Rare Sequence Detection Using Digital PCR

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Overview

Accurate detection of rare nucleic acid sequences in complex backgrounds poses a challenge for PCR assays. The signal-to-noise ratio in PCR samples can hinder both detection and data interpretation for low-abundance targets.

Digital PCR can overcome many of the current limitations to detecting rare sequences. In the Droplet Digital™ PCR (ddPCR™) System, a sample is partitioned into a large number of droplets prior to amplification. After amplification, the fluorescence or lack of fluorescence in each droplet indicates the presence or absence, respectively, of the target amplicon. A Poisson statistical analysis on the total numbers of positive and negative droplets yields absolute quantitation of the initial number of target sequences.

Improved Amplification of Rare Sequences in ddPCR Assays

In standard PCR reactions, a bias against the amplification of low-abundance sequences is often observed. For example, it has been demonstrated in the analysis of microbiomes that highly abundant organisms are over represented in standard real-time PCR assays, whereas low-abundance species are underrepresented (Gonzalez et al. 2012).

In the ddPCR System, after the sample is partitioned into thousands of droplets, a rare sequence will be present in only a few of the droplets in which there will ideally be few or no competing sequences. The partitioning of rare sequences away from the total pool of high-abundance sequences thus facilitates their amplification and subsequent detection, yielding a more accurate estimate of their representation within a sample.

Increased Accuracy in the Measurement of Rare Sequences

ddPCR technology is superior to standard PCR techniques in the quantitation of low-abundance targets:

  • No requirement for a standard curve — the absolute quantification provided by the ddPCR system eliminates the need for standard curves, which are especially difficult to design and control for a low-abundance target in a complex background
  • Higher tolerance to PCR inhibition — since ddPCR data collection occurs at end-point (after 40 cycles), the reaction is less susceptible to PCR inhibition. Matrices that would, in other systems, have led to lower target quantification can be tested by ddPCR without effect on the final result.

When standardizing a test for a rare sequence with a series of increasing dilutions, most PCR-based assay systems have a lower limit of about 1% rare target. In the QX200™ Droplet Digital PCR System , each 20 µl PCR reaction is partitioned into 20,000 water-in-oil droplets, enabling the detection of rare target sequence at an abundance as low as 0.001% — three orders of magnitude greater sensitivity.

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Comparison of sensitivity of qPCR and ddPCR

Comparison of sensitivity of qPCR and ddPCR. A, qPCR assay showing poor linearity below 1% mutant sequence in a wild-type background; B, ddPCR assay with the same template showing linearity down to 0.001% input mutant fraction.
 

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Rare Sequence Detection in Infectious Disease

Current treatment regimens can reduce viral load to levels that are not reproducibly detectable by current diagnostic tests. The increased sensitivity of digital PCR combined with absolute quantification, delivers the ability to measure and monitor very low viral loads. In a recent report (Persaud et al., 2013), ddPCR was used to test the HIV-DNA residual load in the case of an infant who was ultimately deemed "cured" of the infection. With the potential to now cure HIV/AIDS patients, there is a pressing need to determine whether the cure is real, or whether low levels of latent virus remain.

For a more comprehensive discussion of the use of ddPCR for both pathogen detection and microbiome analysis, see Pathogen Detection and Microbiome Analysis Using Digital PCR.

Rare Sequence Detection in Cancer

The ability to identify and measure rare sequences in a wild-type background is crucial for cancer detection, monitoring, and treatment. High sensitivity is required to detect the presence of residual cancer, early stages of recurrence after remission, and new mutations that may arise.

Increased testing sensitivity — in the detection of BCR-ABL transcripts in chronic myeloid leukemia, digital PCR displayed a 2–3 log improvement in sensitivity compared with qPCR. Furthermore, the digital PCR assay detected BCR-ABL transcripts in several patients classified as negative by real-time PCR. The digital PCR assay enabled the continued monitoring of the decline in transcript levels after they became undetectable by real-time PCR (Goh et al. 2011). In a recent article by Dr Jennings and coworkers, the LOD (limit of detection) for the BCR-ABL transcript using the QX100™ System was determined to be as low as 0.0001% (0.001% for the LOQ (limit of quantification) (Jennings et al. 2014).

Human epidermal growth factor receptor 2 (HER2) is overexpressed in about 30% of breast tumors. It has been estimated that approximately 20% of current HER2 genetic testing is inaccurate. In a recent study, formalin-fixed paraffin-embedded (FFPE) human breast tumor samples were analyzed by ddPCR. The authors reported that ddPCR analyses of these samples agreed with conventional pathology assessments using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The results suggested that, in addition to accurately determining amplification of HER2, the ddPCR assay can also provide sensitive, quantitative measurement of copy number variation (Heredia et al. 2013).

Noninvasive cancer testing — the development of more sensitive detection and genotyping of cancers is broadening the scope of noninvasive testing in oncology. Noninvasive testing is particularly valuable for tissues and areas of the body where a biopsy carries a significant risk. The increased ability to detect lower mutant:wild-type sequence ratios will result in greater use of bodily fluids such as plasma, sputum, urine, and stool as sample matrices. Developing more sensitive detection methods, such as digital PCR, for the identification and monitoring of cancer-related markers (and copy number variations) in circulating cell-free (cfDNA) has become a very active field of research. For more information about noninvasive cancer testing see the Liquid Biopsy page.

Detection of new mutations and duplications — cancer cells are genetically unstable, with new mutations and duplication events often arising rapidly. Detecting mutations as they emerge and tailoring treatment to these changes is a major goal of cancer diagnosis and treatment.

