Translational Research | Case Studies
Long PCR for Cost-Effective, Efficient Targeted Next-Gen Sequencing
What happens when a good technique is paired with the right tools? Rapid progress. Just ask researchers in the laboratory of Dr. Kai Wang in the Keck School of Medicine at the University of Southern California in Los Angeles. We had an opportunity to talk with first author Haiying Jia, a visiting scientist in Dr. Wang’s laboratory, while she was attending the American Society of Human Genetics annual meeting in fall 2013. Within a few short months, the research group had published their findings on long-range PCR for NGS using PrimeSTAR GXL polymerase to amplify the entire BRCA1 (83.2 kb) and BRCA2 (84.2 kb) genes from nine subjects and sequence them with an Illumina® MiSeq® system (Jia et al., 2014).
Targeted Sequencing: A Powerful Technique That’s Not Just For Big Labs
Targeted sequencing (also referred to as targeted resequencing) uses the powerful capabilities of next-generation sequencing, or NGS, to interrogate a limited region of interest in a genome—a useful approach for translational researchers wishing to analyze a particular portion of genomic DNA. In a clinical setting, when a small region of the genome is known to contribute to establishment or progression of disease, sequencing of this region can be informative. Alternatively, ultra-deep sequencing of a region of interest across multiple individuals may uncover previously unknown rare variants.
The introduction of “personal” genome sequencers such as the MiSeq (Illumina) instrument has brought NGS approaches within reach of small to medium-sized laboratories. These research groups are at the leading edge of investigations that require maximum flexibility and rapid turnaround time. Reducing the need for reaction optimization is key to evaluating hypotheses quickly.
Proof of Concept: Polymerase Choice Matters
The authors used six different PCR polymerases from four different manufacturers in a trial experiment. Each polymerase was used according to the standard protocol recommended by the manufacturer for amplification of three BRCA1 amplicons with sizes of 12.9 kb, 9.7 kb and 5.8 kb (using the same primer sets for all enzymes). Amplicon Tm values were 54°C, 63.3°C, and 54.5°C, respectively. Of all the enzymes tested, PrimeSTAR GXL polymerase was the only one capable of amplifying all three products (Figure 1, panel D). It was also the only polymerase for which a time-saving two-step PCR protocol was used.
Figure 1. Gel electrophoresis of PCR products from the long-range PCR amplification by six enzymes. The expected product sizes were 12.9 kb (amplicon 1.1), 9.7 kb (amplicon 1.6), and 5.8 kb (amplicon 2.8). (A) and (B): Polymerase 1 (Three amplicons were amplified using amplicon-specific annealing temperature and extension time; see Materials and Methods of Jia et al.). (C): Polymerase 2 (Amplicons for Brca1.1 and 2.8 with similar Tm values were amplified). (D): PrimeSTAR GXL (Three amplicons were amplified using a unified two-step PCR protocol.). (E): Polymerase 4 (Amplicons for Brca1.1 and 2.8 were amplified). (F): Polymerase 5 and Polymerase 6 (Only the amplicon for Brca2.8 was amplified). Figure re-published from Jia et al., 2014 under Creative Commons license 4.0.
The Value of Drama-Free PCR Amplification
The less optimization of PCR conditions for different targets needed, the better. In this respect, the authors noticed a profound difference among the polymerases tested. Most of the enzymes tested required optimization for various targets. One required different cycling conditions for every target, while others failed to amplify product even after optimization was performed. PrimeSTAR GXL polymerase, however, was notably hassle-free. “The PrimeSTAR enzyme can use a unified two-step PCR condition to amplify all three targets, making experimental design and implementation for PCR much easier in real-world settings, as one single thermocycler can be used to amplify all targets simultaneously,” wrote the authors.
The ability to use two-step rather than three-step cycling conditions also saved thermal cycler instrument time. Emphasizing this point, the authors wrote, “In our experiment, we found that the Takara PrimeSTAR GXL DNA Polymerase can amplify all amplicons of BRCA1/2 without altering experimental conditions, which we believe is an key advantage of using this enzyme when resources such as thermocycler is a limiting factor in research and clinical settings.”
Not only was PrimeSTAR GXL polymerase the easiest to work with, it was the least expensive of all six polymerases tested.
Improving Targeted Sequencing Workflows: Sequencing BRCA1 and BRCA2
While trial experiments were helpful, researchers wanted to assess how amenable long-range PCR would be in a more in-depth study. To do so, they amplified the entire genomic regions of BRCA1 and BRCA2 from genomic DNA prepared from nine subjects. Eight of the subjects were controls (breast cancer-free), and the ninth was a patient with hereditary breast cancer. “Our goal is to evaluate if the experimental procedure can work consistently well among a group of samples and if a positive causal mutation can be identified reliably,” wrote the authors.
Long-range PCR with PrimeSTAR GXL polymerase was , in fact, successful (Figure 2). The authors wrote, “For all samples, we were able to generate all the BRCA1/2 amplicons successfully, all of which display a single band with the expected size, without non-specific bands or smear.”
Figure 2. Long-range PCR products produced for targeted sequencing analysis of BRCA1 and BRCA2 with PrimeSTAR GXL DNA Polymerase. Amplicons 1.1–1.9 cover BRCA1 and 2.1–2.8 cover BRCA2. Figure re-published from Jia et al. 2014 under Creative Commons license 4.0.
The amplicons were purified, and sequencing libraries were prepared and quantified. Nine normalized libraries were pooled and sequenced together in one run on the Illumina MiSeq platform. The authors wrote, “The average coverage on the target regions was 2261X (range: 1285X to 3583X), and 93.75% (range: 81.55% to 100.00%) of the target region had coverage of over 10 and 98% (range: 92.53% to 100%) of the target region was covered at least once.” An average of 234 SNVs were identified per sample, most being non-coding variants. A known disease-associated mutation (c.5946delT in BRCA2) was identified in the sample from the patient with hereditary breast cancer. Interestingly, a non-frameshift deletion of unknown significance was identified in a control sample.
The Use of Long-Range PCR in Targeted Sequencing
“When combined with sequencing, long-range PCR can achieve higher sensitivity and provide a faster and more cost effective tool for detecting genetic variations,” wrote Jia et al. Long-range PCR requires a polymerase that is capable of greater processivity than conventional enzymes.“These technical advances [in polymerases] have brought the speed and simplicity of PCR to genomic mapping and sequencing, and have facilitated studies in molecular genetics,” wrote the authors.
Of course, there are PCR-free alternatives for targeted sequencing workflows, such as custom capture arrays. Jia et al. used two different systems to design capture solutions for BRCA1/2, comparing the performance of the custom capture arrays to one another and to long-range PCR. They not only found reduced coverage of intron and exon regions for custom capture systems compared to “...the real-world performance of our long-range PCR method [which] showed that it can get up to 100.00% coverage, even in a multiplex sequencing scenario where uneven sequencing depth exists across samples,” but also noted that the custom capture technique cost four times as much as long-range PCR with PrimeSTAR GXL polymerase.
Concluding Thoughts: A Practical Approach to Targeted Sequencing
The authors concluded that long-range PCR has a valuable role in targeted sequencing, since “...it does not require customized design by commercial vendors, and can be afforded by small laboratories when a small number of samples and continuous regions (such as full gene region including introns) are of interest.” Their goal was to provide pragmatic information about using the method for a wide variety of research applications, including translational research. “Overall, this report provides a practical guide on how to use long-range PCR to perform NGS on large genomic regions, especially when the entire gene regions including introns are of interest.”