NEXT GENERATION SEQUENCING
DIFFERENT TYPES OF DNA SEQUENCING
- Maxam-Gilbert
- Chemical degradation of DNA
- Infrequently used due to toxic compounds and difficulty
- Sequencing by synthesis (SBS)
- Develop by Sanger in 1977
- Uses DNA polymerase in primer extension reaction steps
- Very common approach
- Includes Pyrosequencing or 404, Ion Torrent, Illumina, Pacific
Biosciences and DNBSeq
- Ligation-based
- Sequencing using short probes that hybridise to the template
- SOLiD 2007
- Nanopore based sequencing
- Nanopore sequencing
- Shotgun sequencing - overlapping DNA fragments were cloned and
sequenced separately then assembled into a contig
SANGER SEQUENCING
- Uses non-reversible termination
- If ddATP is used in the reaction, anywhere there's a T in the template strand,
occasionally a ddA will be added to the growing strand
,SECOND GENERATION SEQUENCING
- Infer sequences by measuring pyrophosphate production as each nucleotide
is added
- Pyrosequencing benefits
- Can be performed using natural nucleotides instead of heavily modified
dNTPs
- Can be observed in real time instead of requiring lengthy
electrophoreses
- Major difficulty is finding out how many of the same nucleotide there are in a
row at a given position - homopolymers
THIRD GENERATION SEQUENCING
- Characteristics
- Single molecule sequencing
- Real-time sequencing
- Simple divergence from previous technologies
- Sequencing occurs at the rate of polymerase therefore it produces kinetic
data and can detect modified bases
- Produce very long reads which can be used for de novo genome assembly
NGS TECHNOLOGIES
- NGS technologies differ in their sequencing approaches, technologies
associated with them and the characteristics of the type of data produced
- Is high throughput sequencing
- One main characteristic for all of them is their ability to perform massive
parallel sequencing of DNA molecules
- Can generate large amounts of data
- Also referred to as high throughput sequencing technologies
- Are divided into second generation NGS and third generation NGS
- 5 most popular technologies:
- Ion Torrent
- Illumina
- DNBSeq
- PacBio
- Nanopore
- Second generation
- Illumina
- Ion Torrent
- DNBSeq
- Third generation
- PacBio
- Nanopore
, CATEGORIZING OF THESE TECHNOLOGIES
- By sequencing types:
- Sequencing by synthesis (SBS)
- Involves the use of DNA polymerase
- DNA is synthesised by monitoring changes in signals produced
as the DNA is being synthesised
- There are two types - Cyclic Reversible Termination (CRT) and
Single-Nucleotide Addition (SNA)
- DNBSeq uses CRT and all others except Nanopore use SNA
- Sequencing by ligation (SBL)
- Involves hybridization and ligation of labelled probe and anchor
sequences to a DNA strand
- Two SBL platforms are SOLiD and AllSeq - not widely used
- Sequencing using nanopore pore
- DNA sequenced by the use of nanopores
- DNA strands passed through nanopores which creates
disruptions in the current signal across the pores
- Different base compositions create different current profiles and
these differences are translated (called base calling) into actual
DNA sequence
- By the use of pre-amplification steps:
- All NGS technologies starts with a single molecules of DNA
- Working with a single molecule results in very low signals - difficult to
detect by detectors
- Many NGS technologies use a clonal amplification step (signal
amplification) to create a population of identical molecules before they
are sequenced
- Is one of the main differences between second and third generation
sequencing
- Third generation uses a single molecule and does not involve a clonal
amplification step
- By signal detection type:
- There are three types of signals monitored in different NGS
technologies
- Each of these technologies make us and monitor the changes in one of
these signals
- Signals include changes in pH, changes in colour profiles and changes
in electrical current
- By read length generated:
- All second generation produce read lengths shorter (shorter than
600bp) than traditional Sanger sequencing
- These technologies are referred to as short read sequencing
