Special ReportClinical Chemistry 66:81012–1029 (2020)The dMIQE Group*ABSTRACT: Digital PCR (dPCR) has developed considerably since the publication of the MinimumInformation for Publication of Digital PCRExperiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and ourunderstanding of its technological potential. Yet thesedevelopments also have associated challenges; dataanalysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurementerror. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the communityand reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplifieddMIQE table format to assist researchers in providingkey experimental information and understanding of theassociated experimental process. Adoption ofdMIQE2020 by the scientific community will assist instandardizing experimental protocols, maximize efficientutilization of resources, and further enhance the impactof this powerful technology.IntroductionSince the publication of the guidelines for theMinimum Information for Publication of QuantitativeDigital PCR Experiments (dMIQE) (1), digital PCR(dPCR) has seen considerable technological development with a plethora of new applications. dPCR hasprogressed from an expensive approach with a limitedapplication niche, available to only a few laboratories,towards a mainstream global technology (2) offeringunique advantages and applications to many scientists.To reflect this advancement, we present an updateto dMIQE, dMIQE2020, which builds on the originalguidelines to account for the increase in the number ofapplications and new platforms that have become*Address correspondence to: Jim F. Huggett at National Measurement Laboratory, QueensRd, Teddington TW11 0LY, UK. Fax 44-020-89432767; e-mail: [email protected] March 20, 2020; accepted May 18, 2020.DOI: 10.1093/clinchem/hvaa125available in the last 7 years. We highlight some of the associated advantages and limitations, and these updatedguidelines are written with the support of dPCR instrument manufacturers (see Acknowledgment section).The intention is to enable dMIQE2020 to reflect thismaturing technology by addressing new factors thatneed to be included in publications reporting dPCRdata. We also present a revised simplified dMIQE tableformat (Supplemental Table 1) to aid application andincrease adoption and provide an example of a completed table (Supplemental Table 2).Description of the Method and Brief HistoryA dPCR reaction is performed using limiting dilution toseparate the nucleic acid molecules amongst a largenumber of subreactions, termed partitions (Table 1 provides a summary of the terminology for dPCR). This‘partitioning’ capitalizes on the random distribution ofnucleic acid molecules in solution, so that some of thepartitions contain single (or few) copies of target molecules and, importantly, some contain none. Followingpartitioning, the reaction, comprising all the partitionsto be analyzed, is subjected to PCR. Each dPCR partition contains the core reagents used in a real-time quantitative PCR (qPCR) reaction that will generate afluorescent signal in response to the presence of a targetsequence. A positive partition is identified by an increased fluorescent intensity compared to a negativepartition with baseline signal only. Quantification isperformed by applying Poisson statistics to the proportion of the negative partitions (typically calculated bysubtracting the number of positive partitions [k] fromthe total number [n], see Equation 2 below) to accountfor positive partitions that initially contained more thanone target molecule.The concept of dPCR was developed before qPCR.As early as 1988, just a few years after PCR was described, Saiki et al. (3) applied limiting dilution to thePCR, which was subsequently used to detect HIV DNA(4). In the early 1990s, several articles were publishedapplying the concept mainly in the areas of virology,lymphoid biology, and neoplasia (5). However, following the initial description (6) and subsequent development of qPCR, offering increasingly affordable, precise,high-throughput, and multiplexed measurement ofC American Association for Clinical Chemistry 2020.VThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.1012Downloaded from /66/8/1012/5880117 by Tu Munchen Klinikum R D ISAR - Do not use user on 05 August 2020The Digital MIQE Guidelines Update:Minimum Information for Publication of QuantitativeDigital PCR Experiments for 2020

