Sequencing Technologies

Objectives Understand Sanger sequencing (first generation) Understand Next Gen Sequencing Understand 3rd Generation Nanopore sequencing Review paper

Sequencing Timeline http://www.yourgenome.org/facts/timeline-organisms-that-have-had-their-genomes-sequenced

Sequencing Timeline http://genomicsforeveryone.org/week-1-genetics-and-public-health-genomics/

Cost of sequencing the human genome https://www.genome.gov/sequencingcostsdata/

Nucleic acid hybridization is the basis of sequencing technologies Complementary Base Pairing Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

Modern Sequencing is Automated (a) Standard sequencing machine Figure 20.2 DNA sequencing machines (b) Next-generation sequencing machines Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

Mardis, Elaine R. "DNA sequencing technologies: 2006-2016 Mardis, Elaine R. "DNA sequencing technologies: 2006-2016." Nature Protocols 12.2 (2017): 213-218.

Mardis, Elaine R. "DNA sequencing technologies: 2006-2016 Mardis, Elaine R. "DNA sequencing technologies: 2006-2016." Nature Protocols 12.2 (2017): 213-218.

Overview of Sanger Seqeuncing Steps DNA is denatured Primer is annealed to DNA DNA Polymerase uses a radiolabeled deoxynucleotide triphosphates (normal dNTPs) and a few chain-terminating dideoxynucleotide triphosphates (ddNTPs) to build strand. DNA run on Polyacrylamide gels DNA read via x-ray http://www.csus.edu/indiv/r/rogersa/bio181/seqsanger.pdf

DNA Sequencing Polyacrylamide gel electrophoresis separates ssDNA molecules that differ in length by just one nucleotide Molecules are labelled with a radioactive protein or radioactive isotope, visualized by autoradiography producing a banding pattern http://classroom.sdmesa.edu/eschmid/Lab12%201.jpg https://www.premedhq.com/gel-electrophoresis-and-southern-blotting

Sanger Sequencing Technique DNA (template strand) Primer DNA 3′ T G 5′ C T G A C A 5′ DNA polymerase Figure 20.3a Research method: dideoxy chain termination method for sequencing DNA (part 1) 3′ Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014) http://www.csus.edu/indiv/r/rogersa/bio181/seqsanger.pdf

Deoxyribo- nucleotides Sanger Sequencing Technique DNA (template strand) Primer Deoxyribo- nucleotides Dideoxyribonucleotides (fluorescently tagged) 3′ T G 5′ C T G A C A dATP ddATP 5′ dCTP ddCTP DNA polymerase dTTP ddTTP dGTP ddGTP Figure 20.3a Research method: dideoxy chain termination method for sequencing DNA (part 1) P P P P P P G G 3′ Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

Deoxyribo- nucleotides Sanger Sequencing Technique DNA (template strand) Primer Deoxyribo- nucleotides Dideoxyribonucleotides (fluorescently tagged) 3′ T G 5′ C T G A C A dATP ddATP 5′ dCTP ddCTP DNA polymerase dTTP ddTTP dGTP ddGTP Figure 20.3a Research method: dideoxy chain termination method for sequencing DNA (part 1) P P P P P P G G 3′ Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

Sanger Sequencing Technique DNA (template strand) Labeled strands 5′ 3′ C T G A C A dd G A C T dd A C T G dd C T G A dd T G A dd G A dd A G dd A G A G C T dd 3′ G C T dd C T G C T G C T G C T G C T G C T G C T G Figure 20.3b Research method: dideoxy chain termination method for sequencing DNA (part 2) 5′ 3′ 5′ Shortest Longest Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

Sanger Sequencing Technique Direction of movement of strands Longest labeled strand Detector Laser Shortest labeled strand Results Last nucleotide of longest labeled strand Figure 20.3c Research method: dideoxy chain termination method for sequencing DNA (part 3) G A C T G A C Last nucleotide of shortest labeled strand Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

Human Genome Shotgun Sequencing Bacterial Artificial Chromosome (BAC) Bacterial Artificial Chromosome (BAC) https://www.ndsu.edu/pubweb/~mcclean/plsc411/Genome%20Sequencing%20Complete.pdf http://www.genome.ou.edu/3653/3653-101705.html