In non-small-cell lung cancer, those cancers with activating mutations in epidermal growth factor receptor (EGFR) generally respond well to treatment with tyrosine kinase inhibitors. However, relapses are often observed after treatment due an acquired second mutation, the "gatekeeper resistance" mutation. The ddPCR system was used to analyze both activating EGFR mutations and the acquisition of the resistance mutation. The results demonstrated that the ddPCR system permits the early detection of a failure of the initial treatment with tyrosine kinase inhibitors and shows potential for monitoring disease progression and guiding treatment (Kristoff et al. 2013).

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Noninvasive Prenatal Testing

The analysis of cfDNA provides a noninvasive option for prenatal testing. Between 3 and 13% of the total cfDNA in the plasma of pregnant women is thought to be fetal DNA. New techniques, including next-generation sequencing, have enabled the detection of fetal genomic abnormalities in total maternal cfDNA.

Testing for aneuploidy of chromosomes 13, 18, and 21 is currently approved for clinical use. Digital PCR has been evaluated for prenatal risk assessment for Down syndrome by the detection of trisomy 21 in maternal plasma cfDNA (Tong et al. 2010, Evans et al. 2012) and RNA (Lo et al. 2007). Recent studies have shown that digital PCR analysis of fetal cfDNA can be used for the risk assessment of other common diseases such as sickle cell anemia (Barrett et al. 2012) and hemophilia (Tsui et al. 2011). In a recent article, Gu and coworkers presented their results on the use of the QX200 ddPCR System for noninvasive prenatal diagnosis (NIPD) of methylmalonic acidemia, an inherited metabolic disorder. They conclude that ddPCR is "a cost-effective and noninvasive approach to diagnosing known mutations related to Mendelian disorders in the fetus" (Gu et al. 2014).

Genetically Modified Organism Testing

Genetically modified organisms, or GMOs, have been available for many years. A significant proportion of the global harvest of plants such as maize and soybeans is genetically modified.

As the cultivated acreage of genetically modified plants and the number of modified species increases, it is increasingly important to be able to detect and identify GMO-derived products. Additionally, there is a need to distinguish between authorized and unauthorized use of modified crops as well as requirements for monitoring and validation of product labeling.

Digital PCR can be used for the quantification of a transgenic event relative to an endogenous gene, to measure DNA copy number without a reference target, and to be suitable for certifying GMO copy reference materials in terms of copy number ratio (Corbisier et al. 2010, Burns et al. 2010). A recent study demonstrated that ddPCR is superior to qPCR for GMO quantification in a variety of substrates (Morisset et al. 2013).

Conclusion

The ability to detect and quantify sequences rare sequences constitutes an important challenge for nucleic acid research. The introduction of digital PCR has pushed some of the limits, allowing detection and quantification at significantly lower levels than before. The impact of these advances is likely to be seen in many fields including the detection, monitoring, treatment, and understanding of infectious diseases and cancer, as well as in the fields of GMO testing and environmental studies.

References

Barrett AN et al. (2012). Digital PCR analysis of maternal plasma for noninvasive detection of sickle cell anemia. Clin Chem 58, 1,026–1,032. PMID: 22451622

Burns MJ et al. (2010). The applicability of digital PCR for the assessment of detection limits in GMO analysis. Eur Food Red Technol 231, 353–362.

Corbisier P et al. (2010). Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction. Anal Bioanal Chem 396, 2,143–2,150. PMID: 19816678

Evans MI et al. (2012). Digital PCR for noninvasive detection of aneuploidy: power analysis equations for feasibility. Fetal Diagn Ther 31, 244–247. PMID: 22538702

Goh HG et al. (2011). Sensitive quantitation of minimal residual disease in chronic myeloid leukemia using nanofluidic digital polymerase chain reaction assay. Leuk Lymphoma 52, 896–904. PMID: 21338281

Gonzalez JM et al. (2012). Amplification by PCR artificially reduces the proportion of the rare biosphere in microbial communities. PLOS ONE 7, e29973. PMID: 22253843

Gu W et al. (2014). Noninvasive prenatal diagnosis in a fetus at risk for methylmalonic acidemia. Genet Med Jan 9 PMID: 24406457

Heredia NJ et al. (2013). Droplet Digital™ PCR quantitation of HER2 expression in FFPE breast cancer samples. Methods 59, S20–3. PMID: 23036330

Jennings LJ et al. (2014). Detection and Quantification of BCR-ABL1 Fusion Transcripts by Droplet Digital PCR. J Mol Diagn 16, 174–179. PMID: 24389534

Kristoff J et al. (2013). Absolute quantification of EGFR activation and resistance mutations as well as copy number in circulating nucleic acids by droplet digital PCR. American Association for Cancer Research Annual Meeting. Abstr 3491.

Lo YM et al. (2007). Digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc Nat Acad Sci USA 104:13,116–13,121. PMID: 17664418

Morisset D et al. (2013). Quantitative analysis of food and feed samples with droplet digital PCR. PLOS One 8: e62583. PMID: 23658750

Persaud D et al. (2013). Functional HIV cure after very early ART of an infected infant. 20th Annual Conference on Retroviruses and Opportunistic Infections. Paper #48LB.

Tong YK et al. (2010) Epigenetic-genetic chromosome dosage approach for fetal trisomy 21 detection using an autosomal genetic reference marker. PLOS One 5:e15244. PMID: 21249119

Tsui NB et al. (2011). Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA. Blood 117:3,684–3,691. [Erratum: Blood (2012) 119:4,577] PMID: 21263151

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