technologies
DIFFERENT TYPES OF DNA SEQUENCING
- Maxam-Gilbert
- Chemical degradation of DNA
- Infrequently used due to toxic compounds and difficulty
- Sequencing by synthesis (SBS)
- Develop by Sanger in 1977
- Uses DNA polymerase in primer extension reaction steps
- Very common approach
- Includes Pyrosequencing or 404, Ion Torrent, Illumina, Pacific
Biosciences and DNBSeq
- Ligation-based
- Sequencing using short probes that hybridise to the template
- SOLiD 2007
- Nanopore based sequencing
- Nanopore sequencing
- Shotgun sequencing - overlapping DNA fragments were cloned and
sequenced separately then assembled into a contig
SANGER SEQUENCING
- Uses non-reversible termination
- If ddATP is used in the reaction, anywhere there's a T in the template strand,
occasionally a ddA will be added to the growing strand
,SECOND GENERATION SEQUENCING
- Infer sequences by measuring pyrophosphate production as each nucleotide
is added
- Pyrosequencing benefits
- Can be performed using natural nucleotides instead of heavily modified
dNTPs
- Can be observed in real time instead of requiring lengthy
electrophoreses
- Major difficulty is finding out how many of the same nucleotide there are in a
row at a given position - homopolymers
THIRD GENERATION SEQUENCING
- Characteristics
- Single molecule sequencing
- Real-time sequencing
- Simple divergence from previous technologies
- Sequencing occurs at the rate of polymerase therefore it produces kinetic
data and can detect modified bases
- Produce very long reads which can be used for de novo genome assembly
NGS TECHNOLOGIES
- NGS technologies differ in their sequencing approaches, technologies
associated with them and the characteristics of the type of data produced
- Is high throughput sequencing
- One main characteristic for all of them is their ability to perform massive
parallel sequencing of DNA molecules
- Can generate large amounts of data
- Also referred to as high throughput sequencing technologies
- Are divided into second generation NGS and third generation NGS
- 5 most popular technologies:
- Ion Torrent
- Illumina
- DNBSeq
- PacBio
- Nanopore
- Second generation
- Illumina
- Ion Torrent
- DNBSeq
- Third generation
- PacBio
- Nanopore
, CATEGORIZING OF THESE TECHNOLOGIES
- By sequencing types:
- Sequencing by synthesis (SBS)
- Involves the use of DNA polymerase
- DNA is synthesised by monitoring changes in signals produced
as the DNA is being synthesised
- There are two types - Cyclic Reversible Termination (CRT) and
Single-Nucleotide Addition (SNA)
- DNBSeq uses CRT and all others except Nanopore use SNA
- Sequencing by ligation (SBL)
- Involves hybridization and ligation of labelled probe and anchor
sequences to a DNA strand
- Two SBL platforms are SOLiD and AllSeq - not widely used
- Sequencing using nanopore pore
- DNA sequenced by the use of nanopores
- DNA strands passed through nanopores which creates
disruptions in the current signal across the pores
- Different base compositions create different current profiles and
these differences are translated (called base calling) into actual
DNA sequence
- By the use of pre-amplification steps:
- All NGS technologies starts with a single molecules of DNA
- Working with a single molecule results in very low signals - difficult to
detect by detectors
- Many NGS technologies use a clonal amplification step (signal
amplification) to create a population of identical molecules before they
are sequenced
- Is one of the main differences between second and third generation
sequencing
- Third generation uses a single molecule and does not involve a clonal
amplification step
- By signal detection type:
- There are three types of signals monitored in different NGS
technologies
- Each of these technologies make us and monitor the changes in one of
these signals
- Signals include changes in pH, changes in colour profiles and changes
in electrical current
- By read length generated:
- All second generation produce read lengths shorter (shorter than
600bp) than traditional Sanger sequencing
- These technologies are referred to as short read sequencing
technologies
