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 [email protected]
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
****** ************ ******
******
****** ************ ******
******
+
“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.
Top Related