![How much was the first human genome sequence .
⊹ The total cost of Human genome project (HGP) [ from October 1990 to April 2003 ]
was estimated as ~ $ 2.7 billion .
⊹ The estimated cost of first draft sequence ( i.e.- ~ 90% coverage of genome at
99.9% accuracy ) was ~ $ 300 million worldwide . ( April 1999 - June 2000)
⊹ The finished sequence ( > 95% coverage of genome at 99.99% accuracy ) was
submitted on 2003 ; of which the estimated cost was ~$150 million worldwide .
⊹ NHGRI estimated that the hypothetical 2003 cost to generate a second reference
genome sequence using then-available approaches and technologies was in the
neighborhood of $ 50 million .
⊹ At present , HiSeq X Ten System , released in 2014 , can sequence over 45 human
genomes in a single day for approximately $ 1000 each .
33](https://image.slidesharecdn.com/ngs-mousumee-210611153338/85/NEXT-GENERATION-SEQUENCING-33-320.jpg)
NEXT GENERATION SEQUENCING
- 1. Next Generation DNA Sequencing
- 2. Hello! I am Mousumee Mahapatra Department of Botany Guru Ghasidas Vishwavidyalaya (CU) Bilaspur, Chhattisgarh
- 3. What Is Next Generation Sequencing? ⊹ In 2005 , new era of DNA sequencing have emerged , when a fully automated massively parallel pyrosequencing machine was developed . This led to foundation of Next generation Sequencing. ⊹ It is a choice for large scale genomic and transcription sequencing because of high outputs (Gigabyte per instrument) range. ⊹ It involves the sequencing of small DNA fragments in parallel and then use of bioinformatics analysis to arrange the sequence of those fragments together either by mapping the individual reads to reference genome. ⊹ Thus it is also called massive parallel sequencing or deep sequencing.
- 4. “ ⊹ Work flow of NGS Video source- https.//info.abmgood.com/next-generation- sequencing
- 5. Types of next generation sequencing Pyrosequencing Non-electrophoretic, bioluminescence method that measures the release of inorganic pyrophosphate by propertionally converting it into visible light using a series of enzymatic option. Illumina sequencing Illumina sequencing technology leverages clonal array formation and proprietary reversible terminator technology for rapid and accurate large-scale sequencing. Ion-semiconductor sequencing Ion semiconductor sequencing is a method of DNA sequencing based on the detection of hydrogen ions that are released during the polymerization of DNA. This is a method of "sequencing by synthesis", during which a complementary strand is built based on the sequence of a template strand. 5
- 7. Why Pyrosequencing? 7 Pyros (Greek for “fire,” because light is produced) because genome sequencing is done utilizing light-emitting enzyme- Luciferase. Luciferase emits lights in presence of ATP present in several organisms such as American firefly and poisonous Jack-o-lantern mushroom.
- 8. WORK FLOW OF PYROSEQUENCING 8 Video source- https.//info.abmgood.com/next-generation- sequencing
- 9. How it works? ⊹ In DNA synthesis, a dNTP is attached to the 3’end of the growing DNA strand. The two phosphates on the end are released as pyrophosphate (PPi). ⊹ ATP sulfurylase uses PPi and adenosine 5’- phosphosulfate to make ATP. ⊹ Luciferase is the enzyme that causes fireflies to glow. It uses luciferin and ATP as substrates converting luciferin to oxyluciferin and releasing visible light. – The amount of light released is proportional to the number of nucleotides added to the new DNA strand. ⊹ After the reaction has completed, apyrase is added to destroy any leftover dNTPs. 9
- 10. Procedure involved in pyrosequencing ⊹ DNA is fragmented: To start, the DNA is sheared into 300-800 bp fragments, and the ends are “polished” by removing any unpaired bases at the ends. ⊹ The DNA strand’s ends are made blunt with appropriate enzymes ⊹ “A” and “B” adapters are ligated to the blunt ends using DNA ligase ⊹ The strands are denatured using sodium hydroxide to release the ssDNA template library (sstDNA). The adapters ⊹ The A and B adapters are used as priming sites for both amplification and sequencing since their composition is known. ⊹ The B adapter contains a 5’ biotin tag used for mobilization. ⊹ The beads are magnetized and attract the biotin in the B adaptors. 1. Preparation of Sample DNA http://www.pyrosequencing.com/DynPage.aspx?id=7454
- 11. ⊹ Using water-in-oil emulsion, each ssDNA in the library is hybridized onto a primer coated bead. ⊹ By limiting dilution, an environment is created that allows each emulsion bead to have only one ssDNA. ⊹ Each bead is then captured in a its own emulsion micro-reactor, containing in it all the ingredients needed for a PCR reaction. ⊹ PCR takes place in each of these beads individually, but all in parallel. ⊹ This activity as a whole is emPCR. One adapter contains biotin, which binds to a streptavidin-coated bead. The ratio of beads to DNA molecules is controlled so that most beads get only a single DNA attached to them. Oil is added to the beads and an emulsion is created. PCR is then performed, with each aqueous droplet forming its own microreactor. Each bead ends up coated with about a million identical copies of the original DNA 11 2. Cloning of DNA http://www.pyrosequencing.com/DynPage.aspx?id=7454
- 12. ⊹ Utilizing the A adapter, a primer is added to the ssDNA. ⊹ The beads are now loaded into individual wells created from finely packed and cut fiber-optics (PicoTiterPlate device). ⊹ The size of the wells do not allow more than one ssDNA bead to be loaded into a well. ⊹ Enzyme beads and packing beads are added. Enzyme beads containing sulfurase and luciferase, and packing beads used only to keep the DNA beads in place. ⊹ Above the wells is a flow channel, passing nucleotides and apyrase in a timed schedule. ⊹ Comparing the peak light emission of incorporation of C or T at a CpG side with in the amplicon gives a precis3 measure of amount of methylation at the position within the sample. 12 3. Sequencing https://www.researchgate.net/figure/The-principle-of-Pyrosequencing-a-method-of-sequencing-by-synthesis-from-22- The_fig2_267972290?hcb=1
