BMB838: Introduction to next generation DNA sequencing technology – methods and applications
Students taking the course are expected to:
- Have knowledge of the biochemical mechanisms regulating basic biological processes such as DNA replication and transcription.
- Have knowledge of genome organization in prokaryotic and eukaryotic cells and understand the epigenetic mechanisms controlling genome organization.
- Have knowledge of DNA sequencing technologies.
- Be able to use the most basic laboratory equipment such as micropipettes, centrifuges etc.
- Be able to use the simple bash scripts and R for data analysis
- Give the competence to use NGS technology within life science ranging from microbiology to human biology.
- Give a though introduction to the most used NGS technologies and an updated knowledge on upcoming DNA sequencing technologies.
- Give skills to sequence DNA using Illumina NGS based technology.
- Give knowledge and understanding of the biochemical strategies to enrich the genomes and transcriptomes before sequencing.
- Give knowledge and understanding of the advantages and disadvantages of the different types of NGS technologies used to sequence genomes, epigenomes, cistromes and transcriptiomes.
Expected learning outcome
- Describe the basic principles for the most common NGS platforms such as Illumina, Iontorrent, Roche 454 and Pacific biosciences
- Explain the advantages and disadvantages with the different NGS platforms and describe which of the platforms would be most optimal to study the genome, epigenome and transcriptome.
- Explain the different steps of Illumina sequencing (DNA library synthesis, DNA sequencing and data analysis).
- Describe different biochemical methods applied to enrich different parts of the genome and transcriptome before sequencing.
- Synthesize and quality control DNA libraries for Illumina sequencing – synthesis, purification and multiplexing.
- Understand and apply different types of quality control methods before, during and after Illumina DNA library synthesis.
- Understand the output data files from Illumina sequencing and be able to perform the most basic NGS analysis (demultiplexing, genome alignment and DNA quantification).
- Use different types of NGS analysis tools.
- The course material will include papers and reviews on topics covering the technological development of NGS and the different applications of NGS within life science including cell differentiation, cancer biology and microbiomics. In addition, the course will provide the student with the most updated protocols for synthesis of DNA libraries for Illumina based NGS.
- The theoretical part of the course will provide the student with a general understanding of different NGS platforms and upstream biochemical strategies to enrich the genome and transcriptome.
- The practical part of the course will provide the students with in depth theoretical and practical knowledge on the synthesis of cDNA libraries from eukaryotic RNA and Illumina sequencing of the libraries (RNA-seq). This will include RNA purification, cDNA synthesis, adaptor ligation, DNA purification for Illumina sequencing.
- After sequencing, the student will be introduced to the most basic bioinformatics strategies and will be provided with hands-on experience with demultiplexing, genome alignment, UCSC genome browser visualization and quantification of sequenced DNA fragments.
Exam element a)
Individual written report
Indicative number of lessons
- Intro phase (lectures) - 10 hours
- Training phase: 60 hours, including 20 hours tutorials and 40 hours laboratory
The course is divided into lectures, paper presentations and a laboratory exercise. The lectures introduce the students to the recent NGS technology and application of NGS in research, the clinic and industry. The literature for these lectures is based on recent review papers. The lectures are followed by paper presentations by the student with primary focus on RNA-seq and ChIP-seq analysis. A subsequent laboratory exercise gives the student hands-on experience with the biochemistry used for DNA library construction for NGS and handling NGS technology. The student works in groups of 2-3 students under supervision of an instructor. The student starts by designing an experiment followed by RNA purification from mammalian cells, cDNA synthesis and NGS library construction. The student performs a series of quality control steps to ensure sufficient quality of the DNA libraries before sequencing. Following sequencing the student performs analysis of the RNA-seq data using publically available software (basic bash scripts and R). The student subsequently writes a paper on RNA-seq (from experimental design to quantification of RNA). The paper will be evaluated by an internal censor and graded as passed/fail.
- Student presentation of selected research papers
- Analyse the quality control of NGS libraries
- Analyse NGS data
- Write a rapport on RNA-seq