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 (
Next Generation Sequencing (NGS) has become an important tool in the biological sciences and has a growing number of applications across medical fields. Currently, few undergraduate programs provide training in the design and implementation of NGS applications. Here, we describe an inquiry‐based laboratory exercise for a college‐level molecular biology laboratory course that uses real‐time MinION deep sequencing and bioinformatics to investigate characteristic genetic variants found in cancer cell‐lines. The overall goal for students was to identify non‐small cell lung cancer (NSCLC) cell‐lines based on their unique genomic profiles. The units described in this laboratory highlight core principles in multiplex PCR primer design, real‐time deep sequencing, and bioinformatics analysis for genetic variants. We found that the MinION device is an appropriate, feasible tool that provides a comprehensive, hands‐on NGS experience for undergraduates. Student evaluations demonstrated increased confidence in using molecular techniques and enhanced understanding of NGS concepts. Overall, this exercise provides a pedagogical tool for incorporating NGS approaches in the teaching laboratory as way of enhancing students' comprehension of genomic sequence analysis. Further, this NGS lab module can easily be added to a variety of lab‐based courses to help undergraduate students learn current DNA sequencing methods with limited effort and cost.
more » « less- NSF-PAR ID:
- 10387684
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biochemistry and Molecular Biology Education
- Volume:
- 49
- Issue:
- 4
- ISSN:
- 1470-8175
- Page Range / eLocation ID:
- p. 588-597
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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