Next Generation Sequencing - the basics
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This presentation covers the basics on what Next Generational Sequencing is and some terminology needed in order to understand the process.
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Next Generation Sequencing - the basics
- 1. Next Generation Sequencing The Basics
- 2. What is Sequencing? • Sequencing is determining the order in which nucleotides are arranged in DNA. Image found at https://www.my46.org/intro/how-are-dna-changes-found
- 3. What is NGS? • Tool for discovering, profiling, and quantifying sequences across a transcriptome • Sequences RNA and DNA quicker and more cheaply that the traditional Sanger deoxy-chain- termination method • Catch-all term to describe a number of different modern RNA and DNA sequencing technologies • Illumina (Solexa) sequencing • Roche 454 sequencing • Ion Torrent: Proton / PGM sequencing • Single Molecule Real Time (SMRT) sequencing
- 4. Terminology • DNA Sequencing - process of determining the precise order of nucleotides within a DNA molecule • Genome - complete set of genes or genetic material present in a cell or organism • RNA Sequencing – sequencing of cDNA using whole transcriptome shotgun method • Transcriptome – collection of all RNA transcripts produced in a cell or population of cells • Read – a sequencing of bases that have been “read” by a sequencing machine
- 5. Terminology Cont. • Amplification - Using polymerase chain reaction (Polymerase Chain Reaction/PCR) to generate thousands to millions of copies of a particular DNA sequence • Shotgun Sequencing - An approach used to decode a genome by shredding ("shotgunning") it into smaller fragments of DNA which can then be individually sequenced. • de novo Sequencing – sequencing of a genome that has not been sequenced before or does not have a reference
- 7. Work Cited • Ambry Genetics. Making Sense of NextGent Sequencing. Presented by Kelly Gonzalez, MS, CGC, and Senior Manager of Clinical Genomics. Available at http://www.ambrygen.com/sites/default/files/pdfs/NERRG_4-10- 12_Making_Sense_of_NetGen_Sequencing_KG(3).pdf. Access verified May 21, 2014. • DNA sequencing. (2014, June 20). In Wikipedia, The Free Encyclopedia. Retrieved 14:56, June 27, 2014, from http://en.wikipedia.org/w/index.php?title=DNA_sequencing&oldid=613666 729 • EMBL-EBI. What is Next-Generation DNA Sequencing. Available at http://www.ebi.ac.uk/training/online/course/ebi-next-generation-sequencing- practical-course/what-you-will-learn/what-next-generation-dna-. Access verified May 21, 2014. • iPlant Collaborative. RNA-Seq Tutorial. Available at https://pods.iplantcollaborative.org/wiki/display/eot/RNA-Seq_tutorial. Access verified May 21, 2014.
- 8. Work Cited Cont. • MedicinNet.com. Definition of Shotgun Sequencing. Available at http://www.medterms.com/script/main/art.asp?articlekey=20667. Access verified May 21, 2014 • Omixon. Allen Van Deynze, 2010; Solanaceae Coordinated Agricultural Project. Next Generation Sequencing. Available at http://www.omixon.com/the-basics- of-next-generation-sequencing/. Access verified June 25, 2014. • Reference genome. (2014, June 22). In Wikipedia, The Free Encyclopedia. Retrieved 14:54, June 27, 2014, from http://en.wikipedia.org/w/index.php?title=Reference_genome&oldid=61398 4719 • YouTube. Uploaded by iontorent. See what you've been missing: Explore RNA- Seq for Gene Expression. Available at https://www.youtube.com/watch?v=V_4n8n5Z6I8. Access verified May 21, 2014.
- 9. Next Generation Sequencing Clinical Applications
Editor's Notes
- This video will be covering the basics on what Next Generation Sequencing is and some terminology needed in order to understand the process.
- Image found at https://www.my46.org/intro/how-are-dna-changes-found. Read Slide RNA can also be sequenced by taking RNA and making cDNA with reverse transcriptase. The cDNA is then sequenced the same as the DNA from the nucleus of a cell.
- Next Generation Sequencing is a…read slide. The capillary electrophoresis (CE)-based Sanger method is the first generation sequencing, which is very time and labor extensive. Later videos will go into some detail on the various types of sequencing available including the Sanger, and all the next generation sequencing methods.
- Some terminology needed to understand the process of sequencing, some of which are the following. read slide. + length, speed, and quality of reads depends on the sequencing method used
- + Shotguen sequencing method designed for analysis of DNA sequences longer than 1000 base pairs, up to and including entire chromosomes. This method requires the target DNA to be broken into random fragments. After sequencing individual fragments, the sequences can be reassembled on the basis of their overlapping regions
- This slide demonstrates what the term alignment refers to, which is the alignment of the various fragments DNA or RNA sequences produced from the shotgun method, that need to be put together to find the consensus sequence in which they originally made up. The reference sequence is a digital nucleic acid sequence database, assembled by scientists as a representative example of a species' set of genes. Bioinformatics tools are then used to align the various reads usually come with the sequencing machine. Various bioinformatics tools are needed to further analyze the data.
- This is the Work Cited for this video.
- This is the Work Cited for this video.
- In the next video we will be covering Clinical Applications of the Next Generation Sequencing.