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Table of Contents
- Introduction to Polony sequencing
- Principle of Polony Sequencing
- What are Polonies?
- Process/Steps of Polony Sequencing
- Advantages of Polony Sequencing
- Limitations of Polony Sequencing
- Applications of Polony Sequencing
Introduction to Polony sequencing
Polony sequencing is an open-source DNA sequencing technology that amplifies DNA fragments into polymerase colonies, referred to as polonies.
This method was pioneered by Dr. George Church and his team at Harvard Medical School. It is renowned for its cost-effectiveness and ability to generate highly accurate short reads, making it particularly suited for applications such as genome resequencing and the study of genetic variations.
Polony sequencing serves as a bridge between traditional Sanger sequencing and modern high-throughput technologies. It offers a cost-efficient yet precise approach, ideal for large-scale projects. Its high sensitivity, affordability, and open-source nature have significantly influenced the development of many contemporary high-throughput DNA sequencing techniques.
A notable advancement of this method is multiplex polony sequencing, which builds upon the principles of classical polony sequencing. This enhanced version allows for the simultaneous handling of a larger number of DNA templates, thereby improving both the throughput and accuracy of the sequencing process.
Principle of Polony Sequencing
Polony sequencing operates on the principle of amplifying and analyzing DNA within a solid matrix or on beads, enabling the accurate and simultaneous sequencing of millions of fragments. This technique involves generating tiny clusters of identical DNA molecules, known as polonies, which are immobilized in a matrix and act as templates for sequencing. Each polony corresponds to a single DNA template, ensuring that the amplified sequences remain distinct. This separation prevents cross-contamination or mixing between DNA fragments, thereby maintaining high accuracy. Sequencing is performed directly on these polonies using fluorescently labeled nucleotides to identify each DNA base.
The process begins with the construction of a paired-tag library from genomic DNA, followed by clonal amplification on microbeads through emulsion PCR (ePCR). This step produces discrete polymerase colonies that form the foundation for sequencing. The amplified beads are subsequently enriched and embedded in a polyacrylamide matrix, creating a two-dimensional array. This array facilitates the simultaneous sequencing of millions of DNA templates with precision.
What are Polonies?
- Polonies, or polymerase colonies, are clusters of DNA molecules amplified from a single nucleic acid template, conceptually similar to bacterial colonies growing on an agar plate.
- Each polony consists of identical DNA copies derived from a single template molecule, much like a bacterial colony originates from a single bacterium and contains genetically identical cells.
- These DNA clusters are amplified within an acrylamide gel matrix, offering distinct advantages over traditional in vivo cloning methods.
Process/Steps of Polony Sequencing
1. DNA Isolation and Library Construction
- The process of constructing a sequencing library for polony sequencing begins with the isolation and preparation of genomic DNA.
- The isolated genomic DNA is randomly fragmented into pieces of a specific size. These fragments undergo end repair to convert any damaged ends into blunt-ended DNA, facilitating blunt-end ligation. Additionally, the fragments are subjected to A-tailing, which involves adding an adenine (A) nucleotide to the 3’ ends.
- Next, the DNA fragments are circularized by ligating them to synthetic oligonucleotide sequences containing enzyme recognition sites and sequencing primer sites. The circularized DNA is then amplified through rolling circle replication and subsequently digested into paired-end tags using a restriction enzyme.
- Finally, sequencing primers are ligated to the DNA fragments, and the library is loaded onto beads for amplification and sequencing.
2. Template Amplification using Emulsion PCR
- The DNA library is amplified through emulsion PCR (ePCR), a method that isolates individual DNA molecules within microscopic water-in-oil droplets. Streptavidin-coated beads with biotinylated forward primers are employed for ePCR.
- Each droplet ideally contains one bead and a single DNA template molecule, enabling the independent amplification of DNA without cross-contamination.
- PCR is performed within the droplets, generating millions of clonal DNA fragments bound to individual beads.
- After PCR, the emulsion is broken, and the amplified DNA is recovered on the beads. The beads are then enriched to select those successfully coated with DNA by hybridizing them to larger, non-magnetic capture beads coated with complementary DNA sequences.
- The enriched beads are immobilized on a glass surface within a sequencing flow cell for subsequent sequencing.
