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Next generation-sequencing.ppt-converted
- 1. THE FUTURE TOOLFOR CHOOSING DESIRSBLE BREEDING SELECTIONS NEXT GENERATION SEQUENCING Department of Genetics and Plant Breeding Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur (M.P.) CREDIT SEMINAR ON Submitted by:- SHWETA TIWARI En No.:- 190134008
- 2. INTRODUCTION The procedure todetermine the sequence of nucleotide in the fragment of DNA is termed as DNA sequencing. While the advance version of high throughput sequencing method with more accuracy, enhanced read length, increased speed, reduced time and diminishing cost is called the Next Generation Sequencing. The first generation sequencing in 1977 evolved into second generation sequencing in 2004, over a span of ~30 years and in another 10 year we have third generation of sequencing developed. FIRST GENERATION SEQUENCING SECOND GENERATION SEQUENCING THIRD GENERTAION SEQUENCING THE THREE ERA’S OF SEQENCING :-
- 3. DIFFERENCE BETWEEN TRADITIONAL SEQUENCING METHODS Although, theyare distinct in principle and procedure, but both of them, separate in gel electrophoresis on the basis of their lengths. The sample is loaded into adjacent lane of sequencing gel and the nucleotide order is determined from autoradiography image of the gel. These are called First generation sequencing.
- 4. 01. 700-1000bps ata time 07. Quality of sequence sacrificed :- Initial 15-20 sequence not precise and degradation sequencing after 700bp. 06.Cost infectiveness:- $500 per 1000 bases, compared to less than 0.50 per 1000 bases for next generation sequencing. 03. Difficult to automate 02. Reduced resolution 05. More time requirement 04. Less accuracy Need of NGS as Challenges Faced by Traditional Sequencing Techniques
- 5. NEXT GENERATION SEQUENCING The Progressiveand Improved Version sequence the whole genome with high speed and high throughput sequencing at reduced cost is popularized as NEXT GENERATION SEQUENCING 01. Also known as Massively Parallel Sequencing 02. Faster, Cheaper and required much less time in template preparation
- 6. NGS TIMELINE 454 RocheGS FLX 01 02 03 04 ILLUMINA Genome Analyzer SOLiD Sequencer The Ion Torrent & Pacific Bioscience commercialized SMRT 2004 2006 2007 2010 05 2015Pocket-sized sensing device MinION
- 7. Overview of GeneralProcedure of Next Generation Sequencing A. D. B. C. Emulsion PCR Solid phase amplification NGS Sequencing and imaging Data analysis OVERVIEW OF NGS
- 8. 454 Sequencing GS FLX,GS FLX Titanium series by 454 Life Sciences/ Roche diagnostics Single Molecule Real Time (SMRT) Sequencing By Pacific Bioscience,USA, PACBIO RS Helicos Genetic Analysis System Helicos Tmgenetic Genetic Analysis System by SeqLL,LLC The SOLiD Sequencing SOLiD 5500, SOLiD 5500XL, SOLiD 3 plus Illumina Sequencing Genome Analyzer, HiSeq, MiSeq, NextSeq by Illumina, Inc. Nanopore Sequencing Technologies By Oxford Nanopore’s Complete Genomic by Beijing Genomic Institude and GnuBIO by BioRad SECOND GENERATION SEQUENCING THIRD GENERATION SEQUENCING Ion Torrent Sequencing By Life Technologies Ion Torrent PGM (Personal Genome Machine), Ion Torrent Proton sequencing platform CLASSIFICATION OF NEXT GENERATION SEQUENCING
- 9. 454 DNA SEQUENCING 0301 0402 In 2005by 454 Life sciences (now Roche Diagnostics), USA. First commercialized NGS Genome Sequencer • GS FLX system (~400bases) • GS FLX Titanium series (1Gb) Currently available platform One million beads at a time and one run can takes about 10h, including template preparation. 99.9% Accuracy Processing Sequencing by synthesis : PYROSEQUENCING Principle 1. 454 DNA SEQUENCING
- 10. 1 Sensor record databased on polymerization displayed in form of pyrogram Pyrogram Formed 2 1.LIQIUD PYROSEQUENCING 2. SOLID PYROSEQUENCING Types 3 1. Inaccurate homo-polymer seq. 2. Shorter Read Length 3. Costly Limitations MECHANISM OF ACTION Chemical luminescence enzymatic reaction with accuracy, flexibility, parallel processing and can easily be automated. Incorporation of dNTPs, releases Pyrophosphate (PPi), which convert into ATP, on whose presence luciferase convert luferin into oxyluciferin and illuminate the light.
