Lecture 9a: Overview of Sequencing and NGS UNFINISHED

Question 1

DNA Sequencing

• what is it?
• Developed by who?

Answer 1

• DNA sequencing = determining the nucleotide sequence of DNA.
• Dideoxy sequencing developed by Frederick Sanger in the 1970s.

Question 2

What is SANGER TECHNIQUE?

Answer 2

• Uses dideoxynucleotides (dideoxyadenine, dideoxyguanine, etc)
• These are molecules that resemble normal nucleotides but lack the normal -OH group

Question 3

Dideoxy DNA sequencing -Dideoxy DNA sequencing relies on chain termination:

EXPLAIN THE STEPS OF PROCESS…15

Answer 3

1. DNA template is denatured to single strands.
2. Single DNA primer (3’ end near sequence of interest) is ANNEALED to TEMPLATE DNA and EXTENDED with DNA POLYMERASE
3. FOUR REACTIONS are set up, each CONTAINING:
4. DNA TEMPLATE
5. PRIMER ANNEALED TO TEMPLATE DNA
6. DNA POLYMERASE
7. dNTPS (dATP, dTTP, dCTP, and dGTP)
8. DIFFERENT LABELED DIDEOXYNUCLEOTIDE (ddATP, ddTTP, ddCTP, orddGTP) isADDED TO EACH OF 4 RXN TUBES AT 1/100TH THE CONCENTRATION OF NORMAL dNTPS
9. ddNTPs POSSESS a 3’-H instead of 3’-OH, COMPLETE IN THE REACTION. WITH NORMAL dNTPS, and produce NO PHOSPHODIESTER BOND bond.
10. Whenever the LABELED ddNTPs are INCORPORATED IN THE CHAIN, DNA SYNTHESIS TERMINATES.
11. Dideoxy DNA sequencing also called DYE TERMINATOR sequencing.
12. Each of the FOUR REACTION MIXTURES PRODUCES A POPULATION OF DNA MOLECULES WITH DNA CHAINS TERMINATING AT ALL POSSIBLE POSITIONS.
13. EXTENSION PRODUCTS IN EACH of the four RXN MIXTURES ALSO END WITH A DIFFERENT LABELED ddNTP (depending on the base).
14. Next, each reaction mixture is ‘ELECTROPHORESED’ in a SEPARATE LANES (4 lanes) at HIGH VOLTAGE ON A ‘POLYACRYLAMIDE GEL’ polyacrylamide gel.
15. POLYACRYLAMIDE gels can be thinner –> HIGHER VOLTAGE —> FASTER
16. PATTERN OF BANDS IN OF THE FOUR LANES IS VISUALISED on ‘X-ray film’ or AUTOMATED SEQUENCER
17. LOCATION of “bands” in each of the four lanes INDICATE the SIZE of the FRAGMENT TERMINATING WITH A RESPECTIVE LABELED ddNTP.
18. DNA SEQUENCE IS DEDUCED FROM THE PATTERN OF BANDS UIN THE 4 LANES

Question 4

Dideoxy DNA sequencing relies on chain termination: DIAGRAM

Answer 4

SLIDE 7 AND 8

Question 5

What is pyrosequencing? what is the process? = 6

Answer 5

1. Based on the “sequencing by synthesis” principle instead of chain termination with dideoxy nucleotides.
2. Developed by Pål Nyrén/Mostafa Ronaghi in 1996.
3. . Immobilize a single template DNA molecule on a bead/substrate and synthesize complementary strand.
4. Detect which nucleotide is added at each step. Sequencing
   (polymerization) doesn’t stop…
5. Complex reaction requiring template DNA, primer, DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5’ phosphosulfate (APS), and luciferin.
6. As with dideoxy sequencing, base incorporation is recorded when light is emitted at particular wavelengths.

Question 6

Pyrosequencing reaction diagram

Answer 6

important on slide 10

Question 7

pyrogram result of pyrosequencing

Answer 7

important diagram on slide 10

Question 8

Sanger Sequencing vs pyrosequencing
= 8

Answer 8

1 * Sanger sequencing relies on electrophoretic separation of end-stage PCR products
…2 – Physical limits on number of capillaries
…3 – Relatively expensive

4. • Pyrosequencing VERY expensive
     …5. – Not easy to run many reactions at once
5. • If you can eliminate the need for electrophoresis, new possibilities open up :
     …7 – Sequencing by synthesis
     …8 – Direct scanning of products

Question 9

Next-generation sequencing platforms = 8

Answer 9

1 *HiSeq/MiSeq/NextSeq/NovaSeq (Illumina)

2 * SOLiD2/3/4/5500 (Life TEchnologies)

3 * 454/FLX (Roche Diagnostics)

4 * Ion Torrent PGM/Proton (Life Technologies)

5 * GridIon/MinIon (Oxford Nanopore)

6 * SMRT/Sequel (Pacific Biosciences)

7 * 3rd Generation/Single molecule sequencing

8* Direct in-situ sequencing (?

