The Effect of Input DNA Copy Number on Genotype Call and Characterising SNP Markers in the Humpback Whale Genome Using a Nanofluidic Array

Recent advances in nanofluidic technologies have enabled the use of Integrated Fluidic Circuits (IFCs) for high-throughput Single Nucleotide Polymorphism (SNP) genotyping (GT). In this study, we implemented and validated a relatively low cost nanofluidic system for SNP-GT with and without Specific Target Amplification (STA). As proof of principle, we first validated the effect of input DNA copy number on genotype call rate using well characterised, digital PCR (dPCR) quantified human genomic DNA samples and then implemented the validated method to genotype 45 SNPs in the humpback whale, Megaptera novaeangliae, nuclear genome. When STA was not incorporated, for a homozygous human DNA sample, reaction chambers containing, on average 9 to 97 copies, showed 100% call rate and accuracy. Below 9 copies, the call rate decreased, and at one copy it was 40%. For a heterozygous human DNA sample, the call rate decreased from 100% to 21% when predicted copies per reaction chamber decreased from 38 copies to one copy. The tightness of genotype clusters on a scatter plot also decreased. In contrast, when the same samples were subjected to STA prior to genotyping a call rate and a call accuracy of 100% were achieved. Our results demonstrate that low input DNA copy number affects the quality of data generated, in particular for a heterozygous sample. Similar to human genomic DNA, a call rate and a call accuracy of 100% was achieved with whale genomic DNA samples following multiplex STA using either 15 or 45 SNP-GT assays. These calls were 100% concordant with their true genotypes determined by an independent method, suggesting that the nanofluidic system is a reliable platform for executing call rates with high accuracy and concordance in genomic sequences derived from biological tissue.


Introduction
Single Nucleotide Polymorphism, or SNP, is the most common form of variation which occurs when a single nucleotide (A, T, G or C) in the genome is changed [1]. SNP-genotyping (SNP-GT) is rapidly growing as a useful tool in many scientific disciplines including personalised medicine [2], forensics [3], plant and animal biotechnology [4,5]. Genome-wide association studies utilizing SNPs as markers have enabled identification of genes that underline complex disorders [6]. Depending on the location, a SNP might have consequences at the phenotypic level. Most SNPs are located in the non-coding regions of the genome and have no direct known impact on the phenotype of an individual [7] or cell function [1]. However, some SNPs regardless of their location may pre-dispose the individual to a certain disease or influence their response to drug [3,8]. To identify an association between a SNP and a particular disease or genetic trait, researchers need high throughput, cost effective and accurate approaches to screen vast numbers of samples for numerous SNPs. One such approach is the Fluidigm Dynamic Array TM platform (nanofluidic based genotyping system) which can handle medium throughput multiplexing [9].
The 48.48GT Dynamic Array TM technology (Fluidigm, South San Francisco) allows simultaneous analysis of 48 different SNPs in 48 individual samples using TaqMan H SNP-GT assays. The key to the efficiency of this approach is the chip architecture. The chip consists of a matrix of channels, chambers, and integrated valves finely patterned into layers of silicone in the nanofluidic chip. The valves partition sample / assay combinations into a total of 2,304 (48648) individual reaction chambers prior to thermal cycling.
The genotype for a particular SNP is either homozygous (pp or qq) or heterozygous (pq) and the genotype designated to a SNP following analysis is referred to as the genotype call. The genotype call quality can vary when the amount and/or the quality of the input DNA is not ideal and this may lead to either an incorrect genotype call or No Call for a particular SNP. Specific Target Amplification (STA) can be used prior to genotyping to increase the input DNA copy number. In previous studies STA was performed for 14 cycles in a multiplex format [9,10,11]. Multiplex STA provides simultaneous amplification of many targets of interest in one reaction, thus increasing the assay throughput and allowing more efficient use of each DNA sample [5,11,12]. Multiplex reactions, however, need to be validated to ensure that all reactions are amplified efficiently.
