2Victorian Life Sciences Computation Initiative, The University of Melbourne, Victoria, Australia
3Department of Computing and Information Systems, The University of Melbourne, Victoria, Australia
Current methods for targeted massively parallel sequencing (MPS) have several drawbacks, including limited design flexibility, expense, and protocol complexity, which restrict their application to settings involving modest target size and requiring low cost and high throughput. To address this, we have developed Hi-Plex, a PCR-MPS strategy intended for high-throughput screening of multiple genomic target regions that integrates simple, automated primer design software to control product size. Featuring permissive thermocycling conditions and clamp bias reduction, our protocol is simple, cost- and time-effective, uses readily available reagents, does not require expensive instrumentation, and requires minimal optimization. In a 60-plex assay targeting the breast cancer predisposition genes PALB2 and XRCC2, we applied Hi-Plex to 100 ng LCL-derived DNA, and 100 ng and 25 ng FFPE tumor-derived DNA. Altogether, at least 86.94% of the human genome-mapped reads were on target, and 100% of targeted amplicons were represented within 25-fold of the mean. Using 25 ng FFPE-derived DNA, 95.14% of mapped reads were on-target and relative representation ranged from 10.1-fold lower to 5.8-fold higher than the mean. These results were obtained using only the initial automatically-designed primers present in equal concentration. Hi-Plex represents a powerful new approach for screening panels of genomic target regions.
The advent of massively parallel sequencing (MPS) has heralded a new era for genetic testing (reviewed in (1, 2). There are a number of potential applications for MPS, including its use on genomic regions selectively captured from a DNA sample before sequencing (targeted MPS). Whole-genome sequencing involves considerably higher costs and lower throughput. Targeted MPS is an attractive approach for the screening of large panels of genes or genomic regions, both in research and diagnostic settings. In candidate gene sequence variant discovery projects for instance, targeted MPS provides an alternative to the more costly, time-consuming, and lower-throughput approaches previously used in large-scale case-control mutation screening studies (3, 4). Familial genetic testing for high penetrance cancer genes, such as BRCA1 and BRCA2, has also benefited from the significant cost reduction offered by targeted MPS (5).
Hi-Plex is a streamlined, highly-multiplexed PCR approach for targeted massively parallel sequencing. Our method integrates simple, automated primer design software and simple protocols, requires minimal optimization, does not rely on expensive instrumentation, and uses low-cost, readily available reagents to perform cost-effective and rapid sequencing.
Targeted MPS methods are generally based on PCR amplification or hybridization capture approaches (6-8). Commonly used performance measures for these methods are: (i) percentage of target bases represented by one or more sequence reads (coverage); (ii) percentage of sequences that map to the intended target (on-target); (iii) variability in sequence coverage across target regions (uniformity); (iv) cost; (v) ease of use; and (vi) amount of input DNA required per experiment or per megabase of target. Although unanimously recognized as a highly sensitive, specific, and uniform approach for targeted MPS, PCR-based MPS in its current form also has limitations relating to cost, throughput, and the ability to multiplex to a useful degree. Simultaneous production of many amplicons can lead to differential production of amplicons or nonspecific or failed amplification. Additionally, many PCR-MPS methods require labor-intensive normalization to achieve equimolar pooling of separate PCR products prior to sequencing. Commercial solutions have been developed by several manufacturers recently for library building based on high-multiplex PCR (high-plex PCR), for example, Ion Ampliseq (Life Technologies) (9), TruSeq Amplicon (Illumina), and Haloplex (Agilent) (10). These systems have different constraints in terms of assay design, cost, input material requirements, and turn-around times. Commercial systems have also been developed for miniaturized PCR by microfluidics. The Access Array system (Fluidigm) allows 48 single-plex assays across 48 samples with a relatively modest quantity of input template (50 ng/sample), and the RainStorm technology (RainDance Technologies) is based on the generation of microdroplets in an oil emulsion (11). This approach allows ~4,000 simultaneous amplifications, but its limitations include the requirement for a very high amount of input DNA and sequential processing of individual samples. Microfluidic chips and their associated instrumentation are prohibitively expensive for small research or diagnostic labs. The current approaches each have their own advantages and limitations, and the method of choice is thus dependent on the application, required performance, input material, ease of use, cost, and available instrumentation.
Use of MPS in diagnostic settings also presents specific technical challenges. For example, methods for sequencing DNA extracted from formalin-fixed, paraffin-embedded (FFPE) material have had variable success so far (12). There is great potential for the use of FFPE specimens in cancer studies and clinical diagnostics because pathology departments routinely collect and store them.
To address the limitations described above, we have developed an in-house-customizable PCR-MPS technology that we call Hi-Plex (Figure 1). Hi-Plex panels can be designed and customized in minutes using our simple automated primer design software. Preparation for genetic testing or screening (library building) is performed via a single PCR amplification, followed by size selection, for a fraction of the reagent cost and hands-on time required by other methods. We have tested Hi-Plex in a 60-plex assay using all of the protein-coding and some of the 5′-untranslated and 3′-untranslated regions of the breast cancer predisposition genes PALB2 and XRCC2 (13-15), both on cell line-derived genomic DNA and FFPE tumor-derived DNA.