Competitive multiplex-PCR Amplicon Sequencing Library Preparation

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Blomquist, T. et al 2013. PLoS One.thomas.blomquist@utoledo.edu

Competitive multiplex-PCR Amplicon Sequencing Library Preparation

Sample ofNative Targets(e.g. cDNA or gDNA)

Mixture ofInternal Standards

+

Mix Native Targets (NT) and Internal Standards (IS) in Varying Ratios

******

Competitive multiplex-PCR Amplicon Sequencing Library Preparation

Sample ofNative Targets(e.g. cDNA or gDNA)

Mixture ofInternal Standards

+

Mix Native Targets (NT) and Internal Standards (IS) in Varying Ratios

******

Competitive multiplex-PCR Amplicon Sequencing Library Preparation

Competitive multiplex-PCR Amplicon Sequencing Library Preparation

Sample ofNative Targets(e.g. cDNA or gDNA)

Mixture ofInternal Standards

+

Mix Native Targets (NT) and Internal Standards (IS) in Varying Ratios

******

Competitive multiplex-PCR Amplicon Sequencing Library Preparation

Sample ofNative Targets(e.g. cDNA or gDNA)

Mixture ofInternal Standards

+

Mix Native Targets (NT) and Internal Standards (IS) in Varying Ratios

******

Nati

ve :

Stan

dard

[Internal Standard] loaded

Assess Number of TimesNative : Standard is Sequencedand multiply by [IS] concentrationto determine native concentration

Nati

ve :

Stan

dard

[Internal Standard] loaded

Nati

ve :

Stan

dard

[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.

A B C D E F

109-

108-

107-

106-

105-

104-

103-

102-

Transcript Targets

Rela

tive

Abun

danc

e

Primer ConcentrationLimiting Cap

Before PCR

Native TargetsCompetitive Internal Standards

1,000,000-fold range

Competitive Amplicon Library Preparation Convergence Concept

Internal Standards (IS)

“Abundant” NT108 copies

Native Targets (NT) (e.g. cDNA)

“Rare” NT102 copies

****** ************ ******

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****** ************ ******

******

+

“Abundant” Target IS105 copies

“Rare” Target IS105 copies

A B C D E F

109-

108-

107-

106-

105-

104-

103-

102-

Transcript Targets

Rela

tive

Abun

danc

e

Primer ConcentrationLimiting Cap

Before PCR After PCR

A B C D E FTranscript Targets

Native TargetsCompetitive Internal Standards

1,000,000-fold range1,

000-

fold

rang

e

109-

108-

107-

106-

105-

104-

103-

102-

Competitive Amplicon Library Preparation Convergence Concept

“Rare” Target

“Abundant” Target

A B C D E F

109-

108-

107-

106-

105-

104-

103-

102-

Transcript Targets

Rela

tive

Abun

danc

e

Primer ConcentrationLimiting Cap

Before PCR After PCR

A B C D E FTranscript Targets

Native TargetsCompetitive Internal Standards

1,000,000-fold range1,

000-

fold

rang

e

109-

108-

107-

106-

105-

104-

103-

102-

Competitive Amplicon Library Preparation Convergence Concept

103 reductionin 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.