GENOME – Lecture 1
Next-generation sequencing technology,
sample preparation and applications
Lecture goals
General concepts of NGS
Common NGS chemistries and platforms
Third generation sequencing
How to prepare your sample (library preparation)
Different forms of library preparation
Diversity of applications of NGS
Concept of variant calling and variant frequency
Difference between exome and genome sequencing
Coverage depth analysis and paired-end mapping
ChIP, RNAseq, chromosome conformation and single-cell sequencing concepts
Sequencing technologies: available platforms and how they work
Sample preparation for DNA sequencing
1. DNA fragmentation
2. DNA end-repair
3. Adapter ligation
4. Amplification
Clonal amplification of library molecules
A. Amplification in an emulsion (PCR in one bubble/bead)
B. Local amplification on a carrier (chip/slide) --> cluster of molecules (all the same)
Ion Torrent sequencer
Uses emulsion PCR products
Measures release of hydrogen (when polymerase attaches new base)
Per moment een ander type base toevoegen --> kijk wanneer je signaal meet (verandering in
pH)
Niet zo goed voor meerdere zelfde nucleotides achter elkaar (signaal sterker maar je weet niet
hoeveel sterker)
Illumina cluster formation
Fragments of target DNA, ligated with adaptors and hybridization to carrier. Bridging --> amplification
into clusters, cleave off reverse strands, block ends.
--> clusters with many copies of one library molecule (want 1 molecuul geeft niet genoeg signaal)
Sequencing By Synthesis: incorporation, detection, deblock/fluor removal
(wel geschikt voor meerdere zelfde bases achter elkaar)
Two Channel SBS: twee kleuren voor vier bases (geen kleur is één base
BGI (third generation): DNA nanoballs wherein same sequence is sequenced many times
, Third generation sequencing (single molecule
sequencing)
Longer reads
SMRT (single molecule real time) DNA sequencing
Small holes with polymerases in it. DNA molecule + labeled nucleotides --> zien welk nucleotide
wordt ingebouwd
Nanopore sequencing
DNA through pore in membrane, passing --> change in membrane potential
Next-generation sequencing technology,
sample preparation and applications
Lecture goals
General concepts of NGS
Common NGS chemistries and platforms
Third generation sequencing
How to prepare your sample (library preparation)
Different forms of library preparation
Diversity of applications of NGS
Concept of variant calling and variant frequency
Difference between exome and genome sequencing
Coverage depth analysis and paired-end mapping
ChIP, RNAseq, chromosome conformation and single-cell sequencing concepts
Sequencing technologies: available platforms and how they work
Sample preparation for DNA sequencing
1. DNA fragmentation
2. DNA end-repair
3. Adapter ligation
4. Amplification
Clonal amplification of library molecules
A. Amplification in an emulsion (PCR in one bubble/bead)
B. Local amplification on a carrier (chip/slide) --> cluster of molecules (all the same)
Ion Torrent sequencer
Uses emulsion PCR products
Measures release of hydrogen (when polymerase attaches new base)
Per moment een ander type base toevoegen --> kijk wanneer je signaal meet (verandering in
pH)
Niet zo goed voor meerdere zelfde nucleotides achter elkaar (signaal sterker maar je weet niet
hoeveel sterker)
Illumina cluster formation
Fragments of target DNA, ligated with adaptors and hybridization to carrier. Bridging --> amplification
into clusters, cleave off reverse strands, block ends.
--> clusters with many copies of one library molecule (want 1 molecuul geeft niet genoeg signaal)
Sequencing By Synthesis: incorporation, detection, deblock/fluor removal
(wel geschikt voor meerdere zelfde bases achter elkaar)
Two Channel SBS: twee kleuren voor vier bases (geen kleur is één base
BGI (third generation): DNA nanoballs wherein same sequence is sequenced many times
, Third generation sequencing (single molecule
sequencing)
Longer reads
SMRT (single molecule real time) DNA sequencing
Small holes with polymerases in it. DNA molecule + labeled nucleotides --> zien welk nucleotide
wordt ingebouwd
Nanopore sequencing
DNA through pore in membrane, passing --> change in membrane potential
