1. Competitive multiplex-PCR Amplicon Sequencing Library Preparation Note: Load into slide show mode, and click mouse periodically to play animation show Blomquist, T. et al 2013. PLoS One. thomas.blomquist@utoledo.edu

2. Competitive multiplex-PCR Amplicon Sequencing Library Preparation

6. Native : Standard [Internal Standard] loaded Assess Number of Times Native : Standard is Sequenced and multiply by [IS] concentration to determine native concentration

7. Native : Standard [Internal Standard] loaded Native : Standard [Internal Standard] loaded Complementary DNA (mRNA)Genomic DNA (gDNA) cDNA gives a broad distribution of titration curves compared to gDNA. This is due to the high variation in abundance between transcripts for cDNA, but not between loci in gDNA.

8. A B C D E F 10 9 - 10 8 - 10 7 - 10 6 - 10 5 - 10 4 - 10 3 - 10 2 - Transcript Targets Relative Abundance Primer Concentration Limiting Cap Before PCR Native Targets Competitive Internal Standards 1,000,000-fold range Competitive Amplicon Library Preparation Convergence Concept Internal Standards (IS) “Abundant” NT 10 8 copies Native Targets (NT) (e.g. cDNA) “Rare” NT 10 2 copies ****** + “Abundant” Target IS 10 5 copies “Rare” Target IS 10 5 copies

9. A B C D E F 10 9 - 10 8 - 10 7 - 10 6 - 10 5 - 10 4 - 10 3 - 10 2 - Transcript Targets Relative Abundance Primer Concentration Limiting Cap Before PCR After PCR A B C D E F Transcript Targets Native Targets Competitive Internal Standards 1,000,000-fold range 1,000-fold range 10 9 - 10 8 - 10 7 - 10 6 - 10 5 - 10 4 - 10 3 - 10 2 - Competitive Amplicon Library Preparation Convergence Concept “Rare” Target “Abundant” Target

10. A B C D E F 10 9 - 10 8 - 10 7 - 10 6 - 10 5 - 10 4 - 10 3 - 10 2 - Transcript Targets Relative Abundance Primer Concentration Limiting Cap Before PCR After PCR A B C D E F Transcript Targets Native Targets Competitive Internal Standards 1,000,000-fold range 1,000-fold range 10 9 - 10 8 - 10 7 - 10 6 - 10 5 - 10 4 - 10 3 - 10 2 - Competitive Amplicon Library Preparation Convergence Concept 10 3 reduction in Sequencing Reads* * With one internal standard spike-in. Fewer reads may be required using multiple technical replicates each with different Internal Standard Spike-in ratios relative to the native mixture.