Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing.

Eyal Seroussi

doi:10.3390/genes12020283

Abstract

Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.

Highlights

IntroductionPublisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations
Recent developments in estimating copy-number proportions based on direct Sanger sequencing indicate that such quantification is gaining in scientific acceptance
Frequent indels are associated with outcomes of CRISPR-Cas9 base editing and with analyses of mitochondrial heteroplasmy; and the need to quantify these sparked the development of new software tools to infer copy-number proportions from Sanger trace files

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. A notable commercial application for such quantification is the Mutation Quantifier application of the Mutation Surveyor Software package [15] This application improves the detection of variants with low copy-number proportion (~5%) by comparing the observed and expected peak heights, based on the assumption that intensity ratio of the neighboring same-color peaks is consistent in the samples; this is not always valid, as peak intensities are influenced by their local sequence context [3]. A method termed polymorphism-ratio sequencing (PRS) was developed for this purpose based on microfabricated capillary array electrophoresis and the Sanger protocol [21] This method0 s limit of minor allele frequency detection was 5%, compared to the limit of similar magnitude (5–7.5%) reported for standard. The need to quantify the efficiency of genome-editing enzymes further promoted the development of more sophisticated methods capable of sensitive analyses of indels in Sanger trace files

Base Editing by CRISPR-Cas9 Endonucleases and Nickases

C Methylation

RNA Editing

Findings

Conclusions