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Lecture 5: NGS.

Last generation: (1) Maxam-Gilbert Sequencing – degradation method (diff.) (2) Sanger Sequencing a. Radiographs b. Dye-based --------------------------------------------------------------------------------------------------------------------------------1970s. MAXIM GILBERT SEQUENCING: DNA degradation  Seq degradation method o 4 rxns (G, C; AG, CT)  Covalently modified nt o Radioactive P-32. (P at 5’ end labelled)  Cleavage by piperidine.  Separate = electrophoresis  Read nt up the gel  Max read length: 500 bp.

1985. SANGER: DNA synthesis.  Replaced M-G  DNA synthesis (not DNA degradation)  First method: used radioactively labelled nt + ddNTP in 4 rxns o Radioactive P-32. (P at 5’ end labelled)  Read: autoradiographs (on X-Ray film)  Max read lengths: 200-500 bp routine. Advantage: less radioactive, fewer hazardous chems than M-G.

1990S. SANGER: automated fluorescent dye-labelled Sanger-sequencing.  Second method: used dye labelled nt + ddNTP in 1 rxn only.

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o Each ddNTP associated w/ 1 colour. Max read lengths: 1200 bp.

Advantage: removed radioactivity element of procedure (safer process) … quicker process + longer read lengths. diff peaks for diff colours --------------------------------------------------------------------------------------------------------------------------------

Sequencing Retrospective: DNA sequencing apparatus

Manual or Auto Read Lengths (/day)

1985 – 1995 Manual P/A gel 1200

1996 Semi Auto P/A gel 2400

1998 – 2004 Automated 1 column 14, 400 (14.4k)

2004 – 2010 Automated 8 column 76, 80 (76.8k)

Manual polyacrylamide gel. Automated = no slab gel, used capillary tube instead. Same electrophoresis technique to separate.  More reads, less work = lowered cost  In 2015, ~ $1000 to sequence a whole human genome. -------------------------------------------------------------------------------------------------------------------------------NEXT GENERATION SEQUENCING: 1) Pyrosequencing 2) Ion torrent PGM 3) Illumina MiSeq 4) Nanopore Sequencing 5) SOLiD 5500XL Sequencing by Ligation

Read length Tether

Pyro

Ion torrent

Illumina

1000 Wells

400 Wells - pH meter

600 Flow cell

Nanopore

SOLiD

Tech Base Sequencing by Products used

Machine Emulsion based – fluo readings DNA synthesis DNAP (3’  5’) PPi

Machine Sensor for H+ DNA synthesis DNAP (3’  5’) H+

Machine Sanger dye-based – glass flow cell DNA synthesis DNAP (3’  5’) Dye-terminator nt

Hand-held Forms pore

Machine Emulsion based DNA ligation DNA ligase (5’  3’)

------------------------------------------------------------------------------------------------------------------------------ Sequencing is DNA-synthesis based.  All depend on tethering individual DNA mlcs to a solid phase (not in sol’n , like the 2 earlier methods)  Shorter read lengths than Sanger … but can sequence millions of diff mlcs simultaneously so u get lots of data. Standard Preparation: (slide 9)  Nucleic acid fragments  Fragment into a standard size (< 800 bp)  Polish ends o Make ends clean o Modify end sequences by adding adaptors.  This creates a library of sequences. o Middle is unique o The ends are PREDICTABLE, IDENTIFIABLE, UNIFORM so that we’ve a standardized set of mlcs that can be used in machines. ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PYROSEQUENCING:  Emulsion of: o 1 strand of library DNA o 1 bead (has an adaptor) o Enz/nts.  Vortex mix to create an emulsion which has one bead and one piece of DNA per droplet.  Initiate PCR  DNA fragment is fused to the bead (adaptor)  Fragment gets copied multiple times – each new copy are tethered to the same bead.  Result = bead w many identical copies, fused + bonded to the bead.  

Add the emulsion PCR products to surfaces with many wells Beads fall into wells as the sample passes over the surface

Sequencing:  Melt DNA so only 1 strand is attached to bead (other = washed away)  Add DNAP  Wash entire array of wells, 1 nt @ time (first wash away A, C, G, T)  As each well is washed & the correct nt is added to be properly incorporated into the new strand, a pyrophosphate mlc is released  It can interact w luciferin, which breaks the high nrg bond & releases a photon of light. Detection:  Nt incorporation releases a pyrophosphate  Luciferin breaks high nrg bond in pyrophosphate … releases photon of light. Every time correct nt is passed over array of wells, pyro-phos released --> luciferin produces a fluo outcome.  

Field of the wells – some wells are lit up, others not. o Everywhere N nt is added to growing strand, lights up. Can look sequentially @ each nt that’s added.

Reads of up to: 1000 bp. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ION TORRENT PGM:  Also bead-based tethering system, but detects protons instead of pyrophosphate using luciferin.   

Tiny pH meter sensor associated with each well in an array that makes up chip sequence. Purpose (sensor): to determine when a nt is added to growing DNA strand. What this tells us: which wells are producing a lot of H+ when a particular nt is added to well.

Reads up to: 400 bp. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ILLUMINA MiSEQ:  Most popular – can sequence entire human genome in couple of days for ~ $1000  Tether DNA mlcs to a piece of glass instead of using array of wells.  Similar to dye-terminated ddNTP (in Sangar sequencing): wash glass slides w dye terminated ddNTP.

Process:  Tether DNA to a particular location on the glass  PCR amplify to create daughter DNA next to the original mlc o First strand bends over to the probe/substrate next to it o Daughter strand synthesized o Use RE to dissociate one end of strand o But this strand is inverted  Create many such copies on the glass  Since you’ve strand + its invert, Sequencing same DNA mlc in 2 diff directions gives additional confidence that u sequenced correctly. -------------------------------------------------------------------------------------------------------------------------------------------------------NANOPORE SEQUENCING  DNA dbl helix is melted so  One strand goes through membrane pore  Other strand retained on other side of membrane.  As ssDNA pushed thru pore, an adapter mlc measures size & amt of charge assoc. w/ each nt (along ssDNA strand)  How much that adapter mlc is displaced from the pore center is determined by which nt is part of ssDNA @ that location in seq.  Adapter displacement either inc or dec. (widens/narrows) pore size , allowing larger or smaller flow of ions on either side of DNA mlc.  By det. how many ions can flow thru pore, system can determine which nt was @ that position… and record it.

(!) A protein unzips DNA helix into 2 strands. (@) 2nd protein creates a core in the membrane and holds an adapter mlc. (#) ions flow thru pore, creating current … each base blocks the flow to a different degree, altering current flow. ($) adaptor mlc keeps the bases in place long enough for them to be electronically identified. -------------------------------------------------------------------------------------------------------------------------------------------------------SOLiD: **starts off like Pyro.  DNA fragment  Bind to bead  PCR amplif DNA fragment o Many fragment copies on the 1 bead  Prepare emulsion PCR for solid sequencing.

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Instead of DNA synth, use a set of primers & add many nt @ once.

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Hybridize Ligated Fluorescence measured, dye is cleaved off Free 5’ phosphate for further rxns … keep going until full strand synthesized. Synthesized strand removed

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New primer hybridized, offset by 1 base. Ligation pattern repeated.

Primer reset is repeated for five rounds. Provides dual measurement of each base Increases sequencing accuracy by several bases. **scalable! -------------------------------------------------------------------------------------------------------------------------------------------------------...