DNA gene sequence Group 09 presentation-1.pptx

DNA gene sequence

1. Next Generation Gene Sequencing K.M.B Perera AG/2019096 P.A.M Hansamali AG/2019188 Group 09

2. Gene Sequencing Methods of DNA Sequencing Illumina sequencing Process of Illumina sequencing Library Preparation Cluster generation Sequencing reaction Data analysis Applications of NGS Comparison of NGS methods Outline

3. Gene Sequencing A method used to determine the precise order of the four nucleotide bases – adenine, guanine, cytosine and thymine - that make up a strand of DNA

4. Methods of DNA Sequencing Illumina sequencing Roche 454 ABI SOLiD Maxam and Gilbert sequencing Sanger sequencing First generation DNA sequencing Next generation DNA sequencing

5. Illumina sequencing The Illumina next-generation sequencing method is based on sequencing by synthesis and reversible Illumina terminators that enable the identification of single bases as they are introduced into DNA strands

6. Process of Illumina sequencing

7. Library Preparation Libraries are created using random fragmentation of DNA. Specialized adaptors are added to both ends. Adaptors This should be contain complementary sequences that allow the DNA fragments to bind to the flow cell. Multiplexing When multiple libraries are pooled together unique adaptors “Barcode” are added to each library.

8. Cluster generation The library is loaded into a flow cell Fragments are captured on a lawn of surface-bound oligos complementary to the library adapters ( flow cell contains two different oligo nucleotides that complementary to two adaptors) Unbound DNA washed away. Bounded ssDNA fragments are used as a template to add dNTPs to elongating oligoes. Resulting dsDNA

9. Denaturation – original ssDNA strands are washed off Each fragment is then amplified into distinct, clonal clusters through bridge amplification When cluster generation is complete, the templates are ready for sequencing. Cluster generation cont.

10. Bridge Amplification

11. Bending of the DNA to bind to and adjutant adaptor/ oligo via complementarity DNA replication using the bent ssDNA as template Denaturation of the dsDNA bridge to result in two ssDNA on the flow cell This process continues until all the DNA fragments have been clonally amplified Resulting in a flow cell containing millions of clusters of ssDNA.

12. Primer is binds to the oligonucleotide Chemically modified nucleotides complementary bind to the DNA strand A reversible terminator blocks incorporation of the next base Each nucleotide contains fluorescent tag Florescent signal indicates the added nucleotide Sequencing reaction

13. The instrument software identifies nucleotides Whole process generates millions of reads These reads are overlaid and compared to a reference genome By the overlaying, the whole genome sequence can be generated Data analysis

14. Applications of NGS Rapidly sequence whole genomes Deeply sequence the target regions RNA sequencing (RNA-Seq) ChIP -Seq (Chromatin Immunoprecipitation Sequencing) Analyze epigenetic factors Cancer Genomics Study the human microbiome further Identify novel pathogens Forensic Genomics Infectious Disease Genomics

15. Comparison of NGS methods Illumina Roche 454 ABI SOLiD Sequencing Chemistry Chemically modified nucleotides ie : fluorescently labeled nucleotides pyrosequencing method ligation-based sequencing method Read Length short read lengths 10-100bp longer read length (up to 800 bases) shorter read lengths Error Rates low error rate higher error rate (compared to Illumina) low error rate

16. Illumina Roche 454 ABI SOLiD Data Output high data output lower data output moderate data output (falling between Illumina and Roche 454) Platform HiSeq MiSeq 454 SOLiD Amplification Bridge PCR emPCR (emulsion PCR) emPCR /wildfire Comparison of NGS methods cont.

17. REFERENCES https://www.illumina.com/science/technology/next-generation-sequencing https://www.researchgate.net/figure https://genomebiology.biomedcentral.com/article

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