Genotyping analysis workflow with and without STA.

<p><b>[A]</b> Steps 1–5 (denoted in red arrows) correspond to TaqMan^® SNP-GT protocol without STA or following simplex or multiplex STA. <b>[B]</b> 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^® SNP-GT setup using steps 2–5.</p

Derivation of DNA copy number concentration in the final reaction chamber with and without STA.

<p>Where <i>A</i> =  stock DNA concentration (ng/µL); <i>l</i> =  length in bp (human and whale genomes ∼3×10⁹ bp); V₁ =  DNA sample volume for STA (1.25 µL); V₂ =  STA mixture volume (5 µL); n =  Total number of PCR cycles; V₃ =  Pooled mixture volume, which is derived by multiplying V₂ and the number of SNP-GT assays (V₃ = V₂×SNP-GT assays); D =  Dilution factor (5- or 20- fold); V₄ =  DNA sample volume for genotyping (2.1 µL); V₅ =  GT sample solution volume (5 µL) and V₆ =  Reaction chamber volume (6.75 nL)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039181#pone.0039181-Wang1" target="_blank">[9]</a>.</p><p><i>E_SX</i> was calculated assuming 100% PCR efficiency.</p>†<p>Use equations 1, 5–6 when estimating copies/reaction chamber without STA.</p><p>Use equations 1–6 when estimating copies/reaction chamber with STA in simplex.</p><p>Use equations 1–3, 4^♦ and 5–6 when estimating copies/reaction chamber with STA in Multiplex.</p>*<p>The Avogadro number (6.02214179×10²³) was taken from Mohr et al<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039181#pone.0039181-Mohr1" target="_blank">[20]</a> (CODATA-2006).</p><p>•Average molecular weight of DNA base pair used was 615.8771<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039181#pone.0039181-Doleel1" target="_blank">[21]</a>.</p><p>NA – refers to Not Applicable.</p

Effect of DNA copy number on reliability of genotype call data for a heterozygous human genomic DNA sample, NA17316.

<p> <i>Call rate and call accuracy (%) at different number of copies per reaction chamber and for pq calls were determined from 144 data points.</i></p><p> <i>⁽¹⁾% Call rate [All Calls]  = 100*[(Total number of calls) / (Total number of calls + No Calls)].</i></p><p> <i>⁽²⁾% Call rate [pq Calls]  =  100*[(Correct Calls) / (Total number of calls + No Calls)].</i></p><p> <i>⁽³⁾% Call accuracy [pq Calls]  = 100*[(Correct Calls) / (Total number of calls)].</i></p

Effect of starting copy number on genotype call rate with and without STA.

<p>[A] and <b>[B]</b> Call map view and scatter plots of three human genomic DNA samples showing clustering (<b>pp</b>-‘<b>red</b>’, <b>qq</b>-‘<b>green</b>’ and <b>pq</b> ‘<b>blue</b>’) for a single SNP with and without STA. Black and Grey colors correspond to No Calls. The reaction chambers contained different copies ranging, on average, from approximately 97 to 1 copies(y) without STA and 2.0×10⁴ to 1.6×10² copies with STA. SNP-GT assay (rs513349) was loaded into sixteen separate assay inlets evenly spaced across the 48.48GT array. The remaining inlets were loaded with a NPC as stated in the Methods section.</p

Summary genotype calls obtained for representative whale DNA samples.

<p>Genotype calls obtained for representative whale DNA samples extracted using CTAB or Maxwell^® tissue extraction kit and following simplex or multiplex STA using either 15 [A] or 45 SNP-GT assays [B]. The genotype call (<b>pp</b>, <b>qq</b> and <b>pq</b>) for each reaction are denoted in ‘<b>red</b>’, ‘<b>green</b>’ and ‘<b>blue</b>’, respectively. ‘+’ refers to samples extracted using CTAB method; '*' refers to each sample extracted using both CTAB and Maxwell^® 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^® 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).</p

Estimated DNA copy number in the reaction chamber with (simplex or multiplex) and without STA using whale genomic DNA.

<p> <i>The DNA copy number in the reaction chamber (E_RC) was estimated using equations (1–6) derived in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039181#pone-0039181-t001" target="_blank">Table 1</a>.</i></p>*<p> <i>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.</i></p

Scatter plots showing genotype call clusters for 46 whale samples using one assay (Exonic MALL).

<p>The groups (<b>pp</b>-‘<b>red</b>’, <b>qq</b>-‘<b>green</b>’ and <b>pq</b> ‘<b>blue</b>’) are denoted in circles.</p

Samples analysed using the SNP-GT nanofluidic system.

<p>Samples analysed using the SNP-GT nanofluidic system.</p

Effect of reaction DNA copy number on genotype call accuracy for a heterozygous (pq) sample.

<p>Call map view <b>[A]</b> and scatter plots <b>[B]</b> of the genotype calls from reaction chambers containing predicted 38, 18, 7, 4 and 1 copies(y). The genotype call (<b>pp</b>, <b>qq</b> and <b>pq</b>) for each reaction is denoted in ‘<b>red</b>’, ‘<b>green</b>’ and ‘<b>blue</b>’, respectively. No Call and NPC are denoted in ‘grey’ and NTCs in ‘black’. SNP-GT assay (rs513349) was loaded into sixteen separate assay inlets evenly spaced across the 48.48GT array. The remaining inlets were loaded with a NPC as stated in the Methods section.</p