Next-Gen Sequencing Second Generation Sequencing Technique 1 Genomic DNA is fragmented. 2 Each fragment is isolated with a bead. 3 Using PCR, 106 copies of each fragment are made, each attached to the bead by 5′ end. 4 The bead is placed into a well with DNA polymerases and primers. Figure 20.4a Research method: next-generation sequencing (part 1) Template strand of DNA 3′ 5′ 3′ 5′ Primer A T G C 5 A solution of each of the four nucleotides is added to all wells and then washed off. The entire process is then repeated. Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

Next-Gen Sequencing Technique A T G C A T G C Template strand of DNA C C C C A A dTTP A dATP A T T G G T A PPi T A DNA polymerase G C G C G C G C A G Figure 20.4b Research method: next-generation sequencing (part 2) Primer A G T A T A 6 7 If a nucleotide is joined to a growing strand, PPi is released, causing a flash of light that is recorded. If a nucleotide is not complementary to the next template base, no PPi is released, and no flash of light is recorded. Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

Next-Gen Sequencing Technique 8 A T G C A T G C C A T G C A T G dCTP dGTP C PPi A Figure 20.4c Research method: next-generation sequencing (part 3) 8 The process is repeated until every fragment has a complete complementary strand. The pattern of flashes reveals the sequence. Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

Next-Gen Sequencing Results A 4-mer T G 3-mer C 2-mer 1-mer Figure 20.4d Research method: next-generation sequencing (part 4) Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

Figure 1 454 Workflow: library construction ligates 454-specific adapters to DNA fragments and couples amplification beads with DNA in an emulsion PCR to amplify fragments before sequencing. The beads are loaded into the picotiter plate (PTP). The bottom panel illustrates the pyrosequencing reaction that occurs on nucleotide incorporation to report sequencing by synthesis. http://www.cell.com/trends/genetics/fulltext/S0168-9525(08)00023-1 Trends in Genetics 2008 24, 133-141DOI: (10.1016/j.tig.2007.12.007)

Nanopore Sequencing https://www.nextgenerationsequencing.info/ngs-products/ngs-technologies/oxford-nanopore-technologies http://www2.technologyreview.com/news/427677/nanopore-sequencing/

https://www. slideshare https://www.slideshare.net/AnneliesHaegeman/annelies-haegeman-ngs-basic-principles-and-sequencing-platforms

Multiple sequence alignment Novel BHD missense mutation (K508R). (A) Genomic sequence of control. (B) Genomic sequence of BHDS patient with the c.1978A>G (K508R) mutation. (C) Multiple sequence alignment. The amino acid residues are coloured according to their chemical properties. (D) Degree of conservation. The numerical index reflects the degree of conservation of the physical–chemical properties in the alignment. Star (*) indicates amino acids 100% identical in the alignment (highest score). The next most conserved group is composed of substitutions in amino acids with the same physical chemical class. https://www.mathworks.com/help/bioinfo/ug/viewing-and-aligning-multiple-sequences.html?requestedDomain=www.mathworks.com http://jmg.bmj.com/content/45/6/3210

Multiple sequence alignment Interactive Structure based Sequences Alignment Program Strap Interactive Structure based  Sequences Alignment Program Strap http://www.bioinformatics.org/strap/index2.html

Multiple sequence alignment BLAST: Basic Local Alignment Search Tool http://masscience.com/wp-content/uploads/2015/06/204g_expression-confirmation_blast-EST.jpg

High throughput Technology Qiagen Gene Reader, sequencing by sequence (SBS) Automation Bioinformatics pipeline Identification of mutations and best therapy This is a complete solution https://www.qiagen.com/us/products/ngs/mdx-ngs-genereader/ https://www.youtube.com/watch?v=HQhw5Ihp8IA

Challenges of NGS https://www.slideshare.net/ueb52/introduction-to-next-generation-sequencing-v2?next_slideshow=1

https://www. slideshare https://www.slideshare.net/ueb52/introduction-to-next-generation-sequencing-v2?next_slideshow=1

Summary: High throughput Technology https://www.qiagen.com/us/products/ngs/mdx-ngs-genereader/

Worksheet 2!