2min
Jan 11, 2024
UPDATE (April 5, 2024) - This preprint has now been published in Microbial Genomics, a journal from the Microbiology Society. The paper can be found at https://www.microbiologyresearch.org/content/journal/mgen/10.1099/mgen.0.001228.
ORIGINAL POST
With so many PCR enzymes available today, it can be difficult to know which works best for any specific application. Fortunately, a new preprint from scientists at the Wellcome Sanger Institute and the University of Cambridge offers much-needed information for scientists using next-generation sequencing (NGS) platforms.
The team ran a bake-off to evaluate the performance of more than 20 PCR enzymes in short-read and long-read sequencing, assessing a range of outcomes to help other researchers determine which enzymes would be best for their own work. The independent study included our repliQa HiFi ToughMix, so we were eager to see how it handled. If you don’t have time to read the preprint, here is the authors’ conclusion: “At the time of this report repliQa appears to give the best outcome for a variety of genomes and applications.”
The PCR enzymes were put through their paces and evaluated for coverage uniformity, speed, SNP calling, and more by Michael Quail, Craig Corton, James Uphill, Jacqueline Keane, and Yong Gu. The study, which was a follow-up to a previous analysis performed in 2011, clearly showed how much commercially available PCR enzymes have improved in the past decade. The ultimate goal, of course, is to reduce amplification bias as much as possible to ensure that NGS results are accurate and representative of native biology.
“The results presented demonstrate that there are distinct differences between PCR enzymes in the yield and evenness of amplification of fragments prior to sequencing,” the scientists report, noting that users must be extremely careful in choosing a high-performing enzyme for their NGS workflows to achieve reliable results. “Some enzymes work well, giving even coverage and low bias, in some situations, but few are capable of unbiased amplification of both GC and AT rich templates.”
The study spanned four microbial genomes with a range of GC content — Bordetella pertussis, Escherichia coli, Clostridioides difficile, and Plasmodium falciparum — as well as human DNA from the Genome in a Bottle collection. Sequencing was performed with platforms from Illumina and Pacific Biosciences to assess both short-read and long-read performance.
Our repliQa enzyme achieved top marks across the board. Here are several areas where it outperformed other products:
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Library yield
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Coverage uniformity for both GC-rich and AT-rich genomic regions, in both short-read and long-read sequencing
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Sensitivity and precision for SNP and indel detection
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Fastest amplification
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Long fragment amplification, with longest average subread lengths
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Most contiguous assemblies
Indeed, repliQa’s results for SNP and indel calling were so impressive that the authors call it the “enzyme of choice” for its superior performance. Overall, the scientists determined that using repliQa led to results that “mirrored closely” what would be expected with a PCR-free workflow.
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