Fast sequence-based microsatellite genotyping development workflow.

Olivier Lepais,Cecile F.E Bacles,Emilie Chancerel,Christophe Boury,Abdeldjalil Aissi,Sophie Launey,Erwan Guichoux,Aurélie Manicki,Cyril Dutech,Laura Taillebois,Franck Salin,Françoise Daverat

doi:10.7717/peerj.9085

Abstract

Application of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20–40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

Highlights

Development of high-throughput sequencing (HTS) technologies since the mid 2000’s has greatly widened the scope of genetic applications in research fields such as agronomy, ecology and evolutionary biology where the use of molecular markers is common place
We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped
The easy collection of millions of DNA sequences has facilitated the development of molecular markers such as single nucleotide polymorphism (SNP; (Delord et al, 2018; Garvin, Saitoh, & Gharrett, 2010)) or microsatellites (or single sequence repeats SSR; (Guichoux et al, 2011; Malausa et al, 2011)) for any species of interest

Summary

Introduction

Development of high-throughput sequencing (HTS) technologies since the mid 2000’s has greatly widened the scope of genetic applications in research fields such as agronomy, ecology and evolutionary biology where the use of molecular markers is common place. The easy collection of millions of DNA sequences has facilitated the development of molecular markers such as single nucleotide polymorphism (SNP; (Delord et al, 2018; Garvin, Saitoh, & Gharrett, 2010)) or microsatellites (or single sequence repeats SSR; (Guichoux et al, 2011; Malausa et al, 2011)) for any species of interest. Restriction site-associated DNA polymorphism sequencing allows simultaneous identification and genotyping of SNP in non-model species and interrogation of thousands to hundreds of thousands of loci genome-wide but remains expensive when population samples comprise hundreds of individuals. While marker preference evolves through time with specific markers dominating the genotyping field over a period of time following technological advances (Schlötterer, 2004; Seeb et al, 2011), maintaining our capability to interrogate any kind of polymorphism in the context of rapid HTS technological advances is paramount and should be prioritized

Methods

Results

Conclusion