next generation sequencing

NGS is a new way to examine and explore genes. Sanger's method is still being used in science but has some difficulties. This presentation covers only two types of NGSs but they are good examples of better ways to study genes, genome. NGS are really important nowadays in diagnostics tumors and cancers.

1. Next-generation sequencing By Peter Egorov

2. Next-generation sequencing (NGS) Synonyms : massively parallel or deep sequencing Revolutionized genomic research Fast , more accurate, and less expansive

3. Three general steps: Library preparation: fragmentation ligation with custom linkers Amplification Sequencing

4. Library preparation Fragmentation Addition of adaptors

5. Library preparation A potential problem can occur – pairing of adaptors

6. Library preparation For successful sequencing all fragments are clustered in PCR colonies – “ polonies ”: Copies of particular fragment Attached in parallel and planer fashion Parallel enzymatic manipulation

7. Amplification Is needed for receiving stronger signal in a sequencer for better precision. There are several types of amplification (e.g.): Emulsion PCR Bridge PCR

8. Emulsion PCR Emulsion oil Beads PCR mix Library DNA

9. Bridge PCR

10. Sequencing Several competing methods have been developed by different companies Wikipedia.com

11. Pyrosequencing is DNA sequencing based on : the " sequencing by synthesis " principle, in which the sequencing is performed by detecting the nucleotide incorporated by a DNA polymerase. R elies on light detection based on a chain reaction when pyrophosphate ( PPi ) is released

12. Steps of pyrosequencing (1) Step 1. DNA is broken into pieces and denatured to form single strands Step 2 . I nto wells are added (with ssDNA ): DNA polymerase ATP sulfurylase Luciferase Apyrase APS Luciferin .

13. Steps of pyrosequencing (2) Step 3. Detection of light that is produced due to the chain reaction

14. Reversible terminator Sequencing ( i llumina ) Modified nucleotides are used In amplification Bridge PCR is used (more efficient) Two categories : 3’- O-blocked reversible terminators 3’- unblocked reversible terminators.

15. Modified nucleotides 3’-O-azidomethyl group Fluorescent branch

16. 3’-O-blocked reversible terminators Primers and templates are fixed to solid supports Annealing of nucleotides Solid support is imaged Remove of fluorescent branch and 3’-O-azidomethyl group (with TCEP) as well as extra nucleotides

17. 3’-unblocked reversible terminator (1) 3 main differences from 3’-O-blocked: 3’ position is not blocked (free 3’-OH) One fluorophore for all bases Each base is flowed sequentially rather than at the same time

18. This technique was pioneered by Illumina , with their HiSeq and MiSeq platforms. HiSeq is the cheapest of the second generation sequencers with a cost of $0.02 per million bases. It also has a high data output of 600 Gb per run which takes around 8 days to complete.

19. Potential uses of NGSs in clinical practice NGS captures a broader spectrum of mutations than Sanger sequencing Genomes can be interrogated without bias The increased sensitivity of NGS allows detection of mosaic mutations Microbiology ( NGS was used to reveal and trace an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) on a neonatal intensive care unit in the UK) Oncology ( cancer genomes can now be systemically studied in their entirety )

20. Questions: In your opinion, which sequencing is better from our presentation? What are the advantages of NGS compare to first generation sequencing? What is the main principle of pyrosequencing ?

21. References: Behjati , Sam, and Patrick S Tarpey . “What is next generation sequencing ?”  Archives of Disease in Childhood Education and Practice Edition , BMJ Publishing Group, Dec. 2013, www.ncbi.nlm.nih.gov / pmc /articles/PMC3841808/.  . “ DNA sequencing.”  Wikipedia , Wikimedia Foundation, 8 Nov. 2017, en.wikipedia.org /wiki/DNA_sequencing#Illumina_.28Solexa.29_sequencing . Dr. Fakruddin et al, “ Pyrosequencing – principles and applications ”. International Journal of Life Science & Pharma Research, vol.2, issue 2, Apr-Jun 2012 “Next generation sequencing.”  ATDBio , www.atdbio.com /content/58/Next-generation-sequencing#Sanger-sequencing-and-Next-generation-sequencing . Youtube.com ( pyrosequencing )