Functional Genomics
NWI-BB064B
6 EC
Year 3
Quarter 3
Biology – Bachelor
,L1 – Sequencing & Genomics
The human genome sequencing project (HGP) aimed to sequence all of the around 3 billion bases in
the human genome.
The workhorse of the HGP was the Sanger Sequencing method
Sanger sequencing:
1. DNA chains elongate at -OH group on ribose
2. DNA ‘chain terminator’ is a ddNTP with a fluorescent group attached to it
3. Mix of 4 ddNTPs with different fluorophores allows automated Sanger sequencing
4. Find variations/mutations
Sanger sequencing is still the diagnostic ‘gold standard’
Although powerful, the Sanger sequencing method is limited in its throughput (numer of
samples/DNA molecules)
Next generation sequencing methods have been developed in the last 2 decades.
‘DNA Sequencing by synthesis’ → Illumina sequencing
For sequencing of random (or selected) fragments (200-300 bp) of dsDNA
1. dsDNA is fragmented (e.g., by sonication or restriction digestion)
2. ‘Adapter sequences’ (primers) are ligated to the ends of the DNA
3. These adapters are used to capture the DNA (single stranded) on a glass surface (by their
complementarity to small DNA oligonucleotides on the glass (flow cell))
4. These attached molecules are locally amplified in a ‘solid phase PCR’ to form ‘clusters’
5. Like in Sanger sequencing, this procedure requires a primer, DNA polymerase and
fluorescent chain terminators to incorporate the complementary base. A key difference is
that these chain terminators are chemically reversible, enabling cycling of the reaction
6. After each cycle, the glass surface is imaged (4 colors) (glass surface imaged from the top)
7. Reverse chain terminator (which cleaves of the fluorophore)
8. Initiate next cycle, etc. This process is repeated a number of times
, 9. All the images are analysed, because we know which base has which color, we also know the
sequence of each ‘cluster’ (clusters are at fixed positions). A full flow cell (glass surface) can
sequence >400 million clusters (DNA fragments) in parallel
These are short read sequences that are aligned to the reference genome
- But how do we now know which part of the genome that piece of DNA came from?
- For this we make use of the results of the Human Genome Project → the reference genome
- Mapping reference genome to the sequencing reads
Question: How many different sequences of 4 nucleotides are there?
→ 4 nucleotides, 4 positions = 44 = 256 molecules
Thus, any stretch of 4 nucleotides repeats itself (on average) every 256 bases
Question: How many cycles would you need to perform minimally to make sure that your sequence
read can be placed uniquely on the human genome (around 3 billion bases)?
→ Statistics tell you minimally 20 cycles. In the lab we routinely do 43 cycles
20 cycles (complexity: around 1*1012)
If you want to know the size of the reads → paired end sequencing
Most people in the field use ‘sequencing by synthesis’ Illumina for the large-scale sequencing
But there are other techniques as well
Ion semiconductor sequencing (Ion Torrent):
- Ion semiconductor sequencing is based on the fact that every base added, an H+ is released
- In contrast to sequencing by synthesis methods, each cycle of sequencing contains only one
nucleotide
- When more than one nucleotide is attached the signal increased proportionally
- Advantage: very fast → 4-hour run
- Disadvantage: limited number of reads per semiconductor chip (1 read/well, every read
needs its own well)
, Single Molecule Real Time sequencing (PacBio):
- SMRT sequencing uses mixed dNTPs attached to fluorophores
- In contrast to sequencing by synthesis and Semiconductor methods, SMRT sequencing used a
polymerase fixed to the bottom of the well which pulls the template DNA through
- The retention time of the fluorescent dNTP at the bottom of the well serves as the read-out
- Advantages: very long reads (1-20 kb), fast, can detect modified DNA modifications
- Disadvantages: expensive, moderate throughput
Nanopore sequencing (Oxford Nanopore):
- 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 to the other
- Base sequence is determined using changes in current over the membrane
- Recently: a full human genome was sequenced using a MinIon device
- The Oxford Nanopore MinION is a hand-held genome sequencer that directly connects with
your laptop (via USB). For instance, for field studies/diagnostics at remote locations
- Advantages: potentially long read length, ‘plug and play’
- Disadvantage: moderate accuracy (thus far), starting to become operational in recent years
NWI-BB064B
6 EC
Year 3
Quarter 3
Biology – Bachelor
,L1 – Sequencing & Genomics
The human genome sequencing project (HGP) aimed to sequence all of the around 3 billion bases in
the human genome.
