Next generation sequencing

It contains information about- DNA Sequencing; History and Era sequencing; Next Generation Sequencing- Introduction, Workflow, Illumina/Solexa sequencing, Roche/454 sequencing, Ion Torrent sequencing, ABI-SOLiD sequencing; Comparison between NGS & Sangers and NGS Platforms; Advantages and Applications of NGS; Future Applications of NGS.

1. NEXT GENERATION SEQUENCING (NGS) PREPARED BY ARUNDHATI MEHTA © Arundhati Mehta, 2017

2. DNA SEQUENCING DNA Sequencing is Figuring out the order of DNA nucleotides, or bases (A T G C ), in a genome that make up an organism’s DNA. F. Sangar Sangar Sequencing

3. History of DNA sequencing

4. ERA OF SEQUENCING 1 st Generation sequencing Sequence many identical molecules Sequencing in large gels or capillary tubing limits scale ABI PRISM 377

5. 5 Intro to NGS, 11.30.2016 1 st Generation Sequencing Sequence many identical molecules Sequencing in large gels or capillary tubing limits scale 2nd Generation Sequencing Sequence millions of clonally amplified molecules per run Using a reversible, stepwise sequencing chemistry Immobilized on a surface ERA OF SEQUENCING QIAGEN GeneReader Life Technologies/Applied Biosystems; SOLID 5500 Illumina MiSeq Roche / 454 Pyrosequencer

6. NEXT GENERATION SEQUENCING High throughput DNA Sequencing Technique. Employs Micro and Nanotechnologies Reduce sample size. Low Reagent cost Less Time Massive Parallel Sequencing Sequence thousands of sequences at once. Produce enormous amount of data .

7. NGS WORKFLOW Clonal Amplification by Bridge PCR Sequencing-by-ligation ( SOLiD Platform ) Clonal Amplification by Emulsion PCR Pyrosequencing (454 Sequencing) Sequencing-by-synthesis ( Solexa Technology) Sample Extraction , DNA fragmentation and invitro adapter ligation

8. NGS WORKFLOW Create DNA fragments Add platform-specific adapter sequences to every fragment. Flowcell b inding Flowcell b inding Adapter l igation p oint Adapter molecule Adapter molecules : Bind library to a flowcell or bead; Add sequence primer binding sites & Add barcodes for multiplexing. Adapter molecule bound to DNA .

9. Adapter Binding Flowcell b inding Flowcell b inding Adapter l igation p oint DNA

10. Cluster Amplification: Bridge PCR DNA fragments are flanked with adaptors ( library ) A solid surface is coated with primers complementary to the two adaptor sequences Isothermal amplification, with one end of each “bridge” tethered to the surface Clusters of DNA molecules are generated on the chip. Each cluster is originated from a single DNA fragment, and is thus a clonal population.

11. Cluster Amplification : Emulsion PCR Fragments with adaptors (the library) are PCR amplified within a water drop in oil. One PCR primer is attached to the surface of a bead. DNA molecules are synthesized on the beads in the water droplet. Each bead bears clonal DNA originated from a single DNA fragment Beads (with attached DNA) are then deposited into the wells of sequencing chips – one well, one bead

12. Sequencing & Imaging Technologies: Chain Reversible Termination Sequencing by Cyclic Reversible Termination (CRT): CRT uses reversible terminators in a cyclic method that comprises nucleotide incorporation, fluorescence imaging and cleavage. 100-150bp reads are used.

13. Sequencing & Imaging Technologies: Sequencing by Ligation Sequencing by Ligation (SBL) uses the enzyme  DNA ligase to identify the nucleotide present at a given position in a DNA sequence. 

