Development of DNA Sequencing Technologies

Question 1

a procedure that reveals stained DNA via ultraviolet transillumination (280–320nm)

Answer 1

Agarose Gel Electrophoresis (AGE)

Question 2

DNA Sequencing Technologies

Answer 2

First Generation Sequencing
o Maxam-Gilbert Sequencing
o Sanger Sequencing

Second Generation Sequencing
o 454 Sequencing
o Illumina Sequencing
o SOLiD Sequencing
o Ion Torrent Sequencing

Third Generation Sequencing
o PacBio RS System
o Nanopore Sequencing Technology

Question 3

First Generation Sequencing Technologies

Answer 3

o Maxam-Gilbert Sequencing
o Sanger Sequencing

Question 4

Second Generation Sequencing Technologies

Answer 4

o 454 Sequencing
o Illumina Sequencing
o SOLiD Sequencing
o Ion Torrent Sequencing

Question 4

Third Generation Sequencing Technologies

Answer 4

o PacBio RS System
o Nanopore Sequencing Technology

Question 5

❑sequencing by chemical degradation requiring chemical modifications of the DNA and further cleavage and electrophoresis

Answer 5

1st Gen: Maxam-Gilbert Sequencing

Question 6

1st Gen: Maxam-Gilbert Sequencing

Steps:
❑Radioactive labeling of the 5’-P ends of dsDNA with 32P-dATP
❑Denature with DMSO at 90°C then electrophoresis
❑Modifications of the nitrogenous bases
❑Chemical cleavage of the ssDNA at the 5’-P side
❑Electrophoresis and autoradiography
❑Infer DNA sequence

Question 7

Disadvantages of 1st Gen: Maxam-Gilbert Sequencing:

Answer 7

❑Uses hazardous chemicals
❑Technically challenging
❑Difficult to scale-up

Question 8

❑a nucleic-acid sequencing approach by enzymatic synthesis, aka chain termination method

Answer 8

1st Gen: Sanger Sequencing

Question 9

1st Gen: Sanger Sequencing

Steps:
❑Denature the dsDNA
❑Anneal with primers
❑Extend with dNTPs and ddNTPs
❑Segregation via gel or capillary electrophoresis
❑Interpret virtual electropherogram or electrofluorogram

Question 10

Disadvantages of 1st Gen: Sanger Sequencing

Answer 10

❑Expensive (equipment, reagents, workforce)
❑Time consuming
❑Not suitable for genomic sequencing

Question 11

❑emulsion-PCR technology generating the
longest reads (from 200 to 1000 b)

Answer 11

2nd Gen: Roch 454 Pyrosequencing

Question 12

2nd Gen: Roch 454 Pyrosequencing

Steps:
❑Library construction = nebulization, adapter ligation, biotin-tagged DNA attaches to streptavidin in beads
❑Emulsion PCR
❑DNA-enriched beads to picotiter wells + smaller beads containing enzymes (ATP sulfurylase, luciferase, and apyrase) ❑Single nucleotide is flown, signal detection (base calling), wash/degrade remaining bases
❑Sequencing = Image/signal processing

Question 13

Disadvantages of 2nd Gen: Roch 454 Pyrosequencing

Answer 13

❑Homopolymeric regions → InDel errors
❑Challenging sample prep

Question 14

❑reversible-terminator sequencers generating shorter reads (35-150b) but larger genome coverages

Answer 14

2nd Gen: Illumina Sequencing

Question 15

2nd Gen: Illumina Sequencing
Steps:
❑Library construction = fragmentation, ligation of “Y” adapters, immobilization
❑Cluster generation = PCR or bridge amplification
❑Sequencing = reversible-terminator sequencing using fluorescent nucleotides
❑Data Analysis = assemble the reads into contigs

Question 16

Disadvantages of 2nd Gen: Illumina Sequencing

Answer 16

❑Substitution errors
❑Signal decay (lower accuracy at ends)

Question 17

❑Sequencing by Oligonucleotide Ligation and Detection (SOLiD) can also generate a high coverage, yet with short reds (25–75 bases)

Answer 17

2nd Gen: SOLiD Sequencing

Question 18

2nd Gen: SOLiD Sequencing
Steps:
❑Library construction → emPCR → DNA immobilized in glass surface
❑Sequencing = five universal sequencing primers (with 2 interrogating bases) → ligate → fluorescent probes = repeated in n-1, n-2, n-3, & n-4
❑Data Analysis = assemble the reads

Question 19

Disadvantages of 2nd Gen: SOLiD Sequencing

Answer 19

❑Highly complex
❑Challenging sample prep

Question 20

❑based on the detection of the protons generated on the polymerization reactions of nucleic acids

Answer 20

2nd Gen: Ion Torrent Sequencing

Question 21

2nd Gen: Ion Torrent Sequencing
Steps:
❑Library construction → emPCR → DNA immobilized in picowells
❑Sequencing = one-by-one dNTP-flush extension detecting the proton released
❑Data Analysis = assemble the reads

Question 22

Disadvantages of 2nd Gen: Ion Torrent Sequencing

Answer 22

❑Still relies on clonally amplified template
❑Errors in homopolymeric regions

Question 23

❑ Single-molecule real-time (SMRT) sequencing using special loop adapters and zero mode waveguide design (ZMW)

Answer 23

3rd Gen: PacBio SMRT Sequencing

Question 24

3rd Gen: PacBio SMRT Sequencing
Steps:
❑Library construction = follows strand displacement amplification (rolling circle)
❑Sequencing = fluorescently labeled base (P) → light emission
❑Data Analysis = assemble the reads

Question 25

Disadvantages of 3rd Gen: PacBio SMRT Sequencing

Answer 25

❑Very high error rates
❑Requires high molecular weight template
❑Costly

Question 26

❑based on the detection of the nitrogenous bases as they move through the pores embedded in a lipid bilayer on microwells

Answer 26

3rd Gen: Nanopore Sequencing