In this study, we validated a relatively low cost nanofluidic system for SNP-GT. As proof of principle, we first evaluated the effect of input DNA copy number on genotype call rate using well characterised digital Polymerase Chain Reaction (dPCR)quantified human genomic DNA samples with three different genotypes for a single SNP. Accurate quantification of DNA samples using dPCR combined with sample gravimetric dilutions prior to mixing with other reagents, enabled dispensing of a predicted number of DNA copies into each reaction chamber. The sourced human genomic DNA samples and the SNP assay have been previously used by Wang et al [9]. For samples with low starting input DNA concentration, we validated both simplex and multiplex STA prior to genotyping. This validation approach was then applied to twelve DNA samples that had been extracted from epidermis biopsies of humpback whales, Megaptera novaeangliae. STA initially used fifteen SNP-GT assays previously validated by Polanowski et al. [13] and was subsequently adapted for 45 SNP-GT assays. Simplex STA was designed to test the specificity of each SNP assay. The genotype of each sample determined using the nanofluidic platform at the National Measurement Institute (NMI), was compared for concordance with the true genotypes determined by an independent method at the Australian Antarctic Division (AAD).

Results and Discussion
Evaluating Genotype Call Accuracy on Human Genomic DNA The stock concentrations (copies/mL -haploid genome equivalents) of three human genomic DNA samples were determined by dPCR (n = 3 or 4) to be 4.7610 4 copies/mL with a range of 0.3610 4 (NA17313-'qq'), 9.3610 4 copies/mL with a range of 0.25610 4 (NA17316-'pq') and 1.4610 5 copies/mL with a range of 0.046610 5 (NA17317-'pp'), respectively. Note: Range is the difference between the largest and smallest values in a set of data and was used rather than standard deviation.
Preliminary analysis was performed to evaluate the effect of reaction copy number on call rate accuracy prior to TaqMan H SNP-GT. The dPCR-quantified samples with three different genotypes (pp, pq, qq) for a single SNP (rs513349) were gravimetrically diluted so that the estimated final copies per reaction chamber ranged between 1 and 97 copies (haploid genome equivalents). The final copies per reaction chamber were derived using equations 1, 5-6 ( Table 1). A genotype call rate and call accuracy of 100% was observed for reactions that were estimated to contain $ 9 homozygous (pp or qq) copies of the genotype or $ 46 heterozygous (pq) copies of the genotype. Reactions containing less than 9 homozygous copies of the genotype had a No Call rate of 6 to 60% for NA17313 (qq) and 2 to 35% for NA17317 (pp). However, when a genotype was assigned to a chamber containing homozygous DNA, the assigned genotype was correct regardless of the estimated copy number. For the heterozygous sample (NA17316-pq), reaction chambers containing # 9 copies had an incorrect call rate of 7 to 75% and a No Call rate of 4 to 33%. Clustering of genotype data points was tight for a homozygous call but was more widespread for a heterozygous call ( Figure 1A).
In contrast, a call rate and a call accuracy of 100% were achieved when the same gravimetrically diluted samples were subjected to STA and then diluted 20-fold prior to genotyping resulting in 2.0610 4 to 1.6610 2 copies per reaction chamber. Clustering of the genotype calls for all three human genomic DNA samples was much tighter following STA than in the absence of STA ( Figure 1B and 1A), suggesting that target enrichment improves the quality of results.
Based on the preliminary findings, the effect of copy number on the heterozygous (pq) call accuracy was further investigated in more detail, by lowering the copies per reaction chamber and by increasing the total number of replicates for each dilution. Sample NA17316 (pq) was gravimetrically diluted resulting in 1 to 38 copies per reaction chamber. A call rate and a call accuracy of 100% were achieved when 38 copies per reaction chamber corresponding to ,45 ng/mL of human genomic DNA solution used in sample preparation (,90 ng in 2.1 mL of DNA in GTsample solution) were present. At 18 copies per reaction chamber, which equates to 22 ng/mL, a call rate of 98% with a call accuracy of 99% was achieved ( Table 2). As the number of copies per reaction chamber decreased below 18, the number of incorrect calls (pp, qq) and No Calls increased ( Figure 2A) and the tightness of genotype clusters on the scatter plot decreased ( Figure 2B). The call accuracy dropped from 88% at 7 copies to 33% at 1 copy ( Table 2). The number of No Calls increased from 2 to 38% when the copies per reaction chamber decreased from 18 to 1 copy, suggesting low input DNA copy number affects the quality of data generated, in particular for a heterozygous (pq) sample. With a heterozygous locus, where two alleles are present, unequal sampling of the alleles at very low reaction copy numbers can result in failure to detect one (allele-drop out) or both of the alleles (locus drop-out) [14] leading to an incorrect or No Call. This would explain the observed increase in the number of incorrect and No Calls assigned for the heterozygous sample. Therefore, in order to minimise call error rate, it is important to maintain the allelic balance. Loss of heterozygosity (LOH), which is a common form of allelic imbalance, has been used to identify genomic regions that harbor tumor suppressor genes and to characterize tumor stages and progression [15]. To avoid misinterpretation of such data, it is critical that sufficient copies of the heterozygous locus are present in the genotyping assay.
