Next-generation sequencing (NGS) has revolutionized genomic research. NGS is faster, more accurate, and less expensive than traditional Sanger sequencing. The general steps for NGS involve library preparation through fragmentation and ligation of DNA with adapters, amplification, and sequencing. Several NGS platforms use reversible terminators to sequence DNA in a massively parallel fashion, identifying nucleotides based on detection of distinct fluorescent signals. Compared to first-generation sequencing, NGS allows for high-throughput, cost-effective sequencing of entire genomes.

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

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

12. 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.
 Relies on light detection based on a chain
reaction when pyrophosphate (PPi) is released

13. Steps of pyrosequencing (1)
 Step 1. DNA is broken into pieces and denatured to form
single strands
 Step 2. Into wells are added (with ssDNA):
1. DNA polymerase
2. ATP sulfurylase
3. Luciferase
4. Apyrase
5. APS
6. Luciferin.

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

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

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

17. 3’-O-blocked reversible
terminators
1. Primers and templates are
fixed to solid supports
2. Annealing of nucleotides
3. Solid support is imaged
4. Remove of fluorescent branch
and 3’-O-azidomethyl group
(with TCEP) as well as extra
nucleotides

19. 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

21.  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.

22. 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 )

23. 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?

24. 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)