Syllabus
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)
Annotation
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‘.