Previously Wang et al [9] showed that 50 copies per reaction chamber corresponding to ,60 ng/mL of human genomic DNA is required to obtain a call rate of .99%. Further Chan et al [16], highlighted the importance of using purified samples to achieve $98% call rate. They observed low call rates (47.5% to 77.5%) when using unpurified clinical samples. By quantifying sample NA17316 using dPCR and performing gravimetric dilutions prior to genotyping analysis, an accurate starting copy number concentration of the sample was obtained. This enabled us to predict the number of copies of DNA required in each reaction chamber to obtain a call rate of 98-100%.

Validation of Simplex and Multiplex STA Conditions Prior to Genotyping Whale Samples
Twelve whale DNA samples were used for the validation process. The estimated DNA concentration of the samples varied significantly depending on the extraction method (Table 3). It was, therefore, necessary to incorporate an STA step prior to genotyping. The STA step was evaluated using either 15 or 45 SNP-GT assays in both simplex and multiplex format. Under simplex STA with 15 SNP-GT assays, for each sample an average call rate of greater than 99% was achieved on all four dilutions. Each dilution corresponded to different final copies per reaction chamber ranging from 17 to 662 (EG09-004), 58 to 2270 (EG09-012), and 49 to 1900 (Eden08-040), respectively. Similarly, using 45 SNP-GT assays, a call rate of 100% was achieved for both samples, EG09-012 (227 copies per reaction chamber) and Eden08-040 (189 copies per reaction chamber) ( Figure 3A and 3B). The genotype calls showed 100% match with the Illumina transcriptome sequence data obtained at AAD [13].
For multiplex STA, a call rate and a call accuracy of 100% was achieved for all samples when 15 SNP-GT assays were used ( Figure 3A). Similarly, using 45 SNP-GT assays with two samples, EG09-012 (511 copies per reaction chamber) and Eden08-040 (426 copies per reaction chamber), a call rate and a call accuracy of 100% was achieved ( Figure 3B). For samples EG09-012 and Eden08-040, the number of copies per reaction chamber post multiplex STA with 45 SNP-GT assays was higher than post simplex STA, since post simplex STA, a 1:45 dilution was achieved as a result of pooling of all 45 individual PCR reactions, while, post multiplex STA, a 1:20 dilution was performed. The data clustering was better for samples with a higher yield obtained using modified CTAB method compared to samples extracted using the Maxwell H Kit but this did not affect the final genotype call once STA was performed. These results suggest that DNA samples with sub-optimal quantity can be successfully genotyped following multiplex STA.
In this study one additional observation was made, clustering of data points relies on specificity of the probes. The GT call data is represented as clusters on a scatter plot using the standard format of Cartesian display [17]. Cartesian coordinates use the end-point fluorescence intensities acquired for each fluorophore (FAM and VIC) on the X and Y-axis to represent the X and Y allele [17]. During the multiplex validation process using either 15 or 45 different SNP-GT assays, for one particular assay (Exonic MALL), the data point clusters for the three different genotypes (qq, pq and pp) were in very close proximity making it difficult to confidently accept the assigned genotype call (Figure 4). The fluorescent intensities corresponding to all three clusters showed positive calls for both targets. Therefore the assigned homozygous calls were manually changed to heterozygous calls since the fluorescence intensities on the X and Y-axis were similar. However, once the genotype of the sample was revealed, it was evident that the manually assigned call, pq, was actually incorrect based on the sequence data. A possible explanation is that the probes (FAM and VIC) with low specificity could result in crossreactivity leading to an incorrect genotype call (Fluidigm, personal communication).
