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Samenvatting Genome - all lectures

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Course
Advanced Bioinformatics

Institution
Universiteit Utrecht (UU)

Alle hoorcolleges van het onderdeel Genome

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Uploaded on August 18, 2020
Number of pages 12
Written in 2018/2019
Type Summary

bioinformatics
genome
genoomanalyse
advanced bioinformatics

Institution
Universiteit Utrecht (UU)
Education
Biomedische Wetenschappen
Course
Advanced Bioinformatics

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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

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