Lecture 10: Genome sequencing technology

Question 1

What? First generation sequencing:

Answer 1

one sequence at a time

• eg. Sanger sequencing

Question 2

WHAT? Second generation sequencing:

Answer 2

massively parallel sequencing of fragments of different sequences

eg. Illumina sequencing

Question 3

WHAT? Third generation sequencing:

Answer 3

long read massively parallel sequencing

eg. Pacific Biosystems and Nanopore

Question 4

The cost of sequencing has fallen 10,000x in past
decade - MOORE’S LAW

Answer 4

Moore’s Law: the
number of transistors on a chip doubles every two years while the costs are halved. …

SEQUENCE OF TECHNOLOGY DISCOVERY

2005:
- Automation of first generation sequencing,
‘Next generation sequencing’ and Pacific Biosciences

2007:
illumina

2008:
SOLID/454

2010:
Ion torrent

2015:
Nanopore

2022:
Ultima ($100 genome)

Cost per Genome from $100M to $100 (Moore’ Law)
slide 3

Question 5

First generation sequencing: the Sanger method (1970’s on)

• what is required? What occurs?

Answer 5

1. • Based on ACTION of DNA POLYMERASE
   2. • Requires TEMPLATE DNA
   3. DNA PRIMER
   4. POLYMERASE
   5. NUCLEOTIDES
2. • SMALL AMOUNT OF NUCLEOTIDE ANALOG included.
     – The INCORPORATION OF THE ANALOGUE TERMINATES SYNTHESIS

Historical note: the first human genome was sequenced with Sanger at great cost!

Question 6

SANGER SEQUENCING REACTION:

What is it? What is the process? What is needed? - 5

Answer 6

1 * Chain-termination method

2 * Uses ‘dideoxy nucleotides’

3 * WhenADDED IN RIGHT AMOUNT,
the CHAIN IS TERMINATED EVERY TIME THAT BASES APPEARS IN TEMPLATE

4 * Need a reaction for each
base: A, T, C, and G

5. EXAMPLE OF FIRST GEN. SEQUENCING

Question 7

Sanger Sequencing reaction: EXAMPLE

Answer 7

deoxyribose - HO

dideoxyribose - H
- cannot form a bond with the next base

Template
3’ ATCGGTGCATAGCTTGT 5’

Sequence reaction products
5’ TAGCCACGTATCGAACA* 3’
5’ TAGCCACGTATCGAA* 3’
5’ TAGCCACGTATCGA* 3’
5’ TAGCCACGTA* 3’
5’ TAGCCA* 3’
5’ TA* 3’

Question 8

SEQUENCE SEPARATION:
- Gel electrophoresis = 7

Answer 8

1. • TERMINATED chains need to be SEPARATED
2. • Requires ONE-BASE-PAIR RESOLUTION
   3. • See difference between chain of ‘X and X+1 base pairs’
3. • Gel electrophoresis
     5. * Very THIN GEL
     6. * HIGH VOLTAGE
     7.* WORKS WITH RADIOACTIVE OR FLUORESCENT LABELS

figure on slide 6

Question 9

Sanger Sequencing reaction:

‘Capillary electrophoresis’ (1998) - what is it and what does it possess? 4

Answer 9

1. • AUTOMATED SEQUENCERS used very THIN CAPILLARY TUBES
2. • USED FLUORESCENCE, NO RADIOACTIVITY

3 * Run all 4 FLUORESCENTLY TAGGED REACTIONS is SAME CAPILLARY

4 * Can have 384 CAPILLARIES RUNNING at the SAME TIME.

FIGURE ON SLIDE 7:
- Robotic arm and syringe
- load bar
- 96 glass capillaries
- 96-well plate

Question 10

Sanger Sequencing reaction

How to SEQUENCE READING OF FLUORESCENTLY LABELED REACTIONS = 4

Answer 10

1.* Fluorescently labeled
reactions SCANNED BY LASER as a PARTICULAR POINT IS PASSED

2. • COLOUR PICKED UP by
     DETECTOR

3 * OUTPUT sent DIRECTLY to COMPUTER

4. NB. BIG INCREASE IN SEQUENCING EFFICIENCY AND DECREASE IN COST

• figure on slide 8
  1. Dye-labeled dideoxynucleotides are used to generate DNA fragments of different lengths

2. Graph

Question 11

PROS = 2

CONS = 3

FOR SANGER SEQUENCING

Answer 11

• Cons
  1. Requires MANY COPIES OF TEMPLATE (plasmid, or amplified PCR product)

2. Requires a KNOWN SEQUENCE AT THE 5’ or 3’ END (to design a primer against)
3. LIMITED LENGTH for each SEQUENCE RUN (usually max ***‘1kb’ sequenced

PROS
1. CHEAP
2. QUICK

Question 12

Best applications for sanger: 2

Answer 12

1. • ‘SEQUENCE INSERTS’ contained WITHIN PLASMIDS AND AMPLICONS
2. • CHECK for SUCCESSFUL MUTAGENESIS OF KNOWN INSERT.

Question 13

Second generation Sequencing Technologies
(early 2000’s on): 8

Answer 13

1. • Massively PARALLEL SEQUENCING OF DNA FRAGMENTS

2 * Many DIFFERENT STRATEGIES – MOST USE DNA POLYMERASE PRIMER EXTENSION (similar to Sanger)

3. • DIFFERENCES to sanger:
4. TEMPLATE PREPARATION, 5. SEQUENCING CHEMISTRY,
5. DETECTION OF NUCLEOTIDES
6. • ‘Illumina’ is the MAIN PLATFORM USED
7. • Many other platforms have been and gone, and new ones still emerging

Question 14

Second generation Seq Workflow: 5

Answer 14

1 * Next gen sequencing
does NOT REQUIRE DNA TO BE ‘CLONED’
- can you use DNA or RNA–>DNA

2 * DNA is FRAGMENTED

3 * ADAPTERS are ADDED
TO EACH END

4 * PCR is used to make a
LIBRARY
- …SEQUENCE LIBRARY INSERT…READ ALIGNMENT/ASSEMBLY

5 * Massive parallel sequencing of the library

Question 15

Illumina – massively parallel
sequencing in a flow cell… libraries? = 3

Answer 15

1. • DNA libraries are loaded
     onto a ‘flow cell’
2. • Individual DNA molecules are dispersed
3. • These sequences are
     amplified to form clusters
     (each cluster contains
     identical DNA)

Question 16

Illumina - Template Prep
(cluster generation) - process = 7

Answer 16

1. Illumina/Solexa SOLID PHASE AMPLIFICATION
   —- 2. ONE DNA MOLECULE PER CLUSTER
2. Sample preparation DNA (5 microg)
3. TEMPLATE dNTPs and POLYMERASE

5 BRIDGE AMPLIFIFICATION

6. 100-200 MILLION MOLECULAR CLUSTERS
7. CLUSTER GROWTH

Figure on slide 13.

Question 17

THE FULL PROCESS: Illumina sequencing: step by step = 7

Answer 17

1. The sequencing occurs as SINGLE-NUCLEOTIDE ADDITION REACTIONS
   because of a BLOCKING GROUP AT THE 3-OH POSITION OF RIBOSE SUGAR.
2. STEP 1 The nucleotide is added by polymerase,
3. STEP 2 unincorporated nucleotides are washed away
4. STEP 3 the flow cell is imaged to identify each cluster that is reporting a fluorescent signal
5. STEP 4 the fluorescent groups are chemically cleaved
6. STEP 5 the 3-OH is ‘chemically deblocked’
7. This series of steps is repeated for up to ‘150 NUCLEOTIDE ADDITION REACTIONS’

Question 18

Illumina – how the sequencing works with reversible terminators = 4

Answer 18

1. INCORPORATE ALL 4 NUCLEOTIDES, EACH LABEL WITH A DIFFERENT DYE’
2. WASH, 4 COLOUR IMAGING
3. CLEAVE DYE AND TERMINATING GROUPS, WASH
4. ….. REPEAT CYCLES

• LOOK AT FIGURE ON SLIDE 15 FOR PROPER PROCESS

Question 19

Illumina – reversible terminators Detection

Answer 19

Imaging of fluorescent tags over cycles

Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 —>CATCGT

CCCCCC

FIGURE ON SLIDE 16 SHOWS

Question 20

ILLUMINA CAPACITY -

Different machines have different capacity flowcells = 4

Answer 20

1. Different machines have different capacity flowcells.
2. • Simplest models can do ‘25 million per flowcell’

3 * Intermediate models up to ‘250 million per flowcell’

4 * High end ‘20 B reads’

Question 21

Capacity, multiplexing:

Illumina Multiplexing: ‘What is Multiplexing? What is so special about Illumina?’ = 5

Answer 21

1. Multiplexing: Combining different samples in the same flow cell
2. • You probably don’t need 250 million reads for 1 sample!
     …3. Mix 10 samples and get 25 million reads for each
3. • Multiplexing involves adding a unique sequence ‘barcode’ in each library preparation.
     ….5. This marks each read to indicate which sample it belongs to.

Question 22

Single end vs paired end sequencing

WHAT IS SINGLE-ENDED SEQUENCING?

Answer 22

Each species of DNA is represented by one read

• Linear DNA sequenced towards flow cell
  diagram on slide 18

Question 23

Single end vs paired end sequencing

WHAT IS PAIRED-ENDED SEQUENCING?