- 11. 5tt 2. THE SOLIDDNA SEQUENCING THE SOLiD SEQUENCING 0301 0402 The Applied Biosystem US, commercialized the Polony method in 2005 as SOLiD 3.0 COMMERCIALIZATION Sequencing by oligonucleotide ligation detection PRINCIPLE The method is developed by George M. Church at Harvard SCIENTIST Trouble in sequencing pallindromic sequences Short Read Length LIMITATIONS It is very cost effective with 0.13$ per million bases. It read ~50 bases and generate 20 Gb of total sequence per run with time 6-7 days
- 12. PCR produced million copiesof template strand which bound to large polystyrene coated bead and formed Polonies. a & b The template is attached with bead BEAD HYBRIDISATION c & d POLYMERASE COLONIES e & f Supernatant are separated and attached to slide CENTRIFUGATION AND ATTACHMENT g Base 1 & 2 are complementary bases to be sequenced while 3-5 are degenerated & base 6-8 are inosine base. PROBE ANATOMY h PRIMER BIND Primer binding to adapters through ligase
- 13. i& j The probeanneal to primer and fluorescence snapshot PROBE ANNEALING & SNAPSHOT k & l Dye ends cleaved and free 5` phosphate for next round. The entire process repeated four times, each time with primer offset by 1 bases DYE CLEAVED OFF m WHOLE GENOME SEQUENCING n PALLINDROMIC SEQUENCE Palindrome sequences form hairpin loop, which cause difficulties in sequencing
- 14. ILLUMINA SEQUENCING 0301 0402 Illumina,USA commercialized the SolexaNGS technologyin 2007 Most widely used NGS Illumina HiSeq 2000 and Illumina GAIIx are market leaders in 2011, especially in Europe and US Currently available platform Read length ranges from 35 to150 bases and accuracy is greater than 98.5% Processing Sequencing by synthesis Principle Also known as Bridge amplification method. In contrast to bead flow cell with oligonucleotide adapters is used. It reduced the cycle of amplification 3. ILLUMINA SEQUENCING
- 15. ION SEMICONDUCTOR SEQUENCING 0301 0402 Commercialized as Ion TorrentPGM (Personal Genome Machine) and Ion Torrent Proton sequencing platforms. COMMERCIALISATION In illumine and other next generation sequencing; 38- 40hr is required but in this sequencing done in only 3hr. LESS TIME Rapid Techniques as sequencing is done in real time with read length of 200 nucleotides PROCESSING Sequencing by synthesis Principle 4. ION SEMICONDUCTOR SEQUENCING Ion Torrent Sequencing, pH mediated sequencing, Silicon sequencing or Semiconductor sequencing. Also known as
- 16. MECHANISM OF ACTION Themethod use semiconductor sensing device or ion chip senses the Hydrogen ions produce during DNA synthesis by DNA polymerase. The change in pH is detected by sensing layer of micro well translate the chemical signal into digital signal, measured within a second.
