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Molecular Biology > Techniques in Sequencing > Flashcards

Techniques in Sequencing Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

Also called dideoxynucleotide procedure or Sanger sequencing

A

Chain-Termination DNA Sequencing Technique

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2
Q

Lacks a hydroxyl group on the 3’ ribose carbon!

A

Dideoxynucleotide (ddNTP)

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3
Q

When incorporated into the end of the growing DNA chain, ddNTPs terminate DNA synthesis by _____

A

Disabling the formation of phosphodiester bond

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4
Q

Chain-Termination DNA Sequencing Technique requires 4 separate tubes containing one of each ddNTP and these common reaction components:

A
  • Single-stranded DNA template (can be obtained using M13 vectors or phagemids)
  • Primer
  • Klenow Polymerase (or “Sequenase” – a modified polymerase with higher processivity)
  • dNTPs (dATP, dCTP, dGTP, and dTTP)
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5
Q

A sequencing ladder is read from ________

A

the bottom of the gel to the top. The smallest (fastest-migrating) fragment represents the first nucleotide attached to the primer by the polymerase

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6
Q

BASIC STEPS IN SANGER OR DIDEOXYNUCLEOTIDE SEQUENCING:

A
  1. Extension and termination reaction of the DNA fragment
  2. Loading of reaction products into a polyacrylamide gel (via PAGE) or into a capillary gel system
  3. Manual reading of sequence from the gel OR fluorescence detection of sequence (automated
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7
Q
  • PCR with fluorescent, chain-terminating ddNTPs
  • Size separation by capillargy gel electrophoresis
  • Laser excitation & detection by sequencing machine
A

Automated Sanger Sequencing

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8
Q

designed to sequence large numbers of templates carrying millions of bases simultaneously, in a run that takes a few hours

A

Next-Generation Sequencing (NGS) or High-Throughput Sequencing

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9
Q

• This is based on the principle of pyrophosphate (PPi) produced during the addition of complementary nucleotide in the growing DNA strand.
• It relies on the generation of light (luminescence) when nucleotides are added to a growing strand of DNA
• With this system, there are no gels, fluorescent dyes, and ddNTPs needed.
* most useful for short- to moderate sequence analysis

A

First High-Throughput Sequencing: Pyrosequencing

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10
Q

The PPi is converted to ATP by sulfurylase in the presence of ______

A

Adenosine-5’-phosphosulfate (APS)

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11
Q

The ATP is used to generate a luminescent signal by _____

A

Luciferase-catalyzed conversion of luciferin to oxyluciferin

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12
Q

Pyrosequencing requires the following:

A
  • DNA template
  • Primer
  • DNA Polymerase
  • dNTPs (dATPαS, dCTP, dGTP, and dTTP)
  • Adenosine 5’ Phosphosulfate (APS) and Luciferin
  • ATP Sulfurylase and Luciferase
  • Apyrase
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13
Q

Thus, if the added dNTP is incorporated into the DNAstrand, __________

A

Light will be emitted

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14
Q

Otherwise, there will be no light emitted and the added dNTP will be _______

A

degraded by the enzyme, apyrase, before the addition of a new dNTP

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15
Q

the genomic DNA is first fragmented into small pieces and a genomic library is constructed, typically using plasmids and bacteria

A

Shotgun Sequencing

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16
Q

the nucleotide sequence of tens of thousands of individual clones is determined the full genome sequence is then reconstructed by stitching together in silico the nucleotide sequence of each clone, using the overlaps between clones as a guide

A

Shotgun Sequencing

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17
Q

The shotgun method works well for

A

small genomes (such as those of viruses and bacteria) that lack repetitive DNA

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18
Q

Provides an accurate sequence of a large genome that contains repetitive DNA BUT takes additional time and effort

A

Clone Contig Approach

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19
Q

the genome is broken down into several segments of up to 1.5 Mb, and each with a known position on the genome map before sequencing is carried out.

A

Clone Contig Approach

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20
Q
  • Cloning is done using a bacterial artificial chromosome (BAC) or yeast artificial chromosome (YAC)
  • Overlapping clones (those sharing the same nucleotide sequence) are assembled in silico (using a computer and software programs)
A

Clone Contig Approach

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21
Q

High Throughput NGS Strategies

A

a) Massively Parallel Pyrosequencing: Roche/454 Life Sciences
b) Sequencing by Synthesis: Illumina/Solexa
c) Sequencing by Ligation: Applied Biosystems/SOLiD
d) Sequencing by Semiconductor Technology: Compact Personal Genome Machine (PGM) Sequencer

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22
Q

generates hundreds of megabases to gigabases of nucleotide sequence output in a single instrument run, depending on the platform

A

Next-Generation DNA Sequencing (NGS)

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23
Q

MAIN STEPS IN NGS:

A
  1. Library preparation
  2. Sequencing
  3. Data Analysis
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24
Q

