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Samenvatting Hfdst 2 - Nieuwe generatie van sequencing technologieën
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Omvat het hoodstuk over sequencing technologieën in het vak moleculaire genetica deel I
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HOOFDSTUK 2 – NIEUWE GENERATIE SEQUENCING TECHNOLOGIEËNINLEIDING
Evolutie DNA sequencing
- Duur
Vroeger - Tijdrovend
- Eerder beperkt in haar toepassingen
Introductie van # nieuwe evolutionair nieuwe technieken
1. MPS of massieve parallele DNA sequencing
2. NGS of next-generation sequencing
Tijd Vanaf
sanger seq. = sterk verschillend
2005
Eig
-Gigantische toename in sequencing data per run
-Gepaarde dramatische reductie in sequencing kost per
megabase
Snel & relatief goedkoop volledig genoom sequencen
Vandaag
technieken = Dideoxy DNA sequencing of Sanger sequencing
Tot kort enige DNA sequencing methode
Fred Sanger, 1977 nobelprijs
<
- Vertrek amplificatie van individuele DNA sequenties
Opgezuiverd via PCR (polymerase chain reaction)
- Voor elk
geamplificeerd
DNA fragment (=
amplicon)
Werking ‘nested sets’ van
gelabelde DNA
Dideoxy
kopijen
aangemaakt
DNA seq.
- Gescheiden
volgens grootte
adhv. Gel elektroforese
Veel voordelen nog steeds veel gebruikt
- Zeer hoge accuraatheid
Eig. - Genereerd seq. van 100-800 bp
Voordelen NGS : short-read sequencing (75-
300bp)
- Testen van specifieke DNA sequenties
- Testen van aanwezigheid van mutaties in bepaald gen
Toepassing - Bevestiging van een vermoedelijke mutatie
Massieve Of MPS
, = gebasserd op totaal verschillende technologieën
!MPS of NGS drastische toename in
- Sequencing capaciteit
parallel
- Snelheid
Gepaard met daling kost (=running cost)
DNAseq.
Ipv. Sequencing van opgezuiverde DNA fragmenten miljoen DNA
fragmeten aanwezig in complexe DNA sample
werking
Simultaan gesequenced ZONDER nood aan gel
elektroforese
Schaal van sequencing evolueren van exonen & genen
- Genoom sequencing = volledig genoom
- Targeted DNA sequencing v. vooraf gedefinieerde subset van genoom
zoals exoom of alle genen in = ziekte pahtway gelegen
GEVOLG
! mens telt 180,000 exomen +/- 1% van humaan genoom
- Transcriptoom sequencing
nadat total RNA geconverteerd naar cDNA
- Methyl-seq. = DNA methylatie sites te identificeren doorheen het genoom
Sanger dideoxy sequencing MPS
Gel
Integraal deel van proces /
elektroforese
Read lengte Up to 800 nucleotide 35-14,000 nucleotide
Number of DNA 10,000den – biljoenen of DNA fragmenten
1
templates sequenceren tegelijktijdig
Advensed machines laten 96 sequencing
Sequencing
reacties toe per elektroforese run 80Mb-1000Gb/run
throughput
Rond 80Mb van DNA /run
Sequencing
Heel laag Significant hoog individuele fragmeten
error rates
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