Sequencing technologies What is - PowerPoint Presentation

1. Sequencing technologies

2. What is the DNA sequencing?DNA sequencing determines the order of the four chemical building blocks - called "bases" - that make up the DNA molecule. The sequence tells scientists the kind of genetic information that is carried in a particular DNA segment. For example, scientists can use sequence information to determine which stretches of DNA contain genes and which stretches carry regulatory instructions, turning genes on or off. In addition, and importantly, sequence data can highlight changes in a gene that may cause disease.

3. The Sanger method The Sanger method (‘first-generation’ technology) was the primary sequencing technology between 1975 and 2005. The Sanger method relies on an in vitro DNA replication reaction. Its ingredients are similar to those needed for DNA replication in an organism, or for (PCR). They include: 1- strand DNA template 2- DNA primer 3- DNA polymerase 4- The four DNA nucleotides (dATP, dCTP, dGTP, and dTTP) 5- • Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each labeled with a different color of dye

4. Dideoxy nucleotides are similar to deoxy, nucleotides, but with one key difference: 1) They lack a 3’ hydroxyl group which is required for further DNA extension resulting in chain termination once incorporated in the DNA molecule 2) Each di-deoxynucleotide has a unique fluorescent dye attached to it allowing for automatic detection of the DNA sequence.

5. The mixture is first heated to denature the template DNA (separate the strands) Then cooled so that the primer can bind to the single-stranded template. Once the primer has bound, the temperature is raised againThis temperature allowing DNA polymerase to synthesize new DNA starting from the primer. DNA polymerase will continue adding nucleotides to the chain until it happens to add a dideoxy nucleotide instead of a normal one. At that point, no further nucleotides can be added, so the strand will end with the dideoxy nucleotide.

6. Each reaction also contain a mixture of four di-deoxynucleotide one for each DNA base.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17. Human Genome Project (HGP) One of the goals of the Human Genome Project (HGP) is to support advancements in DNA sequencing technology.10 Although the HGP was completed with the Sanger sequencing method, many groups of researchers were already tinkering with new ideas to increase throughput and decrease cost of sequencing prior to the announcement of the first human genome draft in 2001. After years of experimentations, the second DNA sequencing technology revolution finally took off in 2005 and ended Sanger sequencing dominance in the marketplace.

18. Second generation sequencing(NGS) Commercial SGS tools emerged in 2005 in response to the low throughput and high cost of first-generation methods.The short read technologies currently in use are collectively known as massively parallel sequencing and are often also referred to as second-generation sequencing.NGS allowed scientist to sequence thousands to millions of DNA molecules in a single machine run.

19. SGS workflow consists of distinct steps, as presented in the following subsectionsEven though platforms have different biochemistry and arrays, the workflows include similar steps: 1- DNA extraction2- library preparation, which usually includes shearing the DNA either mechanically or enzymatically, adding adaptors and barcodes/indexes and amplification3- Template preparation, either by bridge amplification or emulsion PCR; 4- Automated sequencing

20. Typical whole genome sequencing workflow in a clinical or public health laboratory.

21.

22. Characteristics, strengths and weaknesses of commonly used sequencing platforms 2018Platform \ InstrumentThroughput range (Gb)Read length (bp)StrengthWeaknessSanger sequencingABI 3500/37300.0003Up to 1 kbRead accuracy and lengthCost and throughputIllumiIlluminaMiniSeq1.7- 7.51×175 to ×150Low initial investmentRun and read lengthMiSeq0.3-151×36 to 2×300Read length, scalabilityRun lengthNextSeq10-1201×75 to 2×150ThroughputRun and read lengthHiSeq (2500)10-1000×50 to ×250Read accuracy, throughputHigh initial investment, runNovaSeq 5000/60002000-60002×50 to ×150Read accuracy, throughputHigh initial investment, run

