Long-read NGS—also called third-generation sequencing—is capable of routinely generating read lengths over 20 kb. In comparison, traditional NGS sequencing technologies typically produce ~150–300 bp reads. These short reads are often inadequate for accurately characterizing repetitive regions, structural variants, and long stretches of homopolymers in the genome. While long-read NGS can provide accurate reads of these repetitive regions, PCR amplification of long and complex DNA is often a challenge. This limits the application of long-read sequencing and impedes any research that requires highly specific amplification of long, repetitive, or GC/AT-rich DNA.
To solve this problem, we developed a DNA polymerase optimized for high-specificity amplification of long sequences and GC/AT-rich sequences (up to 80% GC), and then we rigorously tested it.
Results
Ultra long-range amplification of PCR targets up to 53 kb
PrimeSTAR LS successfully amplified human genomic DNA (gDNA) targets ranging in length from 0.5 kb to 53 kb (Figure 1).
Figure 1. PrimeSTAR LS successfully amplifies target sequences from human gDNA ranging in length from 0.5─53 kb. No incomplete or nonspecific bands were observed. Lane M1: 1 kb DNA ladder. Lane 0.5 kb: p53 target. Lane 1 kb: DCLRE1A target #1. Lane 2 kb: DCLRE1A target #2. Lane 4 kb: DCLRE1A target #3. Lane 15 kb: β-globin target #1. Lane 24 kb: β-globin target #2. Lane 30 kb: β-globin target #3. Lane 40 kb: HBB target. Lane 53 kb: SLC30A9 target.
Compared to five major long-range PCR enzymes from other suppliers (Table 1), PrimeSTAR LS resulted in superior amplification of 52–53 kb targets, with more full-length product and fewer incomplete or nonspecific bands (Figure 2).
Table 1. Polymerase abbreviations
Abbreviation
Polymerase
PrimeSTAR LS
Takara Bio PrimeSTAR LongSeq DNA Polymerase
Comp. T
TOYOBO KOD One
Comp. R
Roche KAPA
Comp. N
NEB LongAmp
Comp. Q
Quantabio repliQa
Comp. I
Thermo (Invitrogen) Platinum
Figure 2. PrimeSTAR LS outperforms major long-range PCR enzymes for ultra long-range amplification. PCR was performed to amplify 52–53 kb targets from human gDNA. The PCR products were purified with magnetic beads and analyzed with the Femto Pulse system (Agilent Technologies) using 100 pg of purified sample to confirm amplification. Lane M: Agilent 165 kb DNA Ladder. Lane 1: PUM1 target (52 kb). Lane 2: SLC30A9 target (53 kb).
Amplification of GC/AT-rich targets
GC-rich templates are prone to forming secondary structures like hairpins and have higher melting temperatures. AT-rich regions destabilize the DNA double helix and have lower annealing temperatures. These characteristics often require the optimization of PCR reaction conditions. Multiplex PCR poses an additional challenge, as there may be a range of GC-rich and AT-rich targets.
Under standard reaction conditions, PrimeSTAR LS successfully amplified GC-rich targets with 65–66% GC (17–20 kb) and AT-rich targets with 65–66% AT (16–21 kb; Figure 3). Multiplex PCR with GC-rich and AT-rich targets also produced clean amplification using PrimeSTAR LS (Figure 4).
Figure 3. PrimeSTAR LS successfully amplifies GC/AT-rich targets. Even for GC- and AT-rich templates that are prone to nonspecific amplification, long-range amplification is possible with PrimeSTAR LS without special buffers or reaction conditions.
Figure 4. PrimeSTAR LS delivers clean and reliable amplification in GC/AT-rich multiplex PCR. Multiplex PCR was performed using two pairs of primers to simultaneously amplify a GC-rich target and an AT-rich target. Each primer was used at a final concentration of 0.2 µM. Amplification was confirmed using the 4200 TapeStation System (Agilent Technologies). Lane 1: 7 kb target (GC: 68%) and 21 kb target (AT: 65%). Lane 2: 7 kb target (GC: 68%). Lane 3: 21 kb target (AT: 65%). Lane M: Ladder. Lane 4: 8 kb target (AT: 66%) and 20 kb target (GC: 65%). Lane 5: 20 kb target (GC: 65%). Lane 6: 8 kb target (AT: 66%).