In conclusion, in the current study we successfully demonstrated that genotype call rate is dependant on gene copy number and then implemented the validated method to genotype 45 SNPs in the humpback whale nuclear genome using a nanofluidic IFC based genotyping system. The minimum heterozygous copies required to achieve a call rate of 100% was 38 haploid human genome copies per reaction chamber. This level of accuracy was achieved by first quantifying DNA sample using dPCR to get an accurate starting copy number concentration and then by performing gravimetric dilutions prior to genotyping. This procedure resulted in predicted number of copies dispensed in each reaction chamber. Our results demonstrate that low input copy number affects the quality of data generated, in particular when a heterozygous (pq) sample is used. For samples with low starting input DNA concentrations, incorporation of STA step prior to genotyping improved the call rate and accuracy to 100%. The proposed method validation with STA enables genotyping on the 48.48GT nanofluidic Dynamic Array TM with excellent call rate and accuracy with no less than 1 ng/mL starting input DNA concentration. The genotype calls obtained for twelve whale samples using the validated method showed 100% concordance with the true genotypes determined by an independent method at the AAD. The simple work-flow employed in setting up reactions on the nanofluidic Dynamic Array TM , combined with STA prior to genotyping proved to be an efficient, fast and accurate way for obtaining correct genotype call with high call rate and accuracy.

Sample/Assay Details
For evaluating the effect of input DNA copy number on genotype call rate, three human genomic DNA samples each with a different genotype for a single SNP (rs513349) [NA17317 (pp), NA17316 (pq) and NA17313 (qq)] were purchased from Coriell Cell Repositories, Camden, New Jersey. The SNP assay Reaction chamber (9:1 mixture of sample and assay) copies/reaction chamber Where A = stock DNA concentration (ng/mL); l = length in bp (human and whale genomes ,3610 9 bp); V 1 = DNA sample volume for STA (1.25 mL); V 2 = STA mixture volume (5 mL); n = Total number of PCR cycles; V 3 = Pooled mixture volume, which is derived by multiplying V 2 and the number of SNP-GT assays (V 3 = V 2 6SNP-GT assays); D = Dilution factor (5-or 20-fold); V 4 = DNA sample volume for genotyping (2.1 mL); V 5 = GT sample solution volume (5 mL) and V 6 = Reaction chamber volume (6.75 nL) [9]. E SX was calculated assuming 100% PCR efficiency. For validation of simplex and multiplex STA, total DNA was extracted from 30 mg epidermis tissue biopsies obtained from six adult humpback whales, Megaptera novaeangliae, using two different methods, a Maxwell H tissue DNA extraction Kit (Promega) and a modified CTAB protocol [18] at the Australian Antarctic Division (AAD) ( Table 3). With the Maxwell H Kit, the homogenized tissue was added to the automated DNA purification cartridge and DNA was eluted in 250 mL of 16 TE (10 mM Tris, 1 mM EDTA, pH 8.0). The DNA concentration was assessed on a Nanodrop 3300 (Thermo Fisher Scientific, Australia). The 45 whale SNP-GT assays were previously validated by Polanowski et al. [13]. The genotype identity of the whale samples was not revealed to the scientists undertaking the SNP-GT validation at NMI.

Digital PCR Measurement of Human Genomic DNA Samples
Digital PCR analysis was performed on the BioMark TM System using the 12.765 Digital Arrays TM (Fluidigm, South San Francisco). A Digital Array TM consists of 12 panels, each containing 765 individual reaction chambers. Three human genomic DNA samples were retrieved from 280uC and allowed to thaw at room temperature. The tubes were then incubated at 60uC at 800 rpm for 2 min in Eppendorf thermomixer, cooled to room temperature and briefly centrifuged. An aliquot (,40 mL) was pipetted into a polypropylene microcentrifuge tube (PN: MCT-175-C-S, Axygen INC, Union city, CA) and kept at 4uC overnight. Prior to gravimetric dilutions the samples were retrieved from 4uC and incubated at 60uC at 800 rpm for 2 min in an Eppendorf thermomixer, cooled to room temperature and analysed for UV absorbance at 260 and 280 nm. Such procedure for sample preparation was previously shown in our laboratory to achieve homogenous solution [19]. Based on the concentration  and 16SNP-GT assay (rs513349). To reduce the uncertainty from pipetting, all PCR components, excluding DNA, were pre-mixed and the final reaction mix was prepared gravimetrically by combining the DNA with the PCR pre-mix. Ten mL reaction mix was aliquoted into each sample inlet on the digital array and approximately 4.6 mL of the reaction mix was distributed throughout the partitions within each panel using an automated IFC controller-MX (Fluidigm, South San Francisco). Each DNA preparation was analysed in triplicate or quadruplicate using duplex conditions. The No Template Control (NTC) containing 16 TE 0.1 buffer in place of DNA was analysed in a single panel. PCR was performed using a modified fast thermocycling condition: 2 min at 95uC, followed by 45 cycles of a 2-step amplification profile of 10 s at 95uC and 30 s at 60uC.