The workhorse of the HGP was the Sanger Sequencing method
Sanger sequencing:
1. DNA chains elongate at -OH group on ribose
2. DNA ‘chain terminator’ is a ddNTP with a fluorescent group attached to it
3. Mix of 4 ddNTPs with different fluorophores allows automated Sanger sequencing
4. Find variations/mutations
Sanger sequencing is still the diagnostic ‘gold standard’
Although powerful, the Sanger sequencing method is limited in its throughput (numer of
samples/DNA molecules)
Next generation sequencing methods have been developed in the last 2 decades.
‘DNA Sequencing by synthesis’ → Illumina sequencing
For sequencing of random (or selected) fragments (200-300 bp) of dsDNA
1. dsDNA is fragmented (e.g., by sonication or restriction digestion)
2. ‘Adapter sequences’ (primers) are ligated to the ends of the DNA
3. These adapters are used to capture the DNA (single stranded) on a glass surface (by their
complementarity to small DNA oligonucleotides on the glass (flow cell))
4. These attached molecules are locally amplified in a ‘solid phase PCR’ to form ‘clusters’
5. Like in Sanger sequencing, this procedure requires a primer, DNA polymerase and
fluorescent chain terminators to incorporate the complementary base. A key difference is
that these chain terminators are chemically reversible, enabling cycling of the reaction
6. After each cycle, the glass surface is imaged (4 colors) (glass surface imaged from the top)
7. Reverse chain terminator (which cleaves of the fluorophore)
8. Initiate next cycle, etc. This process is repeated a number of times
, 9. All the images are analysed, because we know which base has which color, we also know the
sequence of each ‘cluster’ (clusters are at fixed positions). A full flow cell (glass surface) can
sequence >400 million clusters (DNA fragments) in parallel
These are short read sequences that are aligned to the reference genome
- But how do we now know which part of the genome that piece of DNA came from?
- For this we make use of the results of the Human Genome Project → the reference genome
- Mapping reference genome to the sequencing reads
Question: How many different sequences of 4 nucleotides are there?
→ 4 nucleotides, 4 positions = 44 = 256 molecules
Thus, any stretch of 4 nucleotides repeats itself (on average) every 256 bases
Question: How many cycles would you need to perform minimally to make sure that your sequence
read can be placed uniquely on the human genome (around 3 billion bases)?
→ Statistics tell you minimally 20 cycles. In the lab we routinely do 43 cycles
20 cycles (complexity: around 1*1012)
If you want to know the size of the reads → paired end sequencing
Most people in the field use ‘sequencing by synthesis’ Illumina for the large-scale sequencing
But there are other techniques as well
Ion semiconductor sequencing (Ion Torrent):
- Ion semiconductor sequencing is based on the fact that every base added, an H+ is released
- In contrast to sequencing by synthesis methods, each cycle of sequencing contains only one
nucleotide
- When more than one nucleotide is attached the signal increased proportionally
- Advantage: very fast → 4-hour run
- Disadvantage: limited number of reads per semiconductor chip (1 read/well, every read
needs its own well)
, Single Molecule Real Time sequencing (PacBio):
- SMRT sequencing uses mixed dNTPs attached to fluorophores
- In contrast to sequencing by synthesis and Semiconductor methods, SMRT sequencing used a
polymerase fixed to the bottom of the well which pulls the template DNA through
- The retention time of the fluorescent dNTP at the bottom of the well serves as the read-out
- Advantages: very long reads (1-20 kb), fast, can detect modified DNA modifications
- Disadvantages: expensive, moderate throughput
Nanopore sequencing (Oxford Nanopore):
- 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 to the other
- Base sequence is determined using changes in current over the membrane
- Recently: a full human genome was sequenced using a MinIon device
- The Oxford Nanopore MinION is a hand-held genome sequencer that directly connects with
your laptop (via USB). For instance, for field studies/diagnostics at remote locations
- Advantages: potentially long read length, ‘plug and play’
- Disadvantage: moderate accuracy (thus far), starting to become operational in recent years
