Abstract
Next-generation sequencing (NGS) approaches are widely used in genome-wide genetic marker discovery and genotyping. However, current NGS approaches are not easy to apply to general outbred populations (human and some major farm animals) for SNP identification because of the high level of heterogeneity and phase ambiguity in the haplotype. Here, we reported a new method for SNP genotyping, called genotyping by genome reducing and sequencing (GGRS) to genotype outbred species. Through an improved procedure for library preparation and a marker discovery and genotyping pipeline, the GGRS approach can genotype outbred species cost-effectively and high-reproducibly. We also evaluated the efficiency and accuracy of our approach for high-density SNP discovery and genotyping in a large genome pig species (2.8 Gb), for which more than 70,000 single nucleotide polymorphisms (SNPs) can be identified for an expenditure of only $80 (USD)/sample.
Highlights
Genetic variants, single nucleotide polymorphisms (SNPs), are the basis of genetics and enable study of the genetic mechanism underlying human diseases and agriculturally important traits
The procedure of library preparation for genotyping by genome reducing and sequencing (GGRS) approach was improved based on restriction site-association DNA sequencing (RAD-seq) and genotyping by sequencing (GBS) to adapt genotyping for outbred populations
The variation of reads number between individuals was considerably lower than that achieved by the multiplexed shotgun genotyping (MSG) method and the same as that achieved by the GBS method
Summary
Single nucleotide polymorphisms (SNPs), are the basis of genetics and enable study of the genetic mechanism underlying human diseases and agriculturally important traits. Most NGS methods, such as genotyping by sequencing (GBS) [3], multiplexed shotgun genotyping (MSG) [4] and restriction site-association DNA sequencing (RAD-seq), depend on restriction enzymes to produce a reduced representation of a genome [5]. The GBS and MSG methods, which rely on low sequencing depth (,56/site per individual, on average), are simple and cost-effective approaches for genotyping inbred populations in which the parental genotypes are either known or can be assigned probabilities. Accurate genotype calling is difficult to achieve by low sequencing depth approaches for general outbred populations because of the high degree of heterogeneity and phase ambiguity in the haplotype. The RAD-seq method, which sequences target regions deeply (.206) and enables markers to be genotyped accurately for outbred populations, is expensive and labor-intensive for highthroughput SNP detection because of the high sequencing depths and complex library preparation protocol [6]
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