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Abstract The development of next-generation sequencing (NGS) enabled a shift from array-based genotyping to directly sequencing genomic libraries for high-throughput genotyping. Even though whole-genome sequencing was initially too costly for routine analysis in large populations such as breeding or genetic studies, continued advancements in genome sequencing and bioinformatics have provided the opportunity to capitalize on whole-genome information. As new sequencing platforms can routinely provide high-quality sequencing data for sufficient genome coverage to genotype various breeding populations, a limitation comes in the time and cost of library construction when multiplexing a large number of samples. Here we describe a high-throughput whole-genome skim-sequencing (skim-seq) approach that can be utilized for a broad range of genotyping and genomic characterization. Using optimized low-volume Illumina Nextera chemistry, we developed a skim-seq method and combined up to 960 samples in one multiplex library using dual index barcoding. With the dual-index barcoding, the number of samples for multiplexing can be adjusted depending on the amount of data required, and could be extended to 3,072 samples or more. Panels of doubled haploid wheat lines (Triticum aestivum, CDC Stanley x CDC Landmark), wheat-barley (T.aestivumxHordeum vulgare) and wheat-wheatgrass (Triticum durum x Thinopyrum intermedium) introgression lines as well as known monosomic wheat stocks were genotyped using the skim-seq approach. Bioinformatics pipelines were developed for various applications where sequencing coverage ranged from 1 × down to 0.01 × per sample. Using reference genomes, we detected chromosome dosage, identified aneuploidy, and karyotyped introgression lines from the skim-seq data. Leveraging the recent advancements in genome sequencing, skim-seq provides an effective and low-cost tool for routine genotyping and genetic analysis, which can track and identify introgressions and genomic regions of interest in genetics research and applied breeding programs.
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A beginner's guide to low‐coverage whole genome sequencing for population genomics
Abstract Low‐coverage whole genome sequencing (lcWGS) has emerged as a powerful and cost‐effective approach for population genomic studies in both model and nonmodel species. However, with read depths too low to confidently call individual genotypes, lcWGS requires specialized analysis tools that explicitly account for genotype uncertainty. A growing number of such tools have become available, but it can be difficult to get an overview of what types of analyses can be performed reliably with lcWGS data, and how the distribution of sequencing effort between the number of samples analysed and per‐sample sequencing depths affects inference accuracy. In this introductory guide to lcWGS, we first illustrate how the per‐sample cost for lcWGS is now comparable to RAD‐seq and Pool‐seq in many systems. We then provide an overview of software packages that explicitly account for genotype uncertainty in different types of population genomic inference. Next, we use both simulated and empirical data to assess the accuracy of allele frequency, genetic diversity, and linkage disequilibrium estimation, detection of population structure, and selection scans under different sequencing strategies. Our results show that spreading a given amount of sequencing effort across more samples with lower depth per sample consistently improves the accuracy of most types of inference, with a few notable exceptions. Finally, we assess the potential for using imputation to bolster inference from lcWGS data in nonmodel species, and discuss current limitations and future perspectives for lcWGS‐based population genomics research. With this overview, we hope to make lcWGS more approachable and stimulate its broader adoption.
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- Award ID(s):
- 1756316
- PAR ID:
- 10370252
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Molecular Ecology
- Volume:
- 30
- Issue:
- 23
- ISSN:
- 0962-1083
- Page Range / eLocation ID:
- p. 5966-5993
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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