SubjectsSubjects(version: 945)
Course, academic year 2023/2024
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Molecular markers in systematics and plant population biology II - MB120C45
Title: Molekulární markery v systematice a populační biologii rostlin II
Czech title: Molekulární markery v systematice a populační biologii rostlin II
Guaranteed by: Department of Botany (31-120)
Faculty: Faculty of Science
Actual: from 2021
Semester: winter
E-Credits: 3
Examination process: winter s.:
Hours per week, examination: winter s.:0/1, C [TS]
Capacity: unlimited
Min. number of students: unlimited
4EU+: no
Virtual mobility / capacity: no
State of the course: taught
Language: English
Additional information: https://botany.natur.cuni.cz/fer/markers/practicals/DNA/protocols_manuals2-February2021.pdf
Note: enabled for web enrollment
Guarantor: Mgr. Tomáš Fér, Ph.D.
Teacher(s): Mgr. Tomáš Fér, Ph.D.
Class: Ultrapřesný sonikátor pro štěpení DNA/RNA pro příp
Annotation -
Last update: Mgr. Tomáš Fér, Ph.D. (01.03.2021)
The lectures/practicals are given in English. Specialized practical course of molecular methods in plant systematics and population biology. Two methods will be demonstrated: microsatellites and DNA sequencing. The course includes lab work (2 days) and evaluating the data from sequencer using specialized software (3 days). One day is also focused on the basic analysis of next-generation sequencing (NGS) data.
The course is established within the project CZ.02.2.69/0.0/0.0/18_056/0013322 with the name ‘ESF pro VŠ II an UK‘.
Literature -
Last update: Mgr. Tomáš Fér, Ph.D. (22.04.2012)

Weising K. et al. (2005): DNA fingerprinting in plants. Principles, methods, and applications. 2nd edition.

Caetano-Anollés G. & Gresshoff P.M. (1998): DNA markers. Protocols, applications, and overviews.

Hall B.G. (2001): Phylogenetic trees made easy.

Requirements to the exam -
Last update: Mgr. Tomáš Fér, Ph.D. (07.10.2019)

Write out protocols from the practical part.

Syllabus -
Last update: Mgr. Tomáš Fér, Ph.D. (01.03.2021)

The practical course includes lab work (part A), interpretation of the sequencer data and preparing data matrices (part B), data analysis (part C), and basics of next-generation (NGS) data analysis (part D).


A: lab work - 2 days
A1. DNA sequencing
- PCR amplification of the target DNA region (non-coding cpDNA region, ITS…)
- agarose gel electrophoresis (test of successful PCR amplification)
- cleaning of PCR fragments using the kit
- PCR product quantification (spectrophotometrically)
- cycle sequencing using ABI PRISM BigDye Terminator v3.1 kit
- product precipitation
- sequencing run at ABI 3100xl Avant (Biological section of the Faculty of Science)
A2. microsatellites
- use of specific nuclear primers (PCR reaction)
- use of universal chloroplast primers (PCR reaction)
- agarose gel electrophoresis (test of successful PCR amplification)
- PCR product precipitation, mixing with ROX internal standard
- fragment analysis run at ABI 3100xl Avant (Biological section of the Faculty of Science)
A3. basics of NGS library preparation
- DNA fragmentation using Covaris M220

B: sequencer data interpretation - 1 day
B1. DNA sequencing
- editing of sequences (FinchTV etc.)
- automatic alignment using ClustalX and MAFFT
- alignment editing, manipulation with sequences - BioEdit, FaBox
B2. microsatellites
- data analysis in GeneMarker
- interpreting stutter bands, -A addition etc., the building of data matrices

C: data analysis - 1 day
C1. DNA sequencing
- searching for similar sequences in GenBank (BLAST)
- basic tree building (FastTree)
- indel coding – SeqState
- statistic parsimony network - TCS

- building data matrices for subsequent analyses (NEXUS, PHYLIP format etc.), format conversion
C2. microsatellites
- basic data analyses (MicroSatelliteAnalyser)
- AMOVA (Arlequin)...

D: basic NGS data analysis - 1 day
- FASTQ sequences retrieval from short read archive (fastq-dump)
- data quality evaluation (FastQC), trimming (Trimmomatic), duplicate removal (fastuniq)
- read mapping to the reference (BWA)
- analysis of mapped reads (samtools, Tablet)
- de-novo assembly of chloroplast genome (Velvet)
- automated annotation (web GeSeq)

The course is established within the project CZ.02.2.69/0.0/0.0/18_056/0013322 with the name ‘ESF pro VŠ II na UK‘.

 
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