Special ReportThe dMIQE 2020 Guidelinesgeneration of water-in-oil droplets or using prefabricatedchips that contain solid chambers into which the reaction is loaded.Another development has been the establishment ofinstruments with greater than 10,000 partitions per reaction. These instruments can analyze tens of samples in asingle run typically providing 2- or 3-color formats allowingfor multiplexing, with more colors promised by manufacturers for future instruments. This contributes to improvedprecision (Fig. 1A), increased dynamic range (Fig. 1B), andincreased analytical sensitivity due to the larger volume ofnucleic acids extract analyzed per reaction.In parallel with hardware development, the expansion of software tools (both vendor-specific andTable 1. The meaning of frequently used terminology when discussing dPCR.dPCR termDescriptionAlternative nameprereactionthe initial volume prepared prior to partitioning that contains master mix, dNTPs, assay, and templatepartitionthe subreaction used for limiting dilution and subsequently measured as positive or negative post reactiondroplet, chamberanthe total number of partitions used for quantificationaccepted partitions /droplets/chambersa, analyzable partitions/ droplets/chambersaVpthe volume of each partitionreactionthe total volume of the measured partitions (n x Vp)dead volumedifference between the volume of the prereaction and reactionkthe number of positive partitions in a reaction used forquantificationwthe number of negative partitions in a reaction used forquantificationPoissonthe statistical distribution used to account for the probability of apartition initially containing more than one targetklambda, the average number of target molecules per partitionfluorescenceintensitythe fluorescence of a partitionfluorescence amplitude, endpoint fluorescence, relativefluorescence unitbaselinethe fluorescence of the negative partitionsfluorescence noise/backgroundpeak resolutiona measure of the separation in fluorescence between positive andnegative partitionsseparability score, amplitudethresholdthe line that separates the partition clusters based on amplitudeclusterthe group of partitions that are located in a similar space within ascatter plot based on amplituderainthe partitions that are located within the space between the positive and negative clustershigherordermultiplexingthe term given to multiplexing that can count more targets thanthere are fluorescent detection channelspopulation, dropletpopulationintensity multiplexingaThe term ‘chambers’ is used to define the partitions of instruments that use prefabricated chips, however this term is also used by some manufacturers to describe the reaction vessel. Consequently, the use of the term ‘partition’ to describe dPCR subreactions is recommended as it is not used elsewhere when describing the technology and is agnostic to the partitioning format of a given instrument.Clinical Chemistry 66:8 (2020)1013Downloaded from /66/8/1012/5880117 by Tu Munchen Klinikum R D ISAR - Do not use user on 05 August 2020nucleic acids, resource intensive ‘limiting dilution PCR’was eclipsed.Development of the method did not completelystop, however, with the term ‘digital PCR’ being coinedby Vogelstein and Kinzler in 1999 (7) as a method thatoffered improved analytical sensitivity when measuringmixtures of single nucleotide variants by PCR. Thisconcept was further advanced by developing BEAMing(8) and its potential highlighted with some of the earliest circulating tumor DNA (ctDNA) measurement incancer patients (9). More recently, advances in microfluidics have enabled the development of instrumentsthat can partition in very small volume formats.Partitioning is currently achieved either through the

Special ReportAn Overview of the Applications of dPCRRecent uses of dPCR have spanned numerous DNA,RNA, and epigenetic applications. A popular use of themethod is the detection and quantification of rare genetic variants (e.g., single nucleotide variants) in mixtures of other, more predominant, variants of the samesequence. Such ‘rare’ sequence detection can measureactionable mutations in ctDNA (10), fetal genetic variants in noninvasive prenatal testing (11), polymorphisms of a donor organ as an assessment of potentialgraft rejection (12, 13), as well as rare bacterial genotypes (14) and viral drug resistance (15). An example ofearly direct clinical diagnostic application is the measurement of ctDNA in liquid biopsies to guide the treatment of non-small cell lung cancer (16, 17).dPCR can offer greater precision than qPCR (18)and is far simpler to use for copy number quantificationdue the binary nature in which the partitions arecounted as positive or negative. The increased precisionof dPCR (18) enabled improved measurement of copynumber variants (19, 20), including in gene amplification in neuroblastoma (21) and fetal trisomy by noninvasive prenatal testing (22). dPCR also allows rare eventor trace level detection with high confidence since onlya single or small number of DNA molecules are amplified in each individual partition, regardless of whetheran experiment has 10 or 10 000 target molecules per reaction. While qPCR can detect very low concentrationsof a target, calibration of trace measurements is challenging. This is one of the reasons dPCR has been1014 Clinical Chemistry 66:8 (2020)Fig. 1. Predicted precision and linear range of the Poissondistribution. Each graph is generated mathematically basedon the Poisson distribution for dPCR reactions with 4 different numbers of partitions (n). The majority of dPCR instruments offer reactions where n 10 000. (A) The relativeuncertainty (based on modeling the 95% upper confidencelimit of k) is highest at the extremes of the range (very fewpositive partitions; k/n 