High-throughput and Cost-effective Chicken Genotyping Using Next-Generation Sequencing.

Fábio Pértille,Clarissa Boschiero,Per Jensen,Vinicius Henrique Da Silva,Luiz Lehmann Coutinho,José De Ribamar Da Silva Nunes,Mônica Corrêa Ledur,Carlos Guerrero-Bosagna

doi:10.1038/srep26929

Abstract

Chicken genotyping is becoming common practice in conventional animal breeding improvement. Despite the power of high-throughput methods for genotyping, their high cost limits large scale use in animal breeding and selection. In the present paper we optimized the CornellGBS, an efficient and cost-effective genotyping by sequence approach developed in plants, for its application in chickens. Here we describe the successful genotyping of a large number of chickens (462) using CornellGBS approach. Genomic DNA was cleaved with the PstI enzyme, ligated to adapters with barcodes identifying individual animals, and then sequenced on Illumina platform. After filtering parameters were applied, 134,528 SNPs were identified in our experimental population of chickens. Of these SNPs, 67,096 had a minimum taxon call rate of 90% and were considered ‘unique tags’. Interestingly, 20.7% of these unique tags have not been previously reported in the dbSNP. Moreover, 92.6% of these SNPs were concordant with a previous Whole Chicken-genome re-sequencing dataset used for validation purposes. The application of CornellGBS in chickens showed high performance to infer SNPs, particularly in exonic regions and microchromosomes. This approach represents a cost-effective (~US$50/sample) and powerful alternative to current genotyping methods, which has the potential to improve whole-genome selection (WGS), and genome-wide association studies (GWAS) in chicken production.

Highlights

Next-generation sequencing (NGS) analyses have been increasingly employed in production animals, in chickens
Reduced representation methods can be grouped in three classes: (1) reduced-representation sequencing, which includes methods such as reduced-representation libraries (RRLs) and complexity reduction of polymorphic sequences (CRoPS); (2) restriction-site-associated DNA sequencing (RAD-Seq); and (3) low coverage genotyping, which includes methods such as multiplexed shotgun genotyping (MSG), genotyping by sequencing from Cornell
The present study aims at constructing reduced genome representation sequencing libraries using the CornellGBS approach in chickens

Summary

Introduction

Next-generation sequencing (NGS) analyses have been increasingly employed in production animals, in chickens. Single Nucleotide polymorphisms (SNPs) are the most abundant type of molecular markers and their high genomic density facilitates their interrogation by different genetic approaches These include large-scale genome association analyses, genetic analysis of simple and complex disease states, and population genetic studies[2]. CornellGBS is a simple reproducible method based on the Illumina sequencing platform[13] that requires low input of DNA (100 ng) This method allows for a highly multiplexed approach, which is achieved through the incorporation of unique barcodes that identify individual samples in a DNA pool to be sequenced. This approach avoids the low sequence diversity in which the restriction enzyme overhangs appear at the same position in every read, by employing barcodes of variable lengths[9]. To the best of our knowledge this method has not been applied in chicken

Methods

Results

Conclusion