Subject description
The challenge of current research work presents the integration of knowledge and experimental data sets from different research fields (e.g. genomics, transcriptomics, proteomics) and generation of vast data sets. In the frame of this individual research subject we would like to introduce student using real laboratory experiments or data sets with designated research subjects and examples of data analysis from fields of genomics, transcriptomics and proteomics:
1) DNA/RNA quantification
There are a number of methods available to quantify DNA. The traditional method of DNA quantitation involves measuring the absorbance of the sample on a spectrophotometer. Another method involves the use of a fluorescent dye, is PicoGreen dsDNA quantitation method. But all these techniques are not species-specific, while very precise determination of target genes / DNA / RNA was enabled by methods based on the polymerase chain reaction technology. The latest development in DNA quantitation is based on the technique of real time PCR. Several different approaches of real time quantitation of DNA are based on the principle of fluorescent dye binding double-stranded DNA as it accumulates during the PCR process. As the technique is based on the polymerase chain reaction, DNA quantitation can be undertaken by targeting any specific region of template DNA. Another, improved technology called digital PCR (dPCR), is a refinement of conventional PCR methods that can be used to directly quantify and clonally amplify nucleic acids. dPCR carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually.
Within this course-set the students will gain the knowledge about most widely applicable real-time PCR methods and techniques.
2) Analysis of genomics and transcriptomics NGS data set – applied bioinformatics
The data sets generated by next generation sequencing methodologies (NGS) revolutionized the fields of genomics and proteomics in the last few years. The students will be familiarized by the next topics:
a) Quick overview of relevant NGS technologies
b) Sequence Read Archive maintained by NCBI, data acquisition produced by different NGS platforms and data formatting using SRA Toolkit
c) QC analysis of raw NGS data
d) NGS data trimming
e) Basic NGS formats, properties, their use (FASTQ, SAM, BAM, GFF, VCF, BED)
f) Read mapping approach of sequence reconstruction and de-novo assembly
g) NGS data visualization.
3) Study of proteome
Different omics approaches enable investigation of biological processes at the molecular level. Among them proteomics has an important advantage, it investigates proteins that carry out functions of every living cell. By using different proteomic tools information about protein expression, post-translational modifications and protein interactions can be obtained. In the context of this part students will gain knowledge in the following topics:
– Biological material preparation – sampling and sample preparation for proteome analysis
– Proteome analysis by 2-D electrophoresis
– Analysis of 2-D gel images by using specific computer software
– Evaluation of protein identification results obtained by mass spectroscopy.
The subject is taught in programs
Objectives and competences
The objective of this course is to:
1) introduce the DNA/RNA quantification methods and to perform some practical laboratory experiments and calculations on the basis of the obtained results,
2) acquaint students with basics characteristics of NGS data, their databases for storing and with recommended flow of the analysis
3) introduce proteome analysis from sample preparation , protein separation to proteomic data evaluation
Based on the practical examples and real data sets students will learn how to understand biological processes on the level of genome, transcriptome and proteome. They will also be able to properly design such experiments.
Teaching and learning methods
- Theoretical basic
- Practical lab work or computer work
- Analysis of the results using specialized computer software
Expected study results
Knowledge and understanding:
• To gain knowledge on basic principles of Real-Time PCR assay design
• To understand methods and techniques for determining the amount of target gene in the sample or analyzing the expression of individual targets by the method of real-time PCR.
• Data analysis and evaluation of the results of different methodological approaches.
• Statistical analysis of results and graphical presentation
• Understanding of the basis of applied bioinformatics related to NGS data
• Knowledge of importance of proteomics for understanding biological processes and ability to design proteomic experiment from biological material preparation to proteome analysis by 2-D electrophoresis and data evaluation.
Basic sources and literature
1) Real-time PCR handbook. 2012, Real-Time PCR 2nd Edition, Life Technologies Corporation (6 Chapters and 66 pages)
2) Bioinformatics for High Throughput Sequencing. Rodríguez-Ezpeleta, Naiara, Hackenberg, Michael, Aransay, Ana M. (Eds.) 2012, XI, 255p. 29 illus., 25 illus. in color.
3) Revialni in originalni znanstveni članki s področja/Review and original scientific articles from the field.
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University of Ljubljana, Faculty of Electrical Engineering Tržaška cesta 25, 1000 Ljubljana