TaqMan H SNP-genotyping Protocol SNP-GT reactions were setup by preparing the assays and the samples separately according to manufacturer's instruction [17]. For each SNP-GT assay, 5 mL of a 106 assay reaction (subsequently referred to as 106 SNP-GT assay) was prepared by mixing 2.  Figure 5A).
The 48.48GT Dynamic Array TM was placed on the IFC controller -MX (Fluidigm) for loading, mixing (Step 3, Figure 5A) and partitioning of each sample / assay combination at a 9:1 ratio . The genotype call (pp, qq and pq) for each reaction are denoted in 'red', 'green' and 'blue', respectively. '+' refers to samples extracted using CTAB method; '*' refers to each sample extracted using both CTAB and Maxwell H tissue extraction kit; '¤' refers to concordance with true genotype determined at AAD using an independent method. Note: Regardless of the approach used, genotypes for representative samples using either 15 or 45 SNP-GT assays were the same, as indicated by the same color. Samples EG09-004, EVH09-53, WA07-006 and WA07-003, extracted using CTAB or Maxwell H tissue extraction kit were genotyped using 15 SNP-GT assays with and without STA and showed a 100% call rate and concordance (data not shown). doi:10.1371/journal.pone.0039181.g003 Table 3. Samples analysed using the SNP-GT nanofluidic system.  [17]. However, as a result of partitioning of each sample / assay combination at a 9:1 ratio, this equates to 6.1 nL of sample and 0.7 nL of assay dispensed into individual reaction chamber. The chip was then placed on the BioMark TM instrument for thermal cycling (Step 4, Figure 5A): 2 min at 50 uC, 10 min at 95uC followed by 40 cycles of a 2-step amplification profile of 15 s at 95uC and 1 min at 60uC. The data was analysed using the Fluidigm genotyping analysis software v3.0 which produces a genotype call for each sample / assay combination (Step 5, Figure 5A). In this study, default confidence threshold (65) was used to identify the call error and the spread of data points. Confidence threshold reflects the level of confidence in the display of data points for a particular SNP assay and when a call confidence is less than the threshold, the resulting call is assigned No Call [17].

Specific Target Amplification
STA validation was performed initially in a simplex format for each assay and then under multiplex conditions. For simplex STA, each 406 TaqMan H SNP-GT assay was diluted to a 0.26 concentration. For multiplex STA, all 406 TaqMan H SNP-GT assays were pooled and the mix was diluted to a 0.26 concentration. Five mL simplex or multiplex STA reaction mix consisted of 2.5 mL 26 TaqMan H PreAmp master mix (PN: 4391128, Applied Biosystems), 1.25 mL DNA sample and 1.25 mL of either an individual 0.26 TaqMan H SNP-GT assay or the 0.26 TaqMan H SNP-GT pooled assay mix (Step 6, Figure 5B). In the STA negative control (SNTC) used to monitor for false positives (Step 6, Figure 5B), 16 TE 0.1 was used instead of DNA.
The 5 mL individual STA reaction mixes were pipetted into separate wells of a 96 well plate (Step 7, Figure 5B) and STA was performed on the Eppendorf ep 'S' mastercycler (Step 8, Figure 5B) with 10 min at 95uC followed by 14 cycles of a 2step amplification profile of 15 s at 95uC and 2 min at 60uC. After simplex STA, the individually amplified products were pooled into one tube while after multiplex STA, the reaction was diluted 5-or 20-fold (Step 9, Figure 5B) prior to analysis using the TaqMan H SNP-GT protocol (Steps 2-5, Figure 5A).

Effect of DNA Copy Number on the Genotyping Call Rate for Three Human DNA Samples
To evaluate the effect of input DNA copy number on genotype call rate, sufficient GT-sample solution and 106 SNP-GT assays were prepared. Three human DNA samples from Coriell were each gravimetrically diluted based on the concentration measured by dPCR to achieve approximately 3.8610 4 , 1.8610 4 , 3.3610 3 , 1.6610 3 and 3.1610 2 copies/mL (haploid genome equivalents) which equates to 116, 56, 10, 5 and 1 ng/mL. These dilutions and NTC were loaded into the Dynamic Array TM in triplicate resulting in approximately 97, 46, 9, 4, 1 copies(y), respectively, per reaction chamber (estimated using equations 1-6 in Table 1). Four mL of the 106 SNP-GT assay was loaded into sixteen separate assay inlets evenly spaced across the 48.48GT array. The remaining inlets were loaded with a No Primer/Probe Control (NPC) in which the 406 SNP-GT assay (rs513349) was replaced with 16 TE 0.1 . NPC was used to monitor for cross contamination in the assay mix and also to test for possible leakage of assays between adjacent reaction chambers.