0.1 or very few negative partitions; k/n 0.95). As the number of partitions in a reactionincreases, so does the precision at a given proportion ofpositive partitions. Once n 10 000 the relative uncertainty is 5% for the majority of the range. Horizontal dotted lines correspond to a relative uncertainty of 5% and10%. (B) The dynamic range is proportional to the numberof partitions in a reaction, note the dynamic range isgreater than the number of partitions. The horizontal dotted lines correspond to the copies per reaction for eachgiven n where the relative uncertainty of k is less than50%.explored as a method for trace level measurements inminimal residual disease (23, 24) and latency in viralinfections such as HIV (25–27).The analytical sensitivity of the measurement ofdouble stranded DNA molecules can be further enhanced by denaturing the molecules prior to partitioning (28). Since single strands end up in differentpartitions, the analytical sensitivity is improved by aDownloaded from /66/8/1012/5880117 by Tu Munchen Klinikum R D ISAR - Do not use user on 05 August 2020platform-independent) has given operators greater control of their analysis. Scatter plots that depict the endpoint fluorescence of each partition are now commonlyavailable. These plots also enable quality control checksto be performed in parallel for individual reactions andwhole experiments. Other developments include theability to define thresholds that optimally separate positive and negative partitions, to combine different reactions for greater sensitivity and higher precision, and toexport data to enable further analysis using third partysoftware.Despite the increasing capabilities and use ofdPCR, many biological and clinical research scientistsare unclear as to what the practical advantages are.Placed in the same methodological space as establishedqPCR and the increasingly powerful massively parallel(or next generation) sequencing, this is perhaps unsurprising. The reality is that dPCR provides a number ofunique opportunities. Here we explore recent developments and applications of dPCR, discuss the factors thatshould be considered during the experimental design,and detail considerations to be included in publicationsreporting dPCR results.

The dMIQE 2020 GuidelinesThe potential for dPCR to enable new research opportunities and to support traceability in the wider fieldof molecular genetic measurement should have a majorimpact on the accuracy of nucleic acids measurement asa whole. However, researchers and manufacturers musttread cautiously to ensure that the nuances that may affect these measurements are understood. What followsare some steps to consider on this journey.Considerations for Designing and PerformingdPCR ExperimentsLike qPCR, to maximize the performance of dPCR, specific considerations are required to ensure unbiased andreproducible measurements (50, 53). The following sections outline how best to design and perform dPCRexperiments. Sections may be interdependent: for example, to determine the false positive rate of an assay, notonly assay design, but selection of control materials andpartition classification are important.Following dPCR, the end-point fluorescence oftarget containing positive partitions is higher fluorescence than those without, negative partitions (background). These fluorescence values can be plotted inone-color dimension with the fluorescence intensityagainst partition number (Fig. 2) or as two-colors(Fig. 3A–D) or more (Fig. 3E), with the fluorescenceintensity of the different channels aligned to differentaxes. Visualization of these plots can aid assay optimization and quality control.ASSAY DESIGN AND OPTIMIZATIONdPCR requires the same careful assay design considerations as those required for qPCR (53, 54). Smaller( 150 bp) amplicons are desirable although largerproducts can also be quantified (55). To generate thefluorescent signal by PCR, probe-based chemistries (e.g.,hydrolysis probes, ideally with nonfluorescent quenchers)or DNA binding dyes (e.g., EvaGreen) can be used whencompatible with the dPCR platform.Assay optimization, by varying the annealing temperature (Fig. 2A) as well as primer and probe concentrations (Fig. 2B), is best performed using a templatethat closely matches that of the test template, as performance may vary with different template types (e.g., plasmids, genomic DNA, cell-free DNA, synthetic DNA).Furthermore, while different batch syntheses of probesof the same sequence can change the final fluorescenceintensities, this can have a negligible effect on the measured results (Fig. 2C). Matrix effects and inhibitorsmay also reduce the fluorescence intensity of the positivepartitions. While ‘suboptimal’ reactions may providesimilar results to more optimum conditions (Fig. 2D),reflecting methodological robustness, optimizationClinical Chemistry 66:8 (2020)1015Downloaded from /66/8/1012/5880117 by Tu Munchen Klinikum R D ISAR - Do not use user on 05 August 2020factor of two. Other applications that exploit theunique partitioning of dPCR include cis-trans linkagerelationships between