# Sources of PCR-induced distortions in high-throughput sequencing datasets

## TL;DR

This paper investigates how PCR introduces biases and errors into high-throughput sequencing data, finding that stochastic amplification has the biggest impact.

## Contribution

The study quantitatively models PCR-induced biases and identifies stochasticity as the dominant source of sequence misrepresentation.

## Key findings

- PCR stochasticity is the main factor skewing sequence representation in sequencing libraries.
- PCR errors are frequent in later cycles but affect only low-copy sequences.
- Template switches during PCR are rare and have minimal impact on overall sequence distribution.

## Abstract

PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error — bias, stochasticity, template switches and polymerase errors — on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules.