Temperature effect on polymerase fidelity
Xue, Yuan; Braslavsky, Ido; Quake, Stephen (2021), Temperature effect on polymerase fidelity, Dryad, Dataset, https://doi.org/10.5061/dryad.76hdr7stv
The discovery of extremophiles helped enable the development of groundbreaking technology such as polymerase chain reaction. Temperature variation is often an essential step of these technology platforms, but the effect of temperature on the error rate of polymerases from different origins is under-explored. We applied high-throughput sequencing to profile the error rates of DNA polymerases from psychrophilic, mesophilic, and thermophilic origins with single-molecule resolution. We found that reaction temperature substantially increases substitution and deletion error rates of both psychrophilic and mesophilic DNA polymerases. Our motif analysis shows that the substitution error profiles cluster according to phylogenetic similarity of polymerases, not reaction temperature, thus suggesting that reaction temperature increases global error rate of polymerases independent of sequence context. Intriguingly, we also found that the DNA polymerase I of a psychrophilic bacteria exhibits higher polymerization activity than its mesophilic ortholog across all temperature ranges, including down to -19oC which is well below water's freezing temperature. Our results provide a useful reference for how reaction temperature, a crucial parameter of biochemistry, can affect DNA polymerase fidelity in organisms adapted to a wide range of thermal environments.
We purified homogeneous (< 10-9 single nucleotide polymorphism per base) pUC19 plasmid template from a single clone of NEB-5α (New England Biolabs) transformed with pUC19 grown in LB supplemented with ampicillin antibiotic. About 1 µg of purified pUC19 was co-digested with 10 units of SapI and NdeI at 37oC for 24 minutes. Digested pUC19 template fragment was purified twice with 0.35X (V:V) of Ampure XP beads. Concentration and purity of the co-digested pUC19 template was determined on Bioanalyzer using high-sensitivity dsDNA quantification kit. Applying UMI barcoding strategy to measure polymerase errors on single-molecule level has been reported previously by the Xie group25. We adapted their strategy to measure polymerase replication at different reaction temperatures. All reactions were conducted in a buffer made up of 10 mM MOPS pH 8.50, 30% glycerol, 1.5 mM MgCl2, 0.1 mg/mL BSA, 50 mM KCl unless otherwise stated. We used MOPS buffer instead of Tris-based buffer as it has a much smaller ΔpKa/ΔT scaling which minimizes changes in buffer acidity at different temperatures. We increased the UMI length to 15 nucleotides on both ends, effectively increasing the combinatorial space to 430 which minimizes the barcode collision rate between UMIs. After annealing UMI index primer to pUC19 template (100:1 molar ratio), we pre-incubated the reaction mixture in 96 well plate at the desired reaction temperature for 2 minutes prior to the addition of polymerase (volume of reaction mixture to volume of polymerase is 19:1). Reaction was quenched with 50 mM EDTA after giving sufficient time to complete the reaction. Reaction time for each polymerase and condition was determined using fluorometric activity assays and qPCR measurement. We then purified the reaction mixture with Ampure beads and synthesized the complementary strand using the reverse UMI primer with Q5 DNA polymerase in its native reaction buffer (NEB) by incubating at 72oC for 10 minutes which is followed by EDTA quenching. We performed a second round of DNA purification and quantified molecular concentration of the resulting products using qPCR. A total input of 20000 barcoded molecules from each reaction was amplified in two rounds of 13 PCR cycles, with Ampure XP purification between each successive round of amplification. Agilent Bioanalyzer dsDNA kit was used to assess purity of the amplified product.
Library was sequenced on MiSeq using V3 2 x 300 bp chemistry kit. Each read is sorted into groups according to reaction indices. Then each read is trimmed to minima of 150 length and Q score of 20 (Phred+33 quality score), ensuring that the composite reads will cover the entirety of template sequence. Reads with the same reaction indices are then grouped according to their UMI barcodes and the barcodes are then stripped. Reads originating from the same replicated molecule would share the same UMI barcode, thus consensus for each position was determined by 90% majority within the UMI family that shares the same base. Bases with less than 90% majority consensus may contain PCR-induced errors and thus are not included for downstream analysis. Consensus sequence with non-matching bases in the first 5 positions are removed to avoid analysis of mis-primed products. We observed that the apparent error rate for a polymerase decreased and plateaued as consensus number increased, and we picked a minimum of 5 or 10 consensus threshold for each molecule to improve accuracy in replication error calling. Consensus sequence from each UMI family is then aligned to the reference sequence using BWA-MEM26. Substitution, insertion, and deletion errors are determined by comparing the consensus sequence of each UMI molecule to the pUC19 reference sequence without counting ambiguous assignments.
John Templeton Foundation
Chan Zuckerberg Initiative
Hebrew University of Jerusalem