two targets (29–31) and ‘dropoff’ assays for identifying the frequency of a mutationof unknown sequence (32), and evaluation of geneediting efficiency when using approaches like CRISPRCas9 (33).Furthermore, dPCR provides high reproducibilityto the above technical advantages. This is possible whenthe same target is measured in different laboratories (34,35) using different assays or assay formats (36), orinstruments from different manufacturers (37, 38). Thisis also achievable both when measuring purified nucleicacid, but also whole biological samples in which preanalytical steps such as extraction need to be included (36,39). This characteristic has made dPCR a popularmethod to quantify reference materials (40, 41), to support applied molecular testing in clinical diagnostics(42–44) and food testing (45–47).When used to conduct quantitative measurements,molecular genetic methods have historically appliedmass and mole, combined with volume, to calculatecopy number concentration. Mass or mole are arguablynot ideal when considering a large macromolecule suchas DNA, and nucleic acid calibration materials haverarely been traceable to the International System ofUnits (SI) (48). dPCR has the capability of counting allintact (equal or larger than the amplicon) DNA molecules containing a specific target sequence (49), therebypotentially offering SI traceability via counting to theunit one (48). To maximize the potential impact ofsuch a capability, efforts have been made to harmonizeand standardize best practices in dPCR (and qPCR) inthe ISO 20395 standard (50).Quantification accuracy for copy number measurements is dependent on both completeness of molecular count and accurate definition of the unit volumeof sample and total reaction (i.e., number of partitionsof accurately defined volume). Both need to be demonstrated for claims of SI traceability by dPCR to be supported. International collaboration among nationalmetrology institutes, supported by the ConsultativeCommittee for Amount of Substance: Metrology inChemistry and Biology (CCQM), have led researchdemonstrating that dPCR can indeed measure withsufficient accuracy for primary SI-traceability (38, 51).dPCR has provided the first ever nucleic acid referencemeasurement procedure accepted by the JointCommittee on Traceability in Laboratory Medicine(JCTLM) (38) and dPCR is included as an example ofa higher order reference measurement procedure withinthe new edition of the ISO 17511 guideline on metrological traceability of values assigned to calibrators andcontrol materials for diagnostic methods (52).Special Report

Special ReportMULTIPLEX ASSAYSDesign criteria for multiplex dPCR are similar to othermultiplex PCR applications. The increased number ofprimers and probes requires additional consideration ofcomplementary sequences to avoid nonspecific hybridization. Similar to qPCR, multiplexing is performed withfluorescently-labeled probes that are preferentiallydetected in different fluorescence channels (Fig. 3).The simultaneous analysis afforded by multiplexingcan improve resource efficiency, reduce the amount ofsample needed for analysis (important when the specimen is limiting), and allow for direct internal control ofan individual reaction. Multiplex dPCR also has otheradvantages; for copy number variants, the pairing of target and reference genes to determine their ratio arisesnaturally using a duplex approach (Fig. 3A) (57, 58).Similarly, for biallelic variation of single nucleotide variants or small insertion/deletions, typically analyzedwith two hydrolysis probes, a duplex approach visualizedusing two color plots is the favored format (Fig. 3B). Indrop-off assays (59), genotype is determined by counting partition numbers from the single and double positive clusters (Fig. 3C); it is not possible to make thiscalculation accurately using single color analyses.Two color analysis can also be applied to identifysome technical artifacts that may be difficult to discernwith single color analysis. For example, fluorescent bleedthrough (also termed crosstalk or spillover) occurs whensignal from one fluorophore is detected in a channelintended for another. If a duplex experiment is visualized as respective single color plots, fluorescent bleedthrough may appear as additional clusters and can bemistaken for reduced analytical specificity (Fig. 3A; seered arrow). In multiple color plots, fluorescent bleedthrough clearly manifests as a ‘leaning’ or ‘lifting’ of thesingle positive partitions away from the intended axestowards the expected position of the double positivepartitions (Fig. 3A).Fluorescent bleed through can be determined byperforming single probe reactions, using a template thatonly contains the intended target, and visualizing the experiment in two colors. If the clusters ‘lean’ or ‘lift’ intothe other axis, then bleed through is confirmed.Reducing the concentration of the relevant probe,changing the fluorophore, and/or applying a color compensation matrix may reduce the bleed through.Bleed through can often be tolerated as long as itssource is understood. If the ‘lean’ or ‘lift’ is absent whenusing a single color reaction, then it suggests that this1016 Clinical Chemistry 66:8 (2020)may be caused by something else, such as reduced assayspecificity. Probes designed to similar sequences (e.g.,single nucleotide variants) may bind to the alternate variant, reducing specificity (60). While this may be minor,a ‘lean’ or ‘lift’ similar to fluorescence bleed throughmay occur (Fig. 3B), reducing the peak resolution forthe affected reporter. When an alternative variant is predominant, as in rare variants within cell-free DNA, falsepositive signals caused by factors like PCR errors, willultimately limit the lower fractional abundance that canbe measured (see further and Fig. 4).Another artifact with competing probe duplexes(59) is a drop in fluorescence in the double positive partitions (Fig. 3B). When variants of the same moleculeare amplified by a single primer pair in the same partition, competitive PCR or partition-specific competition(PSC) occurs (59). Where different variants are bothpresent in a partition, they will compete for the primers,resulting in a reduced fluorescence intensity when compared to partitions containing a single variant. PSC canoccur even when the reaction is performed in singleplexbecause if other variants (or pseudogenes with sufficienthomology) are present, they will amplify and competewith the variant of interest reducing peak resolution. Ifthe method is performed in duplex and evaluated in atwo-color plot, it is clear that PSC is occurring andthresholds are easier to set (Fig. 3B). In drop-off assays,PSC can cause the double positive partitions to appearas a second cluster with reduced fluorescence intensity(Fig. 3C). The impact of PSC, fluorescent bleedthrough, and other factors can be evaluated using an optimization and quality control protocol outlined previously (59).In addition to multicolor multiplexing, dPCR canspecifically detect more than one target within an individual fluorescent channel. This ‘higher order multiplexing’is achieved by varying the concentrations of differentprobes using the same fluorophore (61) (Fig. 3D) or differently sized amplicons with DNA binding dyes (62).Higher order multiplexing can be performed in a singlecolor, where two different targets are detected within asingle reaction (63), or in multicolor assays that use twoor more probe colors to detect more targets than fluorophores with a single reaction (61).TEMPLATEAs with all other PCR formats, DNA is the only nucleicacid that can be measured by dPCR. Like qPCR, template complexity may impact assay performance as seenwith circular plasmids and high fragment length genomic DNA (37). Restriction digestion to small linearDNA fragments may equalize template differences (36,49, 64), and prevent underestimation of linked targetmolecules (18). However, restriction sites must not beDownloaded from /66/8/1012/5880117 by Tu Munchen Klinikum R D ISAR - Do not use user on 05 August 2020should strive to elevate the positive from the negativepopulations, to maximize the ‘peak resolution’ (56) andreduce the number of partitions in between that aretermed ‘rain’ (see below).

The dMIQE 2020 GuidelinesSpecial ReportClinical Chemistry 66:8 (2020)1017Downloaded from /66/8/1012/5880117 by Tu Munchen Klinikum R D ISAR - Do not use user on 05 August 2020Fig. 2. Examples of the one color plot outputs demonstrating methodological robustness. The same assay and genomic DNAtemplate is used in all parts of the figure. Unless otherwise stated, each reaction contains 900nM of each primer and 250nM ofprobe, annealing temperature: 60C, run for 40 PCR cycles. (A) The effect of the annealing temperature (gradient 65C to 52Cfrom left to right) on the final fluorescence intensity of the positive and negative partitions. Each reaction contains approximately 20000 copies of the gDNA template analyzed using the QX200 (Bio-Rad) with Supermix for probes (no dUTP) (Bio-Rad).The horizontal pink line represents the threshold to separate the positive and negative partitions. (B) The effect of primer andprobe concentrations on the difference in fluorescence intensity between positive and negative partitions. Reactions 1-3 have250, 500, and 1000nM of primer, respectively, all with 50nM of probe. Reactions 4-6 have 250, 500, and 1000nM of primer,respectively all with 250nM of probe. Each reaction contains approximately 40000 copies of the gDNA template analyzed usingthe Naica (Stilla) and Perfecta Multiplex qPCR Toughmix (Quanta BioSciences). The blue horizontal lines represent the thresholdto separate the positive and negative partitions. (C) The difference in fluorescence intensity of positive and negative partitionsbetween 6 different batches of the same assay purchased between 2012 and 2017 from 3 different manufacturers. The genomicDNA template concentration and dPCR platform is the same as that described in (A). (D) Comparison of the copy number concentrations calculated from each of the 18 reactions shown in (A)-(C). Each reaction is represented by a symbol in the order itappears in its respective color plot.

