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Optimize your NGS: work with a polymerase that works
The rapid technological advances in Next Generation Sequencing (NGS) have allowed for tremendous strides in science and medicine, yet several challenges remain. A major challenge is achieving uniform genome coverage, as bias is inevitably present during library preparation. Of the steps that might introduce bias, PCR library amplification is perhaps the most troublesome due to heterogeneity in fragment size and GC content; PCR cycles can potentially amplify bias towards GC-neutral and smaller fragments. While PCR is the industry-established method for amplifying NGS libraries and genomic loci of interest, few polymerases have been developed to allow for unbiased amplification across varying fragment sizes and GC content.
With the expansion of applications for NGS, there is a need to evaluate and understand the viability of enzymes for unbiased PCR amplification. In recent work from Quail et al. 2022, the authors evaluated the performances of a selection of commercially available enzymes in PCR amplification and NGS analyses, including short-read Illumina library preparation and long fragment amplification prior to long-read sequencing.
Minimizing amplification bias
Quail and colleagues examined the ability of 20 PCR enzymes to amplify short read Illumina library fragments that varied in GC content from ~19% to 68%. With standardized templates, the authors calculated a “Low Coverage Index” (LCI), a measure of coverage uniformity and bias. Takara Ex Premier polymerase was one of only three enzymes among those evaluated that provided minimal bias across genomes with varying GC content. This uniform coverage was comparable to PCRfree libraries, the recommended preparation for minimal bias (although not practical due to high DNA mass input requirements). These results were further supported by sequencing data obtained from human genome template amplification, where Takara Ex Premier polymerase had some of the lowest LCI values, suggesting uniform genome coverage.
Detecting SNPs and indels without false negatives and with high sensitivity
Quail and colleagues further tested the accuracy and precision of human genome sequences obtained from each enzyme through SNP and indel detection. Takara Ex Premier polymerase was again highlighted here. Interestingly, the precision of this enzyme was found to be comparable to PCRfree datasets.
Amplifying long read sequencing
Another source of bias when using PCR in conjunction with NGS is long range PCR to generate material for long read sequencing. This is especially difficult in cases of low yield and a bias towards smaller fragments. The authors demonstrated these challenges by analyzing long DNA fragments of S. cerevisiae, where both 12 and 15-cycle PCR generated little to no product. However, a few enzymes were capable of long template amplification with good genome coverage, including Takara Bio’s Terra polymerase, which was among the enzymes that provided even genome coverage. Given these results, the authors conclude that better enzymes for long range PCR continue to be developed.
These data shown by Quail and colleagues highlight the importance of carefully selecting enzymes when amplifying NGS libraries. Of the commercially available enzymes, there is variation in performance, quality, and bias, and scientists should take care when selecting enzymes for their NGS analyses. For short read Illumina library fragment amplification, Takara Ex Premier polymerase is especially good for unbiased, highly sensitive, and precise results.
Quail M. A. et al. Identifying the best PCR enzyme for library amplification in NGS. bioRxiv 514486 [preprint] Nov 5, 2022 [cited May 3, 2023] Available from: https://doi.org/10.1101/2022.10.31.514486
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