14. Sequencing & Imaging Technologies: Pyrosequencing Pyrosequencing : non- electrophoretic , bioluminescence method that measures the release of inorganic pyrophosphate by proportionally converting it into visible light using a series of enzymatic reaction Nucleotide incorporation generates light seen as a peak in the Pyrogram trace

15. Sequencing & Imaging Technologies: Single Molecule-Real Time Sequencing Single Molecule- Real Time (SMRT) is a parallelized single molecule DNA sequencing method. A single DNA polymerase enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template DNA Polymerase ZMW (Zero Mode Waveguide DNA)

16. NGS Tech nologies Overview NGS differs in template preparation, sequencing and imaging, and data analysis Commercially available technologies: Illumina / Solexa Roche/454 Helicos BioSciences Life/APG – SOLiD system Pacific Biosciences Ion Torrent technology

17. Solid-phase amplification can produce 100-200 million spatially separated clusters, providing free ends to which a universal sequencing primer can be hybridized to initiate the NGS reaction ILLUMINA /SOLEXA SEQUENCING Run time: 1 – 10 days Produces: 2–1000 Gb of sequence Read length: 2 x 50 bp – 2 x 250 bp ( paired-end) Cost: $ 0.05–$0.40/Mb Bridge PCR Clustal Amplification

18. Applications DNA sequencing Gene Regulation Analysis Sequencing-based Transcriptome Analysis SNPs and SVs discovery Cytogenetic Analysis ChIP -sequencing Small RNA discovery analysis A whole human genome sequence was determined in 8 weeks to an average depth of ~ 40X, discovering ~ 4 new million SNPs and ~400000 SVs (with an accuracy <1% for both over-calls and under-calls) Over 1800 publications.

19. ROCHE/454 SEQUENCING Sequence much longer reads by sequencing multiple reads at once by reading optical signals as bases are added. The DNA or RNA is fragmented into shorter reads up to 1kb. Uses Emulsion PCR for Clustal Amplificication . PYROSEQUENCING as sequencing approach.

20. Nucleotide incorporation generates light seen as a peak in the Pyrogram trace . All of the sequence reads we get from 454 will be different lengths , because different numbers of these bases will be added with each cycle. Applications Whole genome sequencing Targeted resequencing Sequencing-based Transcriptome Analysis Metagenomics Over 1300 publications...

21. ION TORRENT SEQUENCING Ion torrent and ion proton sequencing do not make use of optical signals. Instead, they exploit the fact that addition of a dNTP to a DNA polymer releases an H+ ion. Run time: 3 h; no termination or deprotection steps Clustal Amplification- Emulsion PCR Read length: 100–300 bp Throughput determined by chip size : 10Mb – 5 Gb Cost: $1–$20/Mb The pH change, if any, is used to determine how many bases (if any) were added with each cycle.

22. LIFE/APG/ABI- SOLiD SEQUENCING AB SOLID TM 3 System generates over 20 gigabases & 400 M tags per run . Library Preparation Emulsion PCR/ Bead Enrichment Bead deposition Sequencing by Ligation Chemical crosslinking to an amino-coated glass surface

23. SANGERS Vs. NGS Features Sanger NGS Sequencing Samples Clones, PCR DNA Libraries Preparation Steps Few, Sequencing reactions clean up Many, Complex procedures Data Collection Samples in plates : 96, 384 Samples on slides 1-16+ Data 1 Read/ Sample Thousands & Millions of Reads/ Samples.

24. Comparison Of NGS Platforms

25. ADVANTAGES OF NGS Sanger Sequencing NGS Sequencing

26. APPLICATIONS OF NGS Mutation discovery Transcriptome Analysis – RNA- Seq Sequencing clinical isolates in strain-to-reference mechanisms. Enabling Metagenomics Defining DNA-Protein interactions – ChIP-Seq Discovering non-coding RNAs Molecular diagnostics for Oncology & Inherited Disease study. Gene Regulation Analysis Whole Genome Sequencing Exploring Chromatin Packaging

27. FUTURE APPLICATIONS OF NGS

28. SUMMARY Next Generation Sequencing has changed the way we carry out molecular biology and genomic studies. It has allowed us to sequence and annotate genomes at a faster rate. It has allowed us to study , variation, expression and DNA binding at a genome – wide level.

29. REFERENCES Elaine R. Mardis (2008) the impact of next-generation sequencing technology on genetics. Cell vol.24 No.3,133-14. Elaine R. Mardis (2009): Next-Generation Sequencing Methods. Annu . Rev. Genomics hum genet. 9:387-402 Jorge S Reis- Filho (2010): Next-Generation Sequencing, Breast Cancer Research 2010, 11( Suppl 3) Some websites – https:// www.ncbi.nlm.nih.gov/pubmed