Makes use of the pyrosequencing technique, emulsion PCR, and microfabricated high density picoliter reactors

A

Massively Parallel Pyrosequencing: Roche/454 Life Sciences

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25
Q

Generates approximately 1106 sequence reads, with read lengths of 400 bases yielding up to 500 Mb of sequence

A

Massively Parallel Pyrosequencing: Roche/454 Life Sciences

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26
Q
  • First introduced in 2005 by 454 Life Sciences

* The advantage of the 454 technology is thus the longer read length which facilitates de novo assembly of genomes

A

Massively Parallel Pyrosequencing: Roche/454 Life Sciences

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27
Q
  • Based on the principle of sequencing-by-synthesis chemistry and uses a novel. fluorescently-labeled reversible terminator nucleotides and a special DNA polymerase
  • 1.5 Gb of single-read data per run, and at least 3 Gb of data in a paired end run recording data from more than 50 million reads per flow cell
A

Sequencing by Synthesis (SBS): Illumina/Solexa

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28
Q

More cost-efficient (than Massively Parallel Pyrosequencing: Roche/454 Life Sciences), high throughput, and accuracy (~98%)

A

Sequencing by Synthesis (SBS): Illumina/Solexa

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29
Q
  • Commercialized in 2006 by Illumina

* Unlike 454 sequencing, DNA templates are amplified in the flow cell by “bridge” amplification or solid phase PCR

A

Sequencing by Synthesis (SBS): Illumina/Solexa

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30
Q

to obtain DNA fragments with adapters attached on both ends

A

Library Preparation

31
Q

Illumina Sequencing Workflow

A
  1. Library Preparation
  2. Cluster Generation
  3. Sequencing by Synthesis
  4. Data Analysis
32
Q

occurs in a flow cell – a thick glass slides with channels or lanes

A

Cluster Generation

33
Q

Materials involved in Cluster Generation

A

a) Hybridize fragment and extend
b) Denature Double Stranded DNA
c) Formation of Single Stranded DNA
d) Bridge Amplification
e) Denature Double Stranded Bridge
f) Bridge Amplification
g) Linearization
h) Reverse Strand Cleavage
i) Blocking
j) Read Primer 1 Hybridization

34
Q

Based on ligation of short oligonucleotides into the DNA strand through DNA ligase

Oligonucleotides are eight nucleotides in octamer length with two known at nucleotides in the 3’ query end, any nucleotides in the next three (degenerate) positions, and a sequence that is common to all of the oligonucleotides at the 5 end

A

Sequencing by Ligation: Applied Biosystems/SOLiD

35
Q

• Has trouble with palindromic sequences

A

Sequencing by Ligation: Applied Biosystems/SOLiD

35
Q

• Has trouble with palindromic sequences

A

Sequencing by Ligation: Applied Biosystems/SOLiD

36
Q

SOLID or ______ System developed by Applied Biosystems

A

Supported Oligonucleotide Ligation and Detection

37
Q

SOLiD Sequencing Workflow

A
  1. Sample Preparation
  2. Amplification via emulsion PCR
  3. Ligation Reactions and Imaging
  4. Data Analysis
38
Q

uses compressed nitrogen to force the DNA through a small hole, shearing it into fragments

A

Nebulization

39
Q

ultrasonic waves shears DNA by resonance vibration

A

Sonication

40
Q

Uses restriction enzymes

A

Digestion

41
Q

requires template strand, primers, oligonucleotide attached to a fluorophore, and ligase

A

Ligation Reactions and Imaging

42
Q

Ligation Reactions and Imaging Process

A

a) Primer binds to template strands
b) Probe hybridization and ligation
c) Fluorescence measured
d) Dye end (3) nucleotides cleaved
e) Steps a to d repeated for 6 or more times

43
Q

was launched by Ion Torrent in 2010 which uses a semiconductor sequencing technology

A

Ion Personal Genome Machine (PGM)

44
Q

Does not require fluorescence and camera scanning, resulting in higher speed, lower cost, and smaller instrument size (it enables 200 bp reads in 2 h)

A

Sequencing by Semiconductor Technology: Compact Personal Genome Machine (PGM) Sequencer

45
Q
  • A change in pH or voltage means a nucleotide/s was/were added
  • Usually used in identification of microbial pathogens
A

Compact Personal Genome Machine (PGM) Sequencer or Ion Personal Genome Machine

46
Q

Also called Single molecule sequencing
No PCR required
Signal is captured in real time

A

Third Generation Sequencer

47
Q

Examples of Third Generation Sequencer

A

a) HeliScope
b) Pacific Biosciences
c) Life Technologies Method
d) Nanopore Sequencing

48
Q

Analyzes many millions of single DNA fragments simultaneously, resulting in sequence throughput in the gigabase range

A

The Helicos Single Molecule Sequencing Device, HeliScope

49
Q

Requires only subnanogram amounts of your DNA samples

Very poor quality DNA, including degraded or modified DNA, can be sequenced!

