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. basics of NGS library preparation- DNA fragmentation using Covaris M220B: 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, FaBoxB2. microsatellites - data analysis in GeneMarker - interpreting stutter bands, -A addition etc., the building of data matricesC: 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 conversionC2. 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‘.
Annotation
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‘.