- 17. Features Roche (454)Illumina SOLiD Chemistry Pyrosequencing (Sequencing by synthesis) Polymerase-based (Sequencing by synthesis) Ligation-based (Sequencing by ligation) Amplification Emulsion PCR Bridge Amp Emulsion PCR Terminators Not Used Used Used Detection based on Light emitted by luciferase Fluorescence from flurophore Fluorescence from flurophore Major error in base calling InDels Base substitution Base substitution Chief cause of error Incorrect deduction of homo- polymorphic length from intensity of luminescence Asynchronous DNA synthesis in the later cycle Bias in fluorescence intensities in later machine cycle. Template DNA fragments attached Beads in microtitier plate well A specific substrate on flow cell Beads in an acrylamide matrix Paired ends/sep Yes/3kb Yes/200bp Yes/3kb COMPARISONS
- 18. Total sequence data/ run 400Mb (GS FLX), ~1 Gb (GS FLX Titanium +) 400Gb (Hi seq 2000) 300 Gb (SOLiD 5500, SOLiD 5500 XL) Time/run 7- 10 h 4- 8 days 5- 7days Read length (short gun sequencing) 400 bases (GS FLX+, 1000 bases) 35-150 bases (up to 250 bases by Hi Seq 2500) 35- 50 bases (SOLiD 3.0) Read accuracy (%) 99.6 98.5 - Template preparation Shotgun, paired end Shotgun, paired end paired end Each base examined Once Once Twice Improved base-calling algorithm Pyrobayes Ibis and BayesCall Rsolid Draft genome preparation Yes Yes - Cost per run (total) $8439 $8950 $17447 Cost per Mb $84.39 $5.97 $5.81 Current platforms GS FLX, GS FLX Titanium Genome Analyzer 1Gb, Hi Seq 600 Gb SOLiD 5500, SOLiD 5500 XL
- 19. The short readlength that need to be assembled with the help of various bioinformatics tools/pipelines into original length template LIMITATIONS OF SGS 01 PCR bias introduced by clone amplification, for detection of base incorporation signal. 02
- 20. THIRD GENERATION SEQUENCING TheThird Generation of high throughput Sequencing develop as remedy to the SGS limitations. Instead of sequencing clonally amplified template, single DNA template is sequenced and this also need to minimal use of biochemical leading to miniaturization of whole process to nano-scale. PACIFIC BIOSCIENCE SMRT HELICOS BIOSCIENCE, USA HELICOS OXFORD NANOSPORE TECHNOLOGY NANOSPORE
- 21. Pacific Biosciences :Single Molecule Real Time (SMRT) Sequencing 1.Pacific Biosciences : Single Molecule Real Time (SMRT) Sequencing Phosphate linked nucleotide Zero-mode waveguide Two Technologies 1. 40kb but at 85% accuracy Error rate 15% Read length 2. Allow multiplexing of1000 Of ZMW in parallel High speed and High Fidelity 3. PACBIO RS Commercialized as 4.
- 22. 2.The Nanopore Sequencing Oxfordnanopore technology enables the identification of broad range of analysis including DNA, RNA, Protein and monitor changes to an electrical current as nucleic acid are passed through a protein nanopore. ERROR RATE 4% GridION Optic signals MiniION USB Stick
- 23. Conclusion The advances innext generation sequencing revolutionized the genetics and help in gaining more knowledge about the living system and the phenotypes emerges out from the system. It has provides great assistance in mastering the large-scale information collection on living systems in diverse application areasuch as treatment of human diseases, development of alternative bio fuels, enhancement of crop yield, ensuring food safety, forensics, etc. However, the First- and Second generation sequencing facilitated, a complete understanding of whole genome sequences and the information encoded therein, a more complete characterization of the methylome and transcriptome and a better understanding of interactions between proteins. But the innovation of new generation single molecule sequencing developed with potential for dramatically longer readlengths, shorter time to result and lower overall cost of DNA
- 24. NEXT GENERATION SEQUENCING THEFUTURE TOOL FOR CHOOSING DESIRSBLE BREEDING SELECTIONS
- 25. NEXT GENERATION SEQUENCING THEFUTURE TOOL FOR CHOOSING DESIRSBLE BREEDING SELECTIONS
- 27.