Introduction to bioinformatics
Hoorcollege 5 – 22 januari 2024
DNA sequencing
Genome sequencing: to determine the order of nucleotides of a DNA molecule
Nitrogenous bases: Adenine, Guanine, Thymine, Cytosine
Sugar and phosphate
Photo 51: molecular structure of nucleic acids
1st Generation Sequencing
- Sanger sequencing “chain termination”
First to use primers
Radiolabeled ddNTP (Dideoxynucleotides)
- Applied Biosystems
Fluorescent instead of radioactive ddNTP
Further improved later by capillarity electrophoresis & laser detection
- Whole Genome Sequencing
Break DNA in small fragments randomly
Sanger sequencing of each fragment
Assemble sequenced fragments together (assembly problem)
Difficult and time consuming
2nd Generation Sequencing
Pyrosequencing
First mass parallelization of sequencing reactions
, Substitution of radio/fluorescent labeled nt by luciferase technique
- Illumina sequencing
Flowcell with adapters complementary to DNA libraries
Bridge amplification produces cluster of clonal population of each original sequence
- SOLiD (Sequencing by Oligonucleotide ligation and detection) by Applied Biosystems
Sequencing by ligation, not by synthesis (less depth and length)
Based on polony sequence method
“Post-light” sequencing (measure PH change after addition of nucleotides)
3rd Generation Sequencing
Long read sequencing, no need to fragment DNA molecule.
ONT (Oxford Nanopore) and PacBio (Pacific Biosciences)
Cost per human genome
A big drop in 2007 they developed a next generating sequencing
Don’t have to know the dates etcetera. Need to know difference between 1/2/3 generation
First-generation methods enabled sequencing of clonal DNA populations.
The second-generation massively increased throughput by parallelizing many reactions.
Third-generation methods allow direct sequencing of single DNA molecules.
Sanger, illumine and ONT sequencing
DNA: Deoxy ribonucleic acid
2 Hydroxy group – Sugar - Phosphate
Hydroxy on 3’ is critical
Sanger sequencing
- Phosphodiester bonds between the 3'-hydroxyl group and the 5'-alpha-phosphate
- dNTPs are added to the 3’ end hydroxyl (5’ – 3’ sense)
- lack of hydroxyl group on 3’ end leads to chain termination
ddNTP: no OH on 3’. This stops the sequencing
Hoorcollege 5 – 22 januari 2024
DNA sequencing
Genome sequencing: to determine the order of nucleotides of a DNA molecule
Nitrogenous bases: Adenine, Guanine, Thymine, Cytosine
Sugar and phosphate
Photo 51: molecular structure of nucleic acids
1st Generation Sequencing
- Sanger sequencing “chain termination”
First to use primers
Radiolabeled ddNTP (Dideoxynucleotides)
- Applied Biosystems
Fluorescent instead of radioactive ddNTP
Further improved later by capillarity electrophoresis & laser detection
- Whole Genome Sequencing
Break DNA in small fragments randomly
Sanger sequencing of each fragment
Assemble sequenced fragments together (assembly problem)
Difficult and time consuming
2nd Generation Sequencing
Pyrosequencing
First mass parallelization of sequencing reactions
, Substitution of radio/fluorescent labeled nt by luciferase technique
- Illumina sequencing
Flowcell with adapters complementary to DNA libraries
Bridge amplification produces cluster of clonal population of each original sequence
- SOLiD (Sequencing by Oligonucleotide ligation and detection) by Applied Biosystems
Sequencing by ligation, not by synthesis (less depth and length)
Based on polony sequence method
“Post-light” sequencing (measure PH change after addition of nucleotides)
3rd Generation Sequencing
Long read sequencing, no need to fragment DNA molecule.
ONT (Oxford Nanopore) and PacBio (Pacific Biosciences)
Cost per human genome
A big drop in 2007 they developed a next generating sequencing
Don’t have to know the dates etcetera. Need to know difference between 1/2/3 generation
First-generation methods enabled sequencing of clonal DNA populations.
The second-generation massively increased throughput by parallelizing many reactions.
Third-generation methods allow direct sequencing of single DNA molecules.
Sanger, illumine and ONT sequencing
DNA: Deoxy ribonucleic acid
2 Hydroxy group – Sugar - Phosphate
Hydroxy on 3’ is critical
Sanger sequencing
- Phosphodiester bonds between the 3'-hydroxyl group and the 5'-alpha-phosphate
- dNTPs are added to the 3’ end hydroxyl (5’ – 3’ sense)
- lack of hydroxyl group on 3’ end leads to chain termination
ddNTP: no OH on 3’. This stops the sequencing
