Figure 1.
Effect of starting copy number on genotype call rate with and without STA.
[A] and [B] Call map view and scatter plots of three human genomic DNA samples showing clustering (pp-‘red’, qq-‘green’ and pq ‘blue’) 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.
Table 1.
Derivation of DNA copy number concentration in the final reaction chamber with and without STA.
Figure 2.
Effect of reaction DNA copy number on genotype call accuracy for a heterozygous (pq) sample.
Call map view [A] and scatter plots [B] of the genotype calls from reaction chambers containing predicted 38, 18, 7, 4 and 1 copies(y). The genotype call (pp, qq and pq) for each reaction is denoted in ‘red’, ‘green’ and ‘blue’, 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.
Table 2.
Effect of DNA copy number on reliability of genotype call data for a heterozygous human genomic DNA sample, NA17316.
Figure 3.
Summary genotype calls obtained for representative whale DNA samples.
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 (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® 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).
Table 3.
Samples analysed using the SNP-GT nanofluidic system.
Figure 4.
Scatter plots showing genotype call clusters for 46 whale samples using one assay (Exonic MALL).
The groups (pp-‘red’, qq-‘green’ and pq ‘blue’) are denoted in circles.
Figure 5.
Genotyping analysis workflow with and without STA.
[A] Steps 1–5 (denoted in red arrows) correspond to TaqMan® SNP-GT protocol without STA or following simplex or multiplex STA. [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.
Table 4.
Estimated DNA copy number in the reaction chamber with (simplex or multiplex) and without STA using whale genomic DNA.