion PCR (ePCR).
• Each droplet ideally contains a single bead and one DNA template molecule which allows individual DNA amplification without cross-contamination.
• PCR is performed within the emulsion droplets to generate millions of clonal DNA fragments bound to individual beads.
• After PCR amplification, the emulsion is broken and the amplified DNA is recovered on beads.
• Amplified beads are enriched to select those successfully coated with DNA by hybridizing them to larger, non-magnetic capture beads coated with complementary DNA sequences. 
• Finally, the beads are immobilized on a glass surface within the sequencing flow cell surface for sequencing. 

3. DNA Sequencing

• Polony sequencing initially used the sequencing-by-synthesis method however, it switched to the sequencing-by-ligation approach which is a significant improvement. 
• First, the DNA on the beads is made single-stranded. Then, an anchor primer is attached to the DNA strand and short fluorescently-labeled fragments called nonamers are ligated to the strand. 
• The fluorescence signals are imaged to identify the DNA sequence. Each base is associated with a unique fluorescent signal allowing the sequence at that position to be determined. 
• To handle large volumes of data, the process has been fully automated with systems including microscopes, flow cells, and high-speed cameras. 

4. Data Analysis

• The sequencing data which consists of millions of reads is processed using open-source software developed by the Church Lab. This converts fluorescent signals into readable DNA sequences.
• Each bead in the array must be tracked across all positions and fluorescence intensity is measured in four color channels for each base. 
• This data is processed to identify the base with the strongest signal. A quality score is assigned based on the accuracy of the signal. 
• Once the images are processed, the resulting sequences are compiled into a file and mapped to a reference genome. This file of individual reads is processed to generate the sequence for the entire genome of interest.

Advantages of Polony Sequencing

• Polony sequencing is known for its high throughput and accuracy in DNA sequencing while using easily available and cost-effective tools.
• Another significant advantage of polony sequencing is its open-access nature which provides freely available software, protocols, and reagent information. This makes it accessible and practical for a wide range of applications.
• It can process large amounts of DNA at once with high precision and low cost which makes it a powerful tool for genomic research and diagnostics.
• Unlike traditional sequencing methods such as Sanger sequencing which requires in vivo cloning and is prone to errors, polony sequencing uses entirely in vitro processes. This removes cloning artifacts, reduces costs, and increases efficiency.

Limitations of Polony Sequencing

• The preparation of polonies involves multiple steps which can be complex and time-consuming.
• Polony sequencing generates shorter reads compared to some modern sequencing technologies which makes it challenging to resolve repetitive regions or assemble complex genomes.
• The lack of uniformity in the amplification of individual targets reduces sequencing efficiency.
• PCR amplification step can introduce biases leading to uneven representation of sequences.
• It has a lower throughput compared to newer high-throughput technologies like Illumina or nanopore sequencing.

Applications of Polony Sequencing

• Polony sequencing is useful in resequencing by comparing a target genome to reference sequences. 
• Polony sequencing can be used in transcriptome analysis to measure gene expression levels. 
• Polony sequencing can be used in genotyping to identify specific genetic variants across genomes with high accuracy. This is useful in studying diseases and understanding genetic traits.
• Polony sequencing is useful in haplotyping to determine haplotypes which are groups of alleles inherited together. This is useful for studying genetic linkage and inheritance patterns. 
• Polony sequencing can sequence nucleic acids directly within fixed cells or tissue sections, preserving spatial information about gene expression. It is used for in situ sequencing.
• Polony sequencing can be used for digital karyotyping which involves mapping genome tags to identify chromosomal amplifications, deletions, and other structural variations.
• Polony sequencing is also applicable in oncology and other fields for identifying mutations in cancer genomes or rare variants in clinical samples.

References

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2. Church, G. M. (2000). Polony Sequencing: DNA sequencing technology and a computational analysis reveals chromosomal domains of gene expression. Retrieved from https://dspace.mit.edu/handle/1721.1/8797
3. Edwards, J. S. (2008). Polony Sequencing: History, Technology, and Applications. Next Generation Genome Sequencing, 57–76. https://doi.org/10.1002/9783527625130.ch5
4. Open source sequencing. (n.d.). Retrieved from https://arep.med.harvard.edu/Polonator/
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7. Shendure, J. A., Porreca, G. J., Church, G. M., Gardner, A. F., Hendrickson, C. L., Kieleczawa, J., & Slatko, B. E. (2011). Overview of DNA sequencing strategies. Current Protocols in Molecular Biology, 96(1). https://doi.org/10.1002/0471142727.mb0701s96