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 polymeraseΦ29 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.