23. Characteristics, strengths and weaknesses of commonly used sequencing platformsPlatform \ InstrumentThroughput range (Gb)Read length (bp)StrengthWeaknessIonTorrentPGM0.08 - 2Up to 400Read length, speedThroughput, homopolymerscS50.6 - 15Up to 400 Read length, speedHomopolymersProton10 - 15Up to 200 Speed, throughputHomopolymers

24. Third-generation sequencing (TGS)There is a third revolution in sequencing technology underway with the commercialization of third generation sequencing technologies such as those from Pacific Biosciences and Oxford Nanopore Technologies. Third-generation methods (TGS), which are beginning to appear, focus on monitoring the natural synthesis of a single DNA molecule without the need for DNA fragmentation and amplification Third generation sequencing is defined as the sequencing of single DNA molecules without the need to halt between read steps, whether enzymatic or otherwise.These technologies directly target single DNA molecules without the need for PCR amplification.

25. Platform \ InstrumentThroughput range (Gb)Read length (bp)StrengthWeaknessPacific BioSciencesPacBio RSII0.5 - 1Up to 60 kbRead length, speed(Average 10 kb, N50 20 kb)High error rate and initialSequel5 – 10 Up to 60 kb Read length, speed(Average 10 kb, N50 20 kb)High error rate Oxford NanoporeMInION0.1 – 1 Up to 100 kb Read length, portabilityHigh error rate, run length,

26. Comparison between commercially available NGS platforms 2020 Platform Sequencing Maximumread lenght(bp)Reads perrun Run timeMaximumoutput Error rateFirst generation Sanger NA9009620 min – 3 h 2.1 Mb0.3%Second generation454GS Junior+ Pyro 700 0.1 M 18 h 70 Mb 1% indelsGS FLXTitanium XL+Pyro 700 1 M 23 h 700 Mb 1% indels

27. Platform Sequencing Maximumread lenght(bp)Reads perrun Run timeMaximumoutput Error rateIlluminaHi SeqaSBS36 (SE) Up to 4 B(SE)<1–3.5 h (HiSeq3000/4000)1500 Gb0.1%substitution125 (PE) Up to 8 B(PE)7 h – 6 d (HiSeq 2500)MiniSeqbSBS150 (PE) 25 M 4–24 h 7.5 Gb<1%substitutionNextSeq 550bSBS75 (SE) Up to400 M(SE)12-30 h120 Gb<1%150 (PE) Up to800 M(PE)substitutionMiSeq (v3)SBS75 (PE)25 M(PE)4–55 h15 Gb0.1%300 (PE)substitution

28. Platform Sequencing Maximumread lenght(bp)Reads perrun Run timeMaximumoutput Error rateIlluminaHi SeqXa SBS 150 (PE) 5.3-6 B <3 d 1800 Gb0.1%substitutionNovaSeq6000c SBS 150 (PE) 20 B 36–44 h 6000 Gb NAIon Torrent PGM SBS 400 (SE) 400000–5.5 M2.3–7.3 h 2 Gb 1% indelsProton SBS Up to 200(SE)60–80 M 2–4 h Up to10 Gb1% indelsS5 SBS 600 (SE) 2–130 M 2.5–4 h 25 Gb 1% indels

29. Platform Sequencing Maximumread lenght(bp)Reads perrun Run timeMaximumoutput Error rateSOLiD (Sequencing by Oligonucleotide Ligation and Detection)5500xl SBL75 (SE)~1.4 B10 d240 Gb 0.01%50 (PE)A-T baisThird generationPacBio (Pacific Bioscience)RS II SMRT >15000(average)Up to5500030 min–4 h 1 Gb 15% indelsSequel SMRT 30000(average)~400000 30 min–20 h 10 Gb 15%

30. Platform Sequencing Maximumread lenght(bp)Reads perrun Run timeMaximumoutput Error rateOxford NanoporeMinION SMRT Up to 900kbUp to1 MUp to 48 h 20 Gb 5–10%

31. THANK YOU FOR YOUR ATTENTION