Downloaded from /66/8/1012/5880117 by Tu Munchen Klinikum R D ISAR - Do not use user on 05 August 2020Fig. 3. Examples of two and three color plot outputs using different multiplex format demonstrating leaning, lifting and partition specific competition.1018 Clinical Chemistry 66:8 (2020)

Special ReportThe dMIQE 2020 Guidelinespresent in the amplicon sequence, and digestion of already fragmented DNA (e.g., from formalin-fixed, paraffin embedded tissues or cell-free DNA) may result in aloss of signal (36).RNA transcripts can only be measured by first converting to complementary DNA (cDNA) in reversetranscriptase digital PCR (RT-dPCR) using either oneor two-step formats. In a one-step strategy, RNA is partitioned with both reverse transcription and PCR occurring sequentially in the same partition. Even if multiplecDNA copies are generated from each RNA molecule,results are not overestimated. In two-step reactions, reverse transcription is first performed in bulk before partitioning the cDNA and subsequent dPCR in a separatereaction. The reverse transcriptase step can be a predominant source of error that should be considered duringexperimental design (65).While newer dPCR formats have greater dynamicranges, they cannot compete with qPCR, which is typically capable of over 6 orders of magnitude.Consequently, in certain situations a prior knowledge ofthe template concentration may be required to avoidsaturating the instrument. When concentrations aresufficiently high, commonly used methods, such asthose that employ fluorometry and spectrophotometry,can be used to quantify nucleic acids and guide the dilution to concentrations for optimal measurement usingdPCR. It should be noted that such methods estimatemass per unit volume of the component nucleic acidbases not the macromolecule. Consequently, determination of genome copies using approaches that measuremass requires knowledge, or assumptions, of templatecomposition, purity, and quality to convert mass tomoles. Users should also be aware of potential interfering factors that may disrupt the accuracy of such opticalmethods. When comparing mass-based nucleic acidquantification with dPCR results, or those from anymethod used to calculate molecular copy number, aclear description of the molecular weight of the genomeused to calculate the genome equivalents must be included, along with the method used to calculate thisvalue.PREANALYTICSWhile dPCR can be accurate, performance depends onthe amount of template added to the reaction. ErrorClinical Chemistry 66:8 (2020)1019Downloaded from /66/8/1012/5880117 by Tu Munchen Klinikum R D ISAR - Do not use user on 05 August 2020Fig. 3. (Continued) Parts A-D of the figure have been generated using the QX200 (BioRad); each part shows a two color scatterplot with the corresponding single color plots orientated along the corresponding axis (channel 1 (Ch1); y-axis, and channel 2(Ch2); x-axis). The horizontal and vertical pink lines represent the thresholds for Ch1 and Ch2, respectively. Part E of the figurehas been generated using the Naica (Stilla); a three color plot is shown with the three axes orientated as the blue channel (xaxis), red channel (y-axis), and green channel (z-axis). The 3 thresholds are not displayed for aesthetic purposes. The corresponding two color plots are presented in Supplemental Fig. S2. (A) Noncompeting duplex reaction (for definition of multiplex formatssee (59)) containing separate primer pairs for each amplicon. The ratio between the 2 targets is 1.6

single run typically providing 2- or 3-color formats allowing for multiplexing, with more colors promised by manufac-turers for future instruments. This contributes to improved precision (Fig. 1A), increased dynamic range ( Fig. 1B), and increased analytical sensiti vity due to the larg