Following the Dynamics of Structural Variants in Experimentally Evolved Populations

Summary

We developed a cost-effective method to follow non-single nucleotide polymorphism allele dynamics that can easily be adapted to experimental evolution frozen archives. A triplet PCR technique was coupled with automated parallel capillary electrophoresis to quantify the relative frequency of an insertion allele over the course of experimental evolution.

Abstract

Structural variants (SVs) (i.e., deletions, insertions, duplications, and inversions) are now known to play an important role in phenotypic variation, and consequently in processes such as disease determination or adaptation to a new environment. However, single-nucleotide variants receive much more attention than SVs, probably because they are easier to detect, and their phenotypic effects are easier to predict. The development of short- and long-read deep sequencing technologies have strongly improved the detection of SVs, but the quantification of their frequency from pooled sequencing (poolseq) data is still technically complex and expensive.

Here, we present a rather simple and inexpensive method, which allows researchers to follow the dynamics of SV allele frequency. As an example of application, we follow the frequency of an insertion sequence (IS) insertion in experimental evolution populations of bacteria. This method is based on the design of triplets of primers around the structural variant borders, such that the amplicons produced by amplification of the wild-type (WT) and derived alleles differ in size by at least 5%, and that their amplification efficiency is similar. The quantity of each amplicon is then determined by parallel capillary electrophoresis and normalized to a calibration curve. This method can be easily extended to the quantification of the frequency of other structural variants (deletions, duplications, and inversions) and to pool-seq approaches of natural populations, including within-patient pathogen populations.

Introduction

Structural variants (SVs) are alterations of the genomic sequence, generally affecting 50 bp or more. The four categories of described SVs are large insertions, large deletions, inversions, and duplications. Until recently, more attention has been devoted to single-nucleotide variants (SNVs) than to structural variants, in terms of their phenotypic effects and their role as genetic determinants of disease, or their contribution to adaptation. This is probably because it is easier to both detect SNVs and predict their phenotypic effects. However, short- and long-read deep sequencing technologies have strongly improved the detection of SVs, at least in single individual....

Protocol

Setting up this protocol requires precise knowledge of the insertion, deletion, inversion, or duplication point within the ancestral sequence. This information is usually obtained by whole-genome sequencing (WGS) of the end or intermediate point samples. In the following protocol, the general principle for the case of an insertion mutation is given for each step, alongside a representative case where the frequency of an IS10 insertion in the mutS gene in an experimental evolution population of E. coli is followed. In this population, WGS of the endpoint population identified the insertion of a 1,329 bp IS10 between positions 2,463 and 2,471, resultin....

Results

Using DNA extracted from an ancestral clone and a hypermutator clone isolated from the S2.11 population at generation 1,000, we established the calibration curve shown in Figure 2. The actual mutant proportions from laboratory-prepared DNA mixes and measured by the parallel capillary electrophoresis instrument were linked by a linear relationship of slope 1.0706, with an R² of 0.9705. Additionally, there was a good agreement between biological replicates; the standard deviation wa.......

Discussion

Here, we have proposed a cost-effective method that allows the dynamics of emerging adaptive SV alleles in experimental evolution populations to be followed. This method couples classic PCR techniques and automated parallel capillary electrophoresis, allowing for the relative quantities of two alleles to be determined. Once set up, it permits the quantification of allele proportions in many samples in parallel, and is much less expensive than WGS. This method can be seen as an equivalent to amplicon sequencing for non-SN.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96 Well Skirted PCR Plate	4titude	4Ti - 0740	PCR
Agarose molecular biology grade	Eurogentec	EP-0010-05	Agarose gel electrophoresis
Agilent DNF-474 HS NGS Fragment Kit Quick Guide for the Fragment Analyzer Systems	Agilent		PDF instruction guide
Buffer TBE	Panreac appliChem	A4228,5000Pc	Agarose gel electrophoresis
Calibrated Disposable Inoculating Loops and Needles	LABELIANS	8175CSR40H	Bacterial culture
Dneasy Blood and Tissue Kit	Qiagen	69506	DNA extraction
Electrophoresis power supply	Amilabo	ST606T	Agarose gel electrophoresis
Fragment Analyzer Automated CE System	Agilent		Parallel capillary electrophoresis
Fragment DNA Ladder	Agilent	DNF-396, range 1-6000bp	Parallel capillary electrophoresis
GENTAMICIN SULFATE SALT BIOREAGENT	Sigma-Aldrich	G1264-1G	Bacterial culture
High Sensitivity diluent marker	Agilent	DNF-373	Parallel capillary electrophoresis
High Sensitivity NGS quantitative analysis kit	Agilent	DNF-474	Parallel capillary electrophoresis
Ladder quick load 1 kb plus DNA ladder	NEB	N0469S	Agarose gel electrophoresis
LB Broth, VegitoneNutriSelect Plus	Millipore	28713	Bacterial culture
Master Mix PCR High Fidelity Phusion Flash	Thermo Fisher Scientific	F548L	PCR
Primers	Eurogentec		PCR
Prosize data analysis software v.4	Agilent	V.4	Parallel capillary electrophoresis
Qubit assays	Invitrogen	MAN0010876	DNA quantification
Qubit dsDNA HS Assay Kit	LIFE TECHNOLOGIES SAS	Q32854	DNA quantification
Thermocycler	Eppendorf	Ep gradients	PCR
UVbox, eBOX VX5	Vilber Lourmat		Agarose gel electrophoresis visualisation
Water for injectable preparation	Aguettant	PROAMP	PCR