Sequence and primer independent stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding

BioRxiv : the Preprint Server for Biology
Katharine BestBenny Chain

Abstract

The polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology. In combination with High Throughput Sequencing (HTS), PCR is widely used to quantify transcript abundance for RNA-seq and especially in the context of analysis of T cell and B cell receptor repertoires. In this study, we combine molecular DNA barcoding with HTS to quantify PCR output from individual target molecules. Our results demonstrate that the PCR process exhibits very significant unexpected heterogeneity, which is independent of the sequence of the primers or target, and independent of bulk experimental conditions. The mechanistic origin of this heterogeneity is not clear, but simulations suggest that it must derive from inherited differences between different DNA molecules within the reaction. The results illustrate that single molecule barcoding is important in order to derive reproducible quantitative results from any protocol which combines PCR with HTS.

Related Concepts

B-Lymphocytes
DNA
Molecular Biology
Polymerase Chain Reaction
T-Lymphocyte
Oligonucleotide Primers
Simulation
DNA Amplification
High Throughput Analysis
Sequence Determinations, RNA

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