When applied to 100 ng FFPE-derived DNA, Hi-Plex resulted in 78.33% (47/60), 91.67% (55/60), and 100% of targeted amplicons represented within 5-fold, 10-fold, and 25-fold of the mean. A total of 2,556,204 reads (97.33%) were on-target, with a mean of 42,603.4 on-target reads per amplicon. The number of on-target reads ranged from 2056 (20.72-fold less than the mean) to 237,945 (5.59-fold higher than the mean) (316 chip).
When the amount of input FFPE-derived DNA was reduced to 25 ng, Hi-Plex resulted in 90% (54/60), 98.33% (59/60), and 100% of targeted amplicons represented within 5-fold, 10-fold, and 12.5 fold of the mean. In this assay, a total of 2,454,875 reads (95.14%) were on-target, with a mean of 40,914.58 on-target reads per amplicon. The number of on-target reads ranged from 4046 (10.11-fold less than the mean) to 235,904 (5.77-fold higher than the mean) (316 chip). Supplementary Figures S1 and S2 show the relative representation of the 60 amplicons in relation to mean primer pair G/C-content and intervening sequence G/C-content observed in the 100 ng and 25 ng FFPE tumor-derived DNA Hi-Plex experiments. The lowest performer in both FFPE runs was the amplicon with the highest primer-intervening sequence G/C-content (74%).
This study has demonstrated the advantages of the Hi-Plex method using Ion Torrent sequencing chemistry. Our system showed minimal amplification biases associated with differential primer efficiencies and eliminates the need for extensive primer concentration optimization, primer redesign, or normalization/pooling of multiple separate PCR products to achieve uniform coverage. We have shown the benefits of Hi-Plex in the context of degraded FFPE specimens, along with low levels of input DNA, without compromising sequencing data quality. That Hi-Plex yielded the narrowest range of relative amplicon representation using 25 ng FFPE-derived material suggests that fragmented template might reduce the potential for off-target amplicons to compete with on-target amplicons and that the system may perform better using relatively low inputs of such material. The use of newer versions of chemistry and software could also have contributed to the improved performance compared with the assay of LCL-derived DNA.
Existing commercial solutions for PCR-based MPS proposed by Life Technologies (Ion Ampliseq), Illumina (TruSeq Amplicon), and Agilent (Haloplex) require expensive reagents, including multiple enzyme formulations and highly specialized oligonucleotide mixtures that are difficult to produce. They also involve laborious protocols with many enzymatic processing and purification steps, and can be limited in the scope of targetable regions. They typically require two days of library preparation involving many hands-on steps, with each step increasing the potential for error, such as the introduction of PCR contamination. Haloplex currently costs in excess of $10,000 AUD (Australian dollars; >$9000 USD) to prepare 96 specimen libraries and is particularly constrained in terms of design due to the need for fortuitous restriction enzyme sites and other local nucleotide sequence. Requirements for particular sequence contexts also constrain the other chemistries, e.g., the Life Technologies design software only allowed ~30% of XRCC2 to be targeted. AmpliSeq is restricted to use with the relatively error-prone Ion Torrent sequencing chemistry that exhibits ~98.2% per base accuracy compared with >99.6% for Illumina sequencing chemistry (22). None of these approaches allow fine control of the product size range, which has implications for relative amplification efficiencies of amplicons and the potential to apply stringent sequencing artifact filtering approaches via paired-end read comparison (6).
Because Hi-Plex performed so well across a wide variety of amplicons without alteration of gene-specific primer sequences or relative concentrations, we envisage that these methods will be broadly applicable across a wide range of targets with minimal optimization. Should fine-tuning be required for given applications, however, redesign of under-performing primer pairs or adjustment of primer concentrations for outlier amplicons remain viable options. Currently, our primer design software implements very basic algorithms. It is likely that future improvements can be made by such measures as taking account of primer secondary structure predictions and avoiding low G/C-content toward the 3′ end of gene-specific primers. Adoption of more sophisticated melting temperature prediction algorithms, such as including nearest-neighbor effects, might confer further benefits (23). More sophisticated existing programs for multiplex PCR primer design do not allow precise definition of the amplicon size, however (24-26). Hi-Plex has been designed so that genes or genomic regions could easily be swapped in and out of a panel, without extensive protocol optimization or primer redesign.
In future experiments, we will test Hi-Plex for considerably higher parallelization, with the aim of achieving robust thousands-plex single-tube multiplexing. The mechanisms underlying Hi-Plex suggest that this should be possible without extensive protocol adjustment. Theoretically, Hi-Plex should be sequencing platform agnostic, able to be transferred across chemistries simply by swapping adapter sequences. The ability to define amplicon size using Hi-Plex should also allow stringent sequencing chemistry artifact-filtering using completely overlapping paired-end reads. When paired reads show discrepancy at given positions, confidence in the call can be assigned more appropriately than would otherwise be possible.
The size selection step could optionally be automated using a system such as the Pippin Prep System (Sage Science). It should not be necessary to conduct separate size selection steps for separate Hi-Plex libraries. Use of specimen encoding, for example barcoded adapters, should allow a large number of Hi-Plex PCR products to be size selected at once. The reagent costs for the Hi-Plex library build (primers, PCR components, and gel extraction), assuming high-throughput, are around one dollar per specimen, rendering this approach orders of magnitude more cost-effective than the alternatives.
Having been successfully demonstrated on both cell line- and FFPE-derived material, Hi-Plex represents an exciting method for a range of molecular screening applications, including diagnostics, disease predisposition profiling, and disease gene discovery.
This work was supported by the Australian National Health and Medical Research Council (NHMRC) (APP1025879 and APP1029974), by a Victorian Life Sciences Computation Initiative (VLSCI) grant number VR0182 on its Peak Computing Facility at the University of Melbourne, an initiative of the Victorian Government, and by the Victorian Breast Cancer Research Consortium (VBCRC). TN-D is a Susan G. Komen for the Cure Postdoctoral Fellow. MCS is a VBCRC Group Leader and Senior Research Fellow of the NHMRC. We thank the Australian Breast Cancer Family Study (ABCFS, Principal Investigator John Hopper) for providing the cell-line derived DNA, and Ee Ming Wong for providing the FFPE tumor-derived DNA.
The authors declare no competing interests.
Address correspondence to Daniel J. Park, Genetic Epidemiology Laboratory, Department of Pathology, Medical Building, The University of Melbourne, Victoria, Australia; E-mail: [email protected]
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