- 13. advantages 13 1 2 3 3 2 1 Larger sequence can be read easily. Approx. 48,000 sequencing can be done per day It is a much accurate Easily automated and there is no need of gel electrophoresis. High reagent costs. Thus not economic friendly. There is high error rate over strings of 6+ homopolymer. The work required is considerable, and special software is required for the instrument and for the analysis. Disadvantages
- 15. Why Illumina sequencing 15 • It is the most successful sequencing system with a claimed >70% dominance of the market. • The Illumina sequencer is different from the Roche 454 (Pyrosequencing) sequencer in that it adopted the technology of sequencing by synthesis using removable fluorescently labeled chain-terminating nucleotides that are able to produce a larger output at lower reagent cost
- 16. Work frame of illumina sequencing 16 Video source- https.//www.illumine.com/next-generation-sequencing
- 17. Procedure involved in illumine sequencing. ⊹ Fragment DNA of interest into smaller strands that are able to be sequenced. • Sonication • Nebulization • Enzyme digestion ⊹ Ligate Adapters. ⊹ Denature dsDNA into ssDNA by heating to 95° C. 1. Preparation of Genomic DNA sample
- 18. ⊹ ssDNA is then bound to inside surface of flow cell channels ⊹ Dense lawn of primer on the surface of the flow cell 2. Attach DNA to surface ⊹ Unlabeled nucleotides and polymerase enzyme are added to initiate the solid phase bridge amplification 3. Bridge amplification
- 19. ⊹ In this step it demonstrates the work done by the sequencing reagents • Primers • Nucleotides • Polymerase enzymes • Buffer 19 4. Fragments become double stranded
- 20. ⊹ The original strand is then washed away, leaving only the strands that had been synthesized to the oligos attached to the flow cell 20 5. Denature the double stranded molecules. Attached Attached 6. Complete amplification Cluster ⊹ Cycle of new strand synthesis and Denaturation to make multiple copies of the same sequence (amplification) • Fragments Become Double Stranded • Denature the Double Strand Molecules
- 21. ⊹ The P5 region is cleaved ⊹ Add sequencing reagents • Primers • Polymerase • Fluorescently labelled nucleotides • Buffer ⊹ First base incorporated 7. Determine first base
- 22. ⊹ Remove unincorporated bases • Detect Signal • Deblock and remove the ⊹ Fluorescent signal new cycle 22 8. Image first base ⊹ Add sequencing reagents • Primers • Polymerase • Fluorescently labeled nucleotides • Buffer ⊹ Second base incorporated 9. Determine second base
- 23. 23 ⊹ The identity of each base of a cluster is read off from sequential images 11. Sequence Reads Over Multiple Chemistry Cycles 10. Image second chemistry cycle ⊹ Remove unincorporated bases • Detect Signal • Deblock and remove the ⊹ Fluorescent signal new cycle
- 24. 24 12. Align data ⊹ After the sequencing is finished they are aligned. ⊹ Each was once one larger sequence that had been fragmented. ⊹ Needs to be realigned to find the original sequence of the larger sequence.
- 25. Application of illumina sequencing Sequence based transcriptome analysis SNPs and SVs discovery and small RNA discovery analysis. Cytogenetic analysis and ChIP sequencing 2 4 DNA sequencing gene regulation analysis 1 3
- 26. Merits and demerits of illumine sequencing Merits : ⊹ Good for sequencing of repetitive and homopolymer sequences . ⊹ Long read length ⊹ Preferable for Whole genome sequencing Demerits : ⊹ Incomplete removal of fluorescent molecules create high background noise . 26
- 28. Why ion-semiconductor sequencing? 28 • It is based on release of proton (H+) after nucleotide incorporation . • Sequencing chip contains many small wells . Ideally , each well contains single bead with clonally amplified DNA (cluster) ( i.e. – amplified in ePCR ) . • Each cluster located directly above a semiconductor transistor which is capable to determine change in pH of solution.
- 29. Work frame of ion-semiconductor sequencing 29 Video source- https.//info.abmgood.com/next-generation-sequencing
- 30. advantages Less reagent cost . 30 No need of chemically modified nucleotides . No need of nucleotides . disadvantages Short reads High error rate over homopolymer
- 31. Cost of sequencing and other specifications of different NGS platforms : 31 * RP = reagent price + sequencing price NA = Not Available Source- https://www.longdom.org/open-access/generations-of-sequencing-technologies-from-first-to- next-generation-0974-8369-1000395.pdf
- 32. COST OF DNA SEQUENCING : 32 Source- https://www.genome.gov/about-genomics/fact-sheets/Sequencing-Human- Genome-cost
- 33. How much was the first human genome sequence . ⊹ The total cost of Human genome project (HGP) [ from October 1990 to April 2003 ] was estimated as ~ $ 2.7 billion . ⊹ The estimated cost of first draft sequence ( i.e.- ~ 90% coverage of genome at 99.9% accuracy ) was ~ $ 300 million worldwide . ( April 1999 - June 2000) ⊹ The finished sequence ( > 95% coverage of genome at 99.99% accuracy ) was submitted on 2003 ; of which the estimated cost was ~$150 million worldwide . ⊹ NHGRI estimated that the hypothetical 2003 cost to generate a second reference genome sequence using then-available approaches and technologies was in the neighborhood of $ 50 million . ⊹ At present , HiSeq X Ten System , released in 2014 , can sequence over 45 human genomes in a single day for approximately $ 1000 each . 33
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