METHODEN IN HET BIOMEDISCH ONDERZOEK 3
3.2.1 DNA SEQUENTIEBEPALING ........................................................................................................................... 3
EVOLUTIE IN METHODEN ................................................................................................................................................. 3
BREDE TOEPASSINGEN ................................................................................................................................................... 3
3.2.1.1 SANGER SEQUENCING = 1ST GENERATION ................................................................................................................ 4
Elektroferogram aflezen ......................................................................................................................................... 4
Verwezenlijkingen .................................................................................................................................................. 5
Gevolgen: .................................................................................................................................................................................. 5
Nieuwe uitdagingen: .................................................................................................................................................................. 5
3..2.1.2 SHORT-READ NEXT-GENERATION SEQUENCING= 2ND GENERATIE SEQUENTIEBEPALING ........................................................ 5
3 pijlers: ................................................................................................................................................................ 5
4 stappen .............................................................................................................................................................. 6
1. Aanmaak DNA library = verzameling van DNA templates .................................................................................................. 6
2. Klonale in vitro amplificatie ............................................................................................................................................. 7
3. Sequencing/ sequentiebepaling ...................................................................................................................................... 8
4. Data analyse ................................................................................................................................................................. 10
Commerciële platformen ..................................................................................................................................... 10
Illumina ................................................................................................................................................................................... 10
Ion torrent ............................................................................................................................................................................... 13
Ultima genomics ..................................................................................................................................................................... 15
Complete genomics ................................................................................................................................................................ 16
Element biosciences ............................................................................................................................................................... 16
Mogelijkheden en beperkingen van 2nd generation sequencing .............................................................................. 17
3.2.1.3 LONG READ DNA SEQUENCING= 3DE GENERATION SEQUENCING ............................................................................. 18
Helicos tSMS ....................................................................................................................................................... 18
PacBio SMRT ....................................................................................................................................................... 19
Nanopore seuencing ............................................................................................................................................ 20
stratos genomics (Roche) ..................................................................................................................................... 20
3.2.1.4 EPIGENETICA SEQUENTIEBEPALING ....................................................................................................................... 21
Methoden............................................................................................................................................................ 22
Voorbeeld ........................................................................................................................................................... 23
Oplossen met .......................................................................................................................................................................... 23
Toepassingen ...................................................................................................................................................... 27
3.2.1.5 SINGLE-CELL SEQUENTIEBEPALING ....................................................................................................................... 28
Methoden voor WGA: ........................................................................................................................................... 28
PCR ......................................................................................................................................................................................... 28
MDA ........................................................................................................................................................................................ 28
MALBAC .................................................................................................................................................................................. 29
Combinatorial indexering ......................................................................................................................................................... 29
3.2.2 DNA GENOTYPEREN ............................................................................................................................... 31
SHORT TANDEM REPEAT GENOTYPERING ........................................................................................................................... 32
TAQMAN GENOTYPERING .............................................................................................................................................. 33
MICROARRAYS ........................................................................................................................................................... 34
HapMap project ................................................................................................................................................... 34
Ilumina= marktleider ............................................................................................................................................ 34
Alfymetrix ............................................................................................................................................................ 35
2 verschillende manieren om SNP uit te lezen .......................................................................................................................... 35
Toepassingen: ..................................................................................................................................................... 36
GWAS ...................................................................................................................................................................................... 36
Imputatie................................................................................................................................................................................. 36
Manhattan plot: ....................................................................................................................................................................... 37
1
, PRS ......................................................................................................................................................................................... 37
CNV ........................................................................................................................................................................................ 37
3.2.3 DNA HYBRIDISEREN ............................................................................................................................................ 38
2
,3.2 DNA
3.2.1 DNA SEQUENTIEBEPALING
EVOLUTIE IN METHODEN
1ste generation 2nd generation 3rd generation
Sanger sequencing Next generation Next-next generation
Cloneren, elektroferese Massieve parallel sequencing van Sequencing van individuele DNA
clonaal in vitro geamplificieerd DNA moleculen (geen amplificatie)
Human genome project Short read NGS Long read NGS
Voor kleine projecten Illumina, ion torrent Nanpore, pacific biosciences
30-100 bp 50 000-1mlj bp
$3 billion $5-10 000 $800 -1000
10 jaar 2 weken uren
BREDE TOEPASSINGEN
De novo genoom sequentie Resequencing Sequentiebepaling als teller
bepaling
Ongekende genomen Individuen tov Aantallen DNA (of RNