Competitive multiplexPCR Amplicon Sequencing Library Preparation Blomquist T

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

Competitive multiplex-PCR Amplicon Sequencing Library Preparation

Competitive multiplex-PCR Amplicon Sequencing Library Preparation Mix Native Targets (NT) and Internal Standards (IS) in Varying Ratios Mixture of Internal Standards Sample of Native Targets (e. g. c. DNA or g. DNA) + ******

Competitive multiplex-PCR Amplicon Sequencing Library Preparation Mix Native Targets (NT) and Internal Standards (IS) in Varying Ratios Mixture of Internal Standards Sample of Native Targets (e. g. c. DNA or g. DNA) + ******

Competitive multiplex-PCR Amplicon Sequencing Library Preparation Mix Native Targets (NT) and Internal Standards (IS) in Varying Ratios Mixture of Internal Standards Sample of Native Targets (e. g. c. DNA or g. DNA) + ******

Competitive multiplex-PCR Amplicon Sequencing Library Preparation Mix Native Targets (NT) and Internal Standards (IS) in Varying Ratios Mixture of Internal Standards + ****** Native : Standard Sample of Native Targets (e. g. c. DNA or g. DNA) Assess Number of Times Native : Standard is Sequenced and multiply by [IS] concentration to determine native concentration [Internal Standard] loaded

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

Competitive Amplicon Library Preparation Convergence Concept Before PCR Native Targets (NT) (e. g. c. DNA) Primer Concentration Limiting Cap 108107 - “Rare” NT 102 copies “Abundant” NT 108 copies 1061, 000 -fold range Relative Abundance 109 - 105104103102 A B C D E F Transcript Targets Native Targets Competitive Internal Standards + Internal Standards (IS) ****** ****** “Abundant” Target IS 105 copies ****** ****** “Rare” Target IS 105 copies

Competitive Amplicon Library Preparation Convergence Concept Before PCR 108 - “Abundant” Target 107106105104103102 A B C D E F Transcript Targets Native Targets Competitive Internal Standards 1, 000 -fold range Primer Concentration Limiting Cap 1, 000 -fold range Relative Abundance 109 - After PCR 109108107106105104 - “Rare” Target 103 A B C D E F Transcript Targets 102 -

Competitive Amplicon Library Preparation Convergence Concept Before PCR 108 - 1, 000 -fold range Primer Concentration Limiting Cap 107106105104103102 A B C D E F Transcript Targets 109108107106 - 1, 000 -fold range Relative Abundance 109 - After PCR 105104103 - 103 reduction in Sequencing Reads* A B C D E F Transcript Targets 102 - Native Targets * With one internal standard spike-in. Fewer reads may be required using multiple technical Competitive Internal Standards replicates each with different Internal Standard Spike-in ratios relative to the native mixture.