High-throughput sequencing (HTS) of reduced representation genomic libraries has ushered in an era of genotyping-by-sequencing (GBS), where genome-wide genotype data can be obtained for nearly any species. However, there remains a need for imputation-free GBS methods for genotyping large samples taken from heterogeneous populations of heterozygous individuals. This requires that a number of issues encountered with GBS be considered, including the sequencing of nonoverlapping sets of loci across multiple GBS libraries, a common missing data problem that results in low call rates for markers per individual, and a tendency for applicability only in inbred line samples with sufficient linkage disequilibrium for accurate imputation. We addressed these issues while developing and validating a new, comprehensive platform for GBS. This study supports the notion that GBS can be tailored to particular aims, and using Zea mays our results indicate that large samples of unknown pedigree can be genotyped to obtain complete and accurate GBS data. Optimizing size selection to sequence a high proportion of shared loci among individuals in different libraries and using simple in silico filters, a GBS procedure was established that produces high call rates per marker (>85%) with accuracy exceeding 99.4%. Furthermore, by capitalizing on the sequence-read structure of GBS data (stacks of reads), a new tool for resolving local haplotypes and scoring phased genotypes was developed, a feature that is not available in many GBS pipelines. Using local haplotypes reduces the marker dimensionality of the genotype matrix while increasing the informativeness of the data. Phased GBS in maize also revealed the existence of reproducibly inaccurate (apparent accuracy) genotypes that were due to divergent copy number variants (CNVs) unobservable in the underlying single nucleotide polymorphism (SNP) data.
- Supplemental Table S1: Details on library preparation for each library - Download Excelxlsx
This table includes detailed protocol information for each library as well...
- Supplemental Table S2: Number of overlapping loci for different size ranges - Download Excelxlsx
This table shows overlapping loci for a set of five libraries that consist...
- Supplemental file S2: Detailed genotyping-by-sequencing protocol - Download Excelxlsx
The detailed GBS protocol used in this study including information on...
- Supplemental file S3: Detailed oligonucleotide information - Download Excelxlsx
This file includes the sequences, purity, and names of adapters that were...
- Supplemental figure S1: Number of unique and repetitive in silico digested NgoMIV-Csp6I loci in maize - Download tiffData
The upper panel shows the number of "repetitive" (less than or equal to 4%...
- Supplemental file S4: Discovered SNPs - Download Excelxlsx
This is a list of B73 reference version 2, 3, and 4 coordinates for all SNPs...
|Release Date|| |
G3: Genes, Genomes, Genetics
|Contact Name|| |
Wisser, Randall J.
|Public Access Level|| |
|Program Code|| |
005:040 - Department of Agriculture - National Research
|Bureau Code|| |
005:18 - Agricultural Research Service