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Lecturer(s)
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Piálek Lubomír, RNDr. Ing. Ph.D.
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Course content
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Content of lectures: This course comprises training of advanced molecular methods applied in current evolutionary research. Based on students' interests and needs it offers two alternative blocks oriented on RAD genotyping and NGS sequencing on the Oxford Nanopore platform. The focus of this course is on practical exercises that will be supplemented by specialized lectures on each topic. Content of practicals: Block A: RAD genotyping 1. RAD-techniques as a method of reduced-representation sequencing of genomes. Double-digest RAD, pros and cons. 2. Preparation of a ddRAD library for sequencing on the Illumina platform. 3. Purification and separation techniques used in preparation of NGS libraries. Purification of products on magnetic beads. Separation on the E-gel platform. DNA size-selection on the Pippin platform. 4. Introduction to processing of obtained ddRAD data. UNIX basic commands, running tasks in the Metacentrum grid. Qualitative filtering of raw data. Preparation of input files and simple scripts for Stacks software. Generation and adaptation of SNP datasets. Block B: Sequencing on the Oxford Nanopore Technologies (ONT) platform 1. Nanopore-sequencing technology on the ONT platform, overview of devices. 2. Preparation of DNA library for whole-genome sequencing (WGS). 3. Sequencing on the MinION system. Performance assessment during a run. 4. Introduction to basecalling and primary analysis of ONT data.
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Learning activities and teaching methods
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Monologic (reading, lecture, briefing), Laboratory
- Class attendance
- 56 hours per semester
- Preparation for classes
- 4 hours per semester
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Learning outcomes
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The objective of the course is familiarization with basics of selected advanced molecular methods applied in current evolutionary research.
Students acquire skills in advanced molecular-biology laboratory techniques used in zoological research like RAD-genotyping and/or sequencing on the Oxford Nanopore platform.
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Prerequisites
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No requirements on previous laboratory experience. Repeated enrolment possible only for the alternative teaching block.
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Assessment methods and criteria
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Systematic student observation
100% presence and active participation in exercises is required.
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Recommended literature
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Andrews KR et al. (2016). Harnessing the power of RADseq for ecological and evolutionary genomics. Nature Reviews Genetics..
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Catchen J et al. (2013). Stacks: an analysis tool set for population genomics. Molecular Ecology..
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Peterson BK et al. (2012). Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species. PLoS ONE..
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Wilson BD et al. (2019). High-Fidelity Nanopore Sequencing of Ultra-Short DNA Sequences. bioRxiv..
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