Question 10

Work flow of conventional

versus

second-generation sequencing

Answer 10

A.
1. DNA FRAGMENTATION
2. IN ‘vivo’ CLONING AND AMPLIFICATION
3. Cycle sequncing
4. Electrophoresis
(1 read/capillary)

B.
1. DNA fragmentation
2. in ‘vitro’ adaptor ligation
3. Generation of polony array
4. Cyclic array sequncing
(>10^6 reads/array)

Diagram on slide 14

Question 11

8 simple steps to NGS

Answer 11

1. Fragment target DNA and ligate UNIVERSAL adaptors
2. Amplify single molecules (beads vs free)
3. Sequence clonal amplicons
   ….4 Each system differs in the way the DNA is sequenced
   ….5 Sequence 1 bp, then read, then repeat
4. Computer assemble the data
   …7 Reference-based assemblies
   ..8 De novo assemblies

Question 12

Adaptors allow : 6

Answer 12

1 – Simplified sample processing

2 – Purifcation steps
…3 * Streptavidin/Biotin
….4 * Magnetic beads

5 – Uniform library quantification by qPCR

6 – Uniform amplification/sequencing

Question 13

NGS Methods….Major platforms available = 3

Answer 13

1 * Roche
– GS-FLX
– GS-Junior

2 * Illumina
– HiSeq, MiSeq, NextSeq, Nova seq

3 * Life Technologies
– SOLiD
– Ion Torrent
– Proton

Question 14

Illumina Sequencing…BRIDGE-PCR

steps = 12

Answer 14

1. Prepare genomic DNA sample…randomly fragment genomic DNA and ligate adapters to both ends of the fragments
2. Attach DNA to the surface…Bind single-stranded fragments randomly to the inside surface of the flow cell channels
3. Bridge amplification …add unlabelled nucleotides and enzyme to initiate solid-phase bridge amplification
4. FRAGMENT BECOME DOUBLE STRANDED… the enzyme incorporates nucleotides to build double-stranded bridges on the solid-phase substrate
5. DENATURE THE DOUBLE-STRANDED MOLECULES…denaturation leaves single-stranded templates anchored to the substrate
6. COMPLETE AMPLIFICATION …Several million dense clusters of double-stranded DNA are generated in each channel of the flow cell
7. DETERMINE FIRST BASE…First chemistry cycle: to initiate the first sequencing cycle, add all 4 labelled reversible terminators, primers and DNA polymerase enzyme to the flow cell.
8. IMAGE FIRST BASE…After laser excitation, capture the image of emitted fluorescence from each cluster on the flow cell. record the identity of the first base for each cluster.
9. DETERMINE SECOND BASE…Second chemistry cycle: to initiate the next sequence cycle, add all 4 labelled reversible terminators and enzymes to the flow cell.
10. IMAGE SECOND CHEMISTRY CYCLE:…after laser excitation, collect the image data as before. record the identity of the second base for each cluster.
11. SEQUENCE READS OVER MULTIPLE CHEMISTRY CYCLES…repeat cycles of sequencing to determine the sequence of bases in a given fragment a single base at a time.
12. ALIGN DATA….Align data, compare to a reference, and identify sequence differences.

Question 15

454/FLX Sequencing

Answer 15

diagram on slide 24 and 25

Question 16

Ion Torrent Sequencing

Answer 16

ion semiconductor sequencing is a method of DNA sequencing based on the detection of hydrogen ions that are released during the polymerization of DNA.

This is a method of “sequencing by synthesis”, during which a complementary strand is built based on the sequence of a template strand

Question 17

Characteristics of Ion Torrent = 9

Answer 17

1 * Post-light
2 – No optics
3 – No fluoresence
4 – No light

5 * Rapid
6 – No chemical reactions other than
polymerase
7– 100Mb in an afternoon. 1Gb in an
afternoon

8* Expandable
9– Mostly solid-state, only need new chips

Question 18

Ion Torrent steps:

Answer 18

1. nucleotide incorporates into DNA
   THEN 2. HYDROGEN ION IS RELEASED

then in
3. Micro-machined wells
4. ion sensitive layer
5. proprietary ion sensor

6. sequencing
7. nucleotide IS not a match
8. then NO HYDROGEN ION RELEASED
9. IF 2 BASES ARE INCORPORATED
10. THEN 2 HYDROGEN IONS ARE RELEASED

Question 19

What’s next? NANOPORE SEQUENCING…3

Answer 19

1. Nanopore sequencing = Under development since 1995; DNA is passed through a nanopore.
2. The bases perturb the charge and the sequence it reads without synthesis or a PCR amplification step, chemical labelling, or optical instrumentation.
3. Oxford Nanopore Technologies – GridION & MinION

Question 20

NANOPORE SEQUENCING WHAT IS IT?

Answer 20

DNA can be sequenced by threading it through a microscopic pore in a membrane

Bases are identified by the way they affect ions flowing through the pore from one side of the membrane to the other.

Question 21

NANOPORE SEQUENCING…steps

Answer 21

1. one protein unzips the DNA helix into two strands
2. a second protein creates a pore in the membrane and holds an “adapter” molecule
3. a flow of ions through the pore creates a current. each base blocks the flow to a different degree, altering the current.
4. the adapter molecule keeps bases in place long enough for them to be identified electronically.