Answer 23

Permits sequencing of each end of one species of DNA

• Arc shape into flow cell
• cut into seq1 and seq 2 (linear) in flow cell

‘Insert length will be equal to the length of the strand between site A1 and A2’

diagram on slide 19

Question 24

Pros/cons of illumina

PROS = 3

CONS = 3

Answer 24

PRO:
1.Massively parallel sequencing

2. Low error rate
3. Multiplexing libraries

CONS:
1.Expensive to buy and run (partly due to lack of competitors)

2. Limited sequence length
3. Library preparation needed

Question 25

Best applications for illumina = 3

Answer 25

1. • Genomic DNA sequencing of known organisms (eg. Human genomes)

2 * RNA-seq (ie human transcriptome)

3 * Small scale sequencing (miseq) to thousands of human genomes at once (Novaseq)

Question 26

Second generation seq - Ion Torrent

‘pH change detection- Life Technologies Inc’

SET UP =

Answer 26

1. Prepared Library
2. Library loaded onto Ion Spheres
3. Ion Semiconductor Sequencing Chip
   ….4. IONS SPHERES CAPTURED IN WELL ON SEMI-CONDUCTOR CHIP
4. Ion PGM
   …6. Sequencing .. SEQUENCE LIBRARY by pH CHANGE DETECTION

Question 27

Ion Torrent sequencing procedure = 4

Answer 27

1. The 4 bases are flooded into the wells, ONE AT A TIME
2. Polymerase Integrates a Nucleotide

3.If the base is incorporated, a pH change is recorded:

4. Hydrogen and Pyrophosphate are RELEASED

DIAGRAM ON SLIDE 22

Question 28

Third generation seq –
‘Pacific biosciences’

WHAT IS IT? WHAT DOES IT DO? = 6

Answer 28

1. • No need to use PCR to make a LIBRARY (single molecule sequencing)
2. • As with second generation, ADAPTERS ADDED TO EACH END of DNA fragments
3. • Specialising in LONG SEQUENCES (~5k long)
4. • A SINGLE LON G MOLECULE isCOPIED BY A POLYMERASE ANCHORED AT THE BOTTOM OF A WELL.
     ….5. EACH WELL has
     ONE POLYMERASE within it.
5. • After a BASE IS ADDED a FLUOROPHORE IS CLEAVED OFF THE BASE, AND THAT IS DETECTED WITHIN THE WELL.

Question 29

How does ‘Pac Bio’ get low sequence error rate? = 3

Answer 29

1. Because of the unique nature of the ‘Bell end’
   adapters….
2. Each INDIVIDUAL MOLECULE gets sequenced an AVERAGE OF 30 TIMES…
3. SO A CONSENSUS SEQUENCE CAN BE BUILT UP

Question 30

How does Pac Bio get low sequence error rate? DIAGRAM = 5

Answer 30

1. Double stranded DNA from ‘SMRT Bell cDNA Library’
2. PacBio Sequencing
3. Template DNA with DNA Polymerase
4. LOW ACCURACY Raw Reads
5. Ligh Consensus accuracy (>98%)

Question 31

Pros/cons of PacBio

PROS = 2

CONS = 3

Answer 31

PROS
1. No Need to Make a Library (Single Molecule Reads)

2. Generates Long Sequences

CONS
1. SLOW

2. NOT WIDELY AVAILIABLE
3. EXPENSIVE

Question 32

Best applications for PacBio = 3

Answer 32

1 * Sequence DNA or RNA of organism with no known reference genome (‘de novo’ sequencing)

2 * Identify new alternative splice forms of transcripts

3 * Sequence transcribed pseudogenes (with only tiny sequence differences to parent gene)

Question 33

Third generation seq - Oxford Nanopore

WHAT IS IT? WHAT DOES IT DO? = 6

Answer 33

1. Worlds first MOBILE DNA SEQUENCER

2 * Plugs into your LAPTOP and runs off a USB

3 * Thousands of PROTEIN PORES IN THE MEMBRANE,
EACH WITH A HOLE IN THE MIDDLE

4 * SINGLE DNA molecules, WITH ADAPTERS at the
end, PASS THROUGH PORE IN MEMBRANE

5. • EACH NUCLEOTIDE SLIGHTLY DIFFERENT CHARGE

6 * CHARGE DETECTED AS NUCLEOTIDES PASS THROUGH MEMBRANE

Question 34

Pros/cons of Nanopore

PROS = 3

CONS = 2

Answer 34

PROS
1. CHEAP

2. QUICK
3. PORTABLE

CONS
1. HIGH ERROR RATE

2. LOW THROUGHPUT

Question 35

Best applications for Nanopore = 3

Answer 35

1 * Detection of alternative spliced isoforms of transcripts

2 * Detect large structural rearrangements

3 * DNA sequencing ‘in the field

Question 36

Current state of Sequencing technology = 3

Answer 36

1 * Illumina is used by most people, it is quick, accurate and easy. BUT it is expensive – the machines and the reagents.

2 * PacBIO is used for people who need very long reads. - This is basically for genomics of organisms with nothing close to a reference genome

3. • Oxford Nanopore/minION has great promise.
     - It’s portability means it could bring DNA sequencing to the masses