Temperature stability
To accommodate common laboratory delays and high-throughput workflows, it is essential for PCR reactions to maintain stability at 4℃ or room temperature for some time after all components have been added but before PCR cycling begins. This temperature stability is especially crucial when setting up PCR reactions with an automated liquid handler. PrimeSTAR LS maintained high specificity after prepared PCR reactions were stored at 4℃ for 17 hr, or at room temperature for 1 hr (Figure 5). PCR reactions for high AT targets demonstrated greater stability at room temperature than reactions for high GC targets.
Figure 5. High specificity maintained at 4℃ for 17 hr and at room temperature for 1 hr. PrimeSTAR LS suppresses nonspecific amplification even when reactions were kept at 4℃ for 17 hr or at room temperature for 1 hr, making it suitable for automated workflows.
Long-read NGS analysis with 20-plex PCR of repetitive regions
PCR amplification of repetitive DNA is often problematic. Repetitive regions are prone to forming hairpin loops, which promote dissociation of the polymerase and lead to incomplete fragments that serve as megaprimers.
PrimeSTAR LS was tested on 20 repeat expansion loci from PacBio’s PureTarget panel (Table 2). These human loci are associated with repeat expansion disorders (Ibañez et al. 2024), including Huntington’s disease and myotonic dystrophy.
Amplicons from multiplex PCR (10–12 kb) of these 20 repetitive DNA sequences were subjected to NGS library preparation using the Native Barcoding Kit 96 V14 (Oxford Nanopore Technologies) and analyzed using GridION (Oxford Nanopore Technologies). Amplicons produced with PrimeSTAR LS resulted in a higher percentage of on-target primary reads than amplicons from other major long-range PCR enzymes (Figure 6). Further analysis revealed excellent sequencing coverage and good depth uniformity for amplicons from PrimeSTAR LS, while amplicons produced by other suppliers resulted in no sequencing coverage or extremely low depth for multiple targets (Figure 7).
With PrimeSTAR LS, it is possible to achieve uniform amplification with less bias and design optimization of multiplex reactions, so analysis with a small amount of sequencing can be expected.
Figure 6. High percentage of on-target primary reads using GridION platform. PrimeSTAR LS showed an on-target amplification rate of 99% or more, indicating that highly specific amplification was achieved even in long-range multiplex PCR.
Figure 7. Uniform sequencing coverage of 20-plex PCR amplicons using GridION platform. Panel A. NGS analysis confirmed that the multiplex products using PrimeSTAR LS (P) successfully amplified a full region of target sequences in all 20 targets uniformly, while other long-range PCR enzymes (N and T) resulted in no depth or extremely low depth for multiple targets. Panel B. Quantitative analysis of depth uniformity further confirmed that full-length sequences were obtained using PrimeSTAR LS for all 20 targets (in duplicate for n = 40), whereas other long-range PCR enzymes were only able to obtain full-length sequences for 53% to 68% of the targets. Increasing the number of sequencing reads did not significantly improve these results (data not shown).
Conclusions
PrimeSTAR LongSeq DNA Polymerase outperforms other long-range PCR enzymes for ultra long-range amplification of PCR targets up to 53 kb. High fidelity was maintained for (1) amplification of GC/AT rich targets, (2) multiplex PCR simultaneously targeting GC-rich and AT-rich targets, and (3) amplification of GC/AT rich targets after reactions were kept at 4℃ for 17 hr or room temperature for 1 hr. For multiplex PCR of 20 repetitive DNA sequences, PrimeSTAR LS produced excellent sequencing coverage and depth of uniformity, delivering notably better results than other commercially available long-range PCR enzymes using long-read NGS analysis with Oxford Nanopore Technologies.
Methods
All PCR was performed according to the relevant manufacturer’s instructions, with a template of human genomic DNA (100 ng/50 μl reaction), on the Clontech PCR Thermal Cycler GP (Cat. # WN400).
References
Ibañez, K. et al. Increased frequency of repeat expansion mutations across different populations. Nature Medicine30, 3357–3368 (2024).
