DOI: 10.5061/dryad.76hdr7stv
title: Temperature effect on polymerase fidelity

description: 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.
description: We purified
 homogeneous (&lt; 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.