A

The Helicos Single Molecule Sequencing Device, HeliScope

50
Q
  • Can also sequence RNA molecules directly
  • Ideal for quantitative applications such as chromatin immunoprecipitation (Chip), RNA expression, and copy number variation, and situations in which sample quantity is limiting or degraded
A

The Helicos Single Molecule Sequencing Device, HeliScope

51
Q

A strand of DNA about 100 200 base pairs is cut into smaller fragments using restriction enzymes and polyA
tails are added

A

HeliScope

52
Q

The shortened strands are then hybridized to the Helicos flow cell plate, which has billions of polyT chains bound to its surface

A

HeliScope

53
Q

Each hybridized template is sequenced at once. Therefore, billions per run can be read Labeling is performed in ______. Fluorescent labeled bases are added, and a laser in the instrument illuminates the label, taking a read of which strands have taken up that particular labeled base

A

Quads consisting of 4 cycles each, for each of the 4 nucleotide bases

54
Q

The label is then cleaved, and the next cycle begins with a new base. After the flow cell has been treated with each base (4 cycles), __________

A

the quad is complete, and a new one begins again with the initial nucleotide base

55
Q

Uses a sequencing by synthesis system, a SMRTcell consisting of millions of zero mode waveguides (ZMWs), a DNA template, called a SMRTbell which is a closed, single-stranded circular DNA that is created by ligating hairpin adaptors to both ends of a target double stranded DNA molecule

A

Pacific Biosciences

56
Q

Sample preparation and turn over rate is fast, average read length is 1300 bp (longer than any 2 nd NGS, but has lower throughput)

Useful in microbiology research to assemble genomes, and to assess the analysis of structural variation, haplotyping metagenomics, and identification of splicing isoforms

A

Pacific Biosciences

57
Q

Direct RNA sequencing with this system is problematic

A

Pacific Biosciences

58
Q

PacBio SMRT Sequencing Workflow

A
  1. Generate DNA amplicon
  2. Ligate adaptors (formation of SMRT bell)
  3. Sequence
  4. Data Analysis
59
Q

Uses the fluorescence resonance energy transfer (FRET) based single molecule sequencing by synthesis technology and a quantum dot labeled polymerase

A

Life Technologies Method

60
Q

Quantum dots are fluorescent semiconducting nanoparticles which are much brighter and less susceptible to bleaching than fluorescent dyes

A

Life Technologies Method

60
Q

Quantum dots are fluorescent semiconducting nanoparticles which are much brighter and less susceptible to bleaching than fluorescent dyes

A

Life Technologies Method

61
Q

The applications of this approach are in genome assembly, structural variation, haplotyping, and metagenomics

A

Life Technologies Method

62
Q

Readout relies on the size difference between all deoxyribonucleoside monophosphates (dNMPs)

DNA fragments are sequenced in wells containing nanopores as disruption of ionic currents are measured

A

Nanopore Sequencing

63
Q

Produces read lengths 5 kbp with speeds of 1 bp/ns

A

Nanopore Sequencing

64
Q

Detection of bases is fluorescent tag free

It is less sensitive to temperature

A

Nanopore Sequencing

65
Q

Instead of sequencing DNA during polymerization, single DNA strands are sequenced through nanopore by means of DNA strand depolymerization

A

Nanopore Sequencing

66
Q

Strand is passed through nanopore and signal is interpreted into sequence data

A nanopore reader and sensor detect digital signal in real time and arrays them in microchips

A

Nanopore Sequencing

67
Q

NGS, reads are much shorter but has a higher throughput than Sanger; thus, involves huge volume of data.

As an example, a single run of 454 GS FLX, Illumina, and SOLiD instruments are typically 15GB, 1TB, and 15TB, respectively.

A

NGS

68
Q

Intensive conversion of image data (raw data) into an actual base sequence

A

Base Calling

69
Q

Base calling is usually done using a __________

A

platform specific data pipeline software

70
Q

Error rates of NGS platforms are estimated in the form of quality values which are affected by factors such as:

A
  • signal to noise levels,
  • cross talk from nearby beads or clusters,
  • and dephasing
71
Q
  • Short reads have the inability to uniquely align large portions of a read set when the read length becomes too short
  • For longer reads, unique alignment and assembly is reduced due to higher probability of repetitive sequences and shared homologies within closely related gene families and pseudogenes
A

Alignment and Assembly

72
Q

NGS Accuracy and Softwares

A
  • Error rates for individual NGS reads are higher compared to conventional sequencing
  • To assemble, align, and analyze NGS data requires an adequate number of overlapping reads, or coverage.
  • Beyond sequence errors, inadequate coverage can cause failure to detect actual nucleotide variation

Molecular Biology (2 decks)

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