3. DNA Sequencing
- Polony sequencing originally employed the sequencing-by-synthesis method but later transitioned to sequencing-by-ligation, which offers improved accuracy.
- To begin, the DNA on the beads is denatured into single strands. An anchor primer is then attached to the DNA strand, and short fluorescently-labeled fragments called nonamers are ligated to the strand.
- The fluorescence signals from these nonamers are captured through imaging to determine the DNA sequence. Each base emits a unique fluorescent signal, enabling its identification at specific positions.
- The sequencing process has been fully automated to manage large volumes of data, utilizing advanced systems including microscopes, flow cells, and high-speed cameras.
4. Data Analysis
- The sequencing data, comprising millions of reads, is processed using open-source software developed by the Church Lab, which translates fluorescent signals into readable DNA sequences.
- Each bead in the array is tracked across all positions, and fluorescence intensity is measured in four color channels for each base. This data is analyzed to identify the base with the strongest signal, and a quality score is assigned based on the signal’s accuracy.
- After processing the images, the resulting sequences are compiled into a file and mapped to a reference genome. This compilation of individual reads is then processed to reconstruct the complete genome sequence of interest.
Advantages of Polony Sequencing
- Polony sequencing is highly regarded for its exceptional throughput and accuracy in DNA sequencing, utilizing readily available and cost-effective tools.
- One of its standout features is its open-access nature, offering freely available software, protocols, and reagent information. This accessibility makes it a practical and versatile option for various applications.
- The method is capable of processing large volumes of DNA with high precision and at a low cost, making it an invaluable tool for genomic research and diagnostics.
- Unlike traditional sequencing methods such as Sanger sequencing, which relies on in vivo cloning and is prone to errors, polony sequencing employs entirely in vitro processes. This eliminates cloning artifacts, reduces costs, and significantly improves efficiency.
Limitations of Polony Sequencing
- The preparation of polonies involves several steps, which can be intricate and time-intensive.
- Polony sequencing produces shorter reads compared to some modern sequencing technologies, making it difficult to resolve repetitive regions or assemble complex genomes.
- The process suffers from a lack of uniformity in the amplification of individual targets, which can reduce sequencing efficiency.
- The PCR amplification step can introduce biases, leading to an uneven representation of sequences in the final data.
- Additionally, polony sequencing has a lower throughput compared to newer high-throughput technologies such as Illumina and nanopore sequencing.
Applications of Polony Sequencing
- Polony sequencing is valuable for resequencing, allowing for the comparison of a target genome to reference sequences.
- It can be utilized in transcriptome analysis to measure gene expression levels accurately.
- In genotyping, polony sequencing is effective for identifying specific genetic variants across genomes with high precision, making it instrumental in studying diseases and understanding genetic traits.
- This technology is also useful in haplotyping, where it determines haplotypes—groups of alleles inherited together—providing insights into genetic linkage and inheritance patterns.
- Polony sequencing enables the direct sequencing of nucleic acids within fixed cells or tissue sections, preserving spatial information about gene expression. This application, known as in situ sequencing, is especially significant in spatial transcriptomics.
- It can also be applied in digital karyotyping, a process that maps genome tags to identify chromosomal amplifications, deletions, and other structural variations.
- Furthermore, polony sequencing is widely used in oncology and other fields to detect mutations in cancer genomes or identify rare variants in clinical samples.
References
- Castiblanco, J. (2013, July 18). A primer on current and common sequencing technologies. In: Anaya JM, Shoenfeld Y, Rojas-Villarraga A, et al. (Eds.), Autoimmunity: From Bench to Bedside [Internet]. Bogotá (Colombia): El Rosario University Press. Available at: https://www.ncbi.nlm.nih.gov/books/NBK459463/
- 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
- Edwards, J. S. (2008). Polony Sequencing: History, Technology, and Applications. In: Next Generation Genome Sequencing (pp. 57–76). https://doi.org/10.1002/9783527625130.ch5
- Open Source Sequencing. (n.d.). Retrieved from: https://arep.med.harvard.edu/Polonator/
- Polony Sequencing. (2022, November 15). Retrieved from: https://encyclopedia.pub/entry/34647
- Porreca, G. J., Shendure, J., & Church, G. M. (2006). Polony DNA sequencing. Current Protocols in Molecular Biology, 76(1). https://doi.org/10.1002/0471142727.mb0708s76
- 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
