SubjectsSubjects(version: 945)
Course, academic year 2023/2024
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Molecular markers in systematics and plant population biology II - MB120C45E
Title: Molecular markers in systematics and plant population biology 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 2020
Semester: summer
E-Credits: 3
Examination process: summer s.:
Hours per week, examination: summer s.:0/1, C [TS]
Capacity: 15
Min. number of students: unlimited
4EU+: no
Virtual mobility / capacity: no
State of the course: taught
Language: English
Additional information: http://botany.natur.cuni.cz/dna/images/stories/pdf/protokoly-praktika/protokoly2.pdf
Note: enabled for web enrollment
Guarantor: Mgr. Tomáš Fér, Ph.D.
Class: Ultrapřesný sonikátor pro štěpení DNA/RNA pro příp
Původní předmět
Annotation -
Last update: Mgr. Michal Štefánek (31.05.2020)
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, evaluation of the data from
sequencer, data analysis and presentation. The basics of next-generation (NGS) sequencing libraries and NGS
data analysis are also demonstrated.

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‘.
Literature -
Last update: Mgr. Michal Štefánek (31.05.2020)

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. Michal Štefánek (31.05.2020)

Write out protocols from the practical part.

Syllabus -
Last update: Mgr. Michal Štefánek (31.05.2020)

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. NGS library preparation

  • DNA sonication using Covaris M220, quality check using gel
  • library preparation using NEBNext Ultra II kit
  • library quantification using Qubit or LabChip

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)

 
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