User:Bioskryber/sandbox/Primary Template Directed Amplification

Primary Template-directed Amplification (PTA) is a whole genome amplification technique used to amplify low picogram DNA into microgram quantity for downstream applications. PTA takes advantage of the high fidelity Φ 29 polymerase with strand displacement activity and generates amplification product from random primers annealed to the primary template. Exonuclease-resistant terminators are incorporated into the reaction to create smaller double-stranded amplification products that undergo limited subsequent amplification. This transforms the reaction from exponential into a quasilinear process with more of the amplification occurring from the primary template1.

Background

Whole genome amplification (WGA) is often required for unbiased sequencing of single cell, forensic, unculturable microbe samples or ancient genomic fragments. WGA methods such as Degenerate Oligonucleotide-primed Polymerase Chain Reaction (DOP-PCR)2,3, Multiple Replacement Amplification (MDA)4, Linear Amplification via Transposon Insertion (LIANTI)5 and Multiple Annealing and Looping Based Amplification Cycles (MALBAC)6 based methods provided useful tools for single cell WGS. However, each of these methods falls short either in genome coverage, coverage uniformity, cell to cell reproducibility or variant calling sensitivity and specificity. By directing and redirecting amplification to the original template through a quasi-linear amplification process, PTA method generates high quality data with >95% genome coverage and unprecedented coverage uniformity, minimized allelic skewing and allele drop out (ADO) rate with excellent cell to cell reproducibility.

Φ29 DNA polymerase

How PTA works.

Φ29 DNA polymerase is a bacteriophage enzyme with high processivity and fidelity. Φ29 DNA Polymerase has two globular domains, an N-terminal exonuclease domain and a C-terminal polymerase domain7.  Its N-terminal exonuclease domain carries out 3’–5' proofreading activity which help to reduce the amplification error rate to 1 in 106−107 bases, two magnitudes better than Taq polymerase8. Moreover, the isothermal reaction catalyzed by Φ29 DNA polymerase occurs at a moderate isothermal condition of 30 °C, forfeiting the requirement of a thermal cycler.

Reaction

PTA reaction initiates with the binding of random primers and Φ29 DNA polymerase to the genomic DNA. Complementary DNA molecules are synthesized through the strand displacing and polymerase activity of Φ29 DNA polymerase. Unlike the MDA method, PTA reactions take advantage of the exonuclease-resistant terminators to restrict the size of the double-stranded amplification products to a range of 100bp to 5kb, which generally do not undergo further amplification. As a result, amplification originates mostly from the primary template and error propagation from daughter amplicons during subsequent amplification is limited.  

Workflow

PTA workflow contains three easy steps: cell lysis, whole-genome amplification, bead-based cleanup of amplified DNA. The cell lysis and whole-genome amplification step takes less than 60 minutes to set up and includes a convenient 10-hour overnight amplification step. WGA products are converted to DNA libraries for multiplexed sequencing with no fragmentation required and are compatible with whole-genome sequencing, targeted sequencing, or multi-omics workflows.

Product quality

PTA products range from 100bp to 5kb with a typical yield of 1-4 µg of DNA from single cells with genome coverage >95%. Products have significantly lower error rate and allelic dropout rate compared to MDA method. PTA is extremely sensitive and can amplify picogram DNA, however, in a well-controlled environment, no template control (NTC) reactions only generates 0-50ng products, indicating a very clean background.

Advantages

PTA is a highly accurate WGA method that generates enough DNA for various downstream applications including multiplexed sequencing on Illumina® or other short-read platforms. The appropriate size distribution of PTA products enables a simplified library preparation workflow with no fragmentation required. PTA minimizes amplification errors that occur during secondary amplification of daughter molecules, making it a reliable tool in the single nucleotide variant (SNV) allele detection and copy number variant (CNV) calling.

Other advantages of PTA include but are not limited to:

·      Greater than 95% genome coverage

·      Excellent coverage uniformity with low ADO rate

·      Great cell to cell reproducibility

·      High sensitivity (up to 85%) and specificity (up to 99%) in SNV calling

·      Simple and scalable workflow

·      Compatible with single cells, multiple cells, and ultra-low input DNA samples

Limitations

PTA does not work with ctDNA or cells fixed with paraformaldehyde-based fixation methods.

Applications

PTA can be used in a wide range of applications involving accurate variant analysis (SNPs, indels, SNVs and CNVs) of single cells and ultra-low input samples that are required in Preimplantation Genetic Testing (PGT), Gene Editing, Cancer Genomics, Microbiome research and Forensic Sciences.

References

1, Veronica Gonzalez, Sivaraman Natarajan, Yuntao Xia, David Klein, Robert Carter, Yakun Pang, Bridget Shaner, Kavya Annu, Daniel Putnam, Wenan Chen, Jon Connelly, Shondra Pruett-Miller, Xiang Chen, John Easton, Charles Gawad (2020). Accurate Genomic Variant Detection in Single Cells with Primary Template-Directed Amplification. BioRxiv. https://doi.org/10.1101/2020.11.20.391961

2, L Zhang, X Cui, K Schmitt, R Hubert, W Navidi, and N Arnheim (1992). Whole genome amplification from a single cell: implications for genetic analysis. PNAS 89 (13) 5847-5851. PMID: 1631067 PMCID: PMC49394 DOI: 10.1073/pnas.89.13.5847

3, Vivian G. Cheung and Stanley F. Nelson (1996). Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA. PNAS 93 (25) 14676-14679. PMID: 8962113 PMCID: PMC26194 DOI: 10.1073/pnas.93.25.14676

4, Frank B. Dean, Seiyu Hosono, Linhua Fang, Xiaohong Wu, A. Fawad Faruqi, Patricia Bray-Ward, Zhenyu Sun, Qiuling Zong, Yuefen Du, Jing Du, Mark Driscoll, Wanmin Song, Stephen F. Kingsmore, Michael Egholm, and Roger S. Lasken (2002). Comprehensive human genome amplification using multiple displacement amplification. PNAS 99 (8) 5261-5266. PMID: 11959976 PMCID: PMC122757 DOI: 10.1073/pnas.082089499

5, Chongyi Chen, Dong Xing, Longzhi Tan, Heng Li, Guangyu Zhou, Lei Huang and X. Sunney Xie (2017). Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI). Science 356 (6334)189-194. PMID: 28408603 PMCID: PMC5538131 DOI: 10.1126/science.aak9787

6, Chenghang Zong, Sijia Lu, Alec R Chapman, X Sunney Xie (2012). Genome-Wide Detection of Single-Nucleotide and Copy-Number Variations of a Single Human Cell. Science 338 (6114)1622-1626. PMID: 23258894 PMCID: PMC3600412 DOI: 10.1126/science.1229164

7, Andrea J Berman, Satwik Kamtekar, Jessica L Goodman, José M Lázaro, Miguel de Vega, Luis Blanco, Margarita Salas, and Thomas A Steitz (2007). Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases. EMBO J. 26 (14) 3494–3505. PMCID: PMC1933411 PMID: 17611604 DOI: 10.1038/sj.emboj.7601780

8, Alexandra M de Paz, Thaddeus R Cybulski, Adam H Marblestone, Bradley M Zamft, George M Church, Edward S Boyden, Konrad P Kording, and Keith E J Tyo (2018) High-resolution mapping of DNA polymerase fidelity using nucleotide imbalances and next-generation sequencing. Nucleic Acids Res. 46(13): e78. PMCID: PMC6061839 PMID: 29718339 DOI: 10.1093/nar/gky296

External links

• https://bioskryb.com/