Effect of DNA Copy Number on Genotyping Call Rate for Heterozygous Human DNA Sample
Human DNA sample NA17316 was gravimetrically diluted based on the dPCR-measured concentration to approximately 38, 18, 7, 4 and 1 copies(y) per reaction chamber. Each dilution of GT-sample solution was loaded into nine replicate inlets. Three sample inlets were treated as NTC, which contained 16 TE 0.1 in place of DNA. Four mL of the 106SNP-GT assay was loaded into sixteen assigned assay inlets evenly spaced across the 48.48GT array. The remaining assay inlets were loaded with NPC. The PCR thermal cycling conditions were the same as described in the TaqMan H SNP-GT protocol.
Effect of DNA Copy Number on the Genotyping Call Rate with STA for Whale DNA Samples The process for validating the STA method in simplex and multiplex format is illustrated in Figure 5B (Steps 6-9). STA initially used fifteen SNP-GT assays previously validated by Polanowski et al. [13] using real-time PCR and was subsequently adapted for 45 SNP-GT assays. Simplex STA validation was conducted using 15 SNP-GT assays on three samples (EG09-004, EG09-012 and Eden08-040) extracted using modified CTAB protocol [18] with input DNA concentrations ranging between 8.75 and 30 ng/mL (2 fold dilution from stock, Table 3). After Steps 1, 6-9 corresponds to STA reaction setup in simplex and multiplex conditions. Post STA, the amplified products are pooled (simplex STA), or further diluted 5 or 20 fold (multiplex STA) prior to performing TaqMan H SNP-GT setup using steps 2-5. doi:10.1371/journal.pone.0039181.g005 STA, 15 individual STA reactions for each sample were pooled and further diluted three-fold to achieve a total dilution of each individual STA reaction of 1:45. This dilution was designed to be equivalent to the dilution that would result from pooling 45 individual STA reactions. The pooled STA reaction for each sample was serially diluted and four dilutions analysed in triplicate using the same 15 SNP-GT assays and the TaqMan H -SNP-GT protocol (Steps 2-5, Figure 5A). The final copies per reaction chamber were derived using equations 1-3, 4 ¤ , 5-6 ( Table 1). Based on equations (1-6), Table 4 illustrates an example of the copy number estimates generated without STA or with either simplex or multiplex STA using 15 SNP-GT assays. The predicted copies per reaction chamber were 17 to 662, 58 to 2270 and 49 to 1900 for EG09-004, EG09-012 and Eden08-040, respectively; the difference in the ranges being attributed to the variable starting DNA concentrations prior to STA (Table 3).
Multiplex STA validation was undertaken on all twelve whale DNA samples (6 samples extracted using Maxwell H tissue DNA extraction Kit and same 6 samples extracted using modified CTAB), one NTC, one NAC, and three positive controls that had been pre-validated during the simplex STA step (EG09-012, EG09-004 and Eden08-040). After multiplex STA, each sample was diluted 20-fold to reduce the concentration of the multiplex primers prior to TaqMan H SNP-GT.
For simplex and multiplex STA validation using 45 SNP-GT assays, the standard STA protocol was followed (Steps 6-9, Figure 5B) using two samples (EG09-012 and Eden08-040). Each reaction was analysed in ten replicates against 45 SNP-GT assays using the TaqMan H SNP-GT protocol (Steps 2-5, Figure 5A). NTC, SNTC or NPC inlets containing 16TE 0.1 buffer in place of DNA and primer/probes were analysed in one or more alternate inlets. Blank inlets were used in order to accommodate the chipsetup; they contained 2.5 mL 26 TaqMan H universal PCR mastermix with AmpErase H UNG (PN: 4304437, Applied Biosystems) and 2.5 mL of 16 TE 0.1 and were different to NTC or SNTC. Table 4. Estimated DNA copy number in the reaction chamber with (simplex or multiplex) and without STA using whale genomic DNA. The DNA copy number in the reaction chamber (E RC ) was estimated using equations (1-6) derived in Table 1. *The copies/reaction chamber post-simplex and multiplex STA PCR is an estimate obtained when using 15 SNP-GT assays with a 5 -fold dilution post STA. doi:10.1371/journal.pone.0039181.t004