Abstract
Next-generation sequencing (NGS) technology is being applied to an increasing number of non-model species and has been used as the primary approach for accurate genotyping in genetic and evolutionary studies. However, inferring genotypes from sequencing data is challenging, particularly for organisms with a high degree of heterozygosity. This is because genotype calls from sequencing data are often inaccurate due to low sequencing coverage, and if this is not accounted for, genotype uncertainty can lead to serious bias in downstream analyses, such as quantitative trait locus mapping and genome-wide association studies. Here, we used high-coverage reference data sets from Crassostrea gigas to simulate sequencing data with different coverage, and we evaluate the influence of genotype calling rate and accuracy as a function of coverage. Having initially identified the appropriate parameter settings for filtering to ensure genotype accuracy, we used two different single-nucleotide polymorphism (SNP) calling pipelines, single-sample and multi-sample. We found that a coverage of 15× was suitable for obtaining sufficient numbers of SNPs with high accuracy. Our work provides guidelines for the selection of sequence coverage when using NGS to investigate species with a high degree of heterozygosity and rapid decay of linkage disequilibrium.
Highlights
Traits, in particular, the accuracy of genotyping SNP sites has a significant influence on the results obtained
This is because, compared with the low-coverage sequencing for an inbred line of rice, which could be considered as a diploid, it is very difficult to identify the genotypes for heterozygous SNP sites with very low covered reads
Using two different SNP calling pipelines, we found that a coverage of 15×was suitable for NGS analysis to obtain a sufficient number of SNPs called with high accuracy
Summary
Traits, in particular, the accuracy of genotyping SNP sites has a significant influence on the results obtained. For some non-model organisms, higher coverage sequencing for a large population would be expensive and beyond the means of many researchers Another approach, low-coverage sequencing assisted by genotype imputation methods, is a possible solution for population studies of some organisms. The aforementioned methods have not been successfully applied to a large group of organisms with a high degree of heterozygosity and rapid decline in linkage disequilibrium (LD). This is because, compared with the low-coverage sequencing for an inbred line of rice, which could be considered as a diploid, it is very difficult to identify the genotypes for heterozygous SNP sites with very low covered reads. On the basis of our findings, we provide guidelines for the selection of sequencing coverage for NGS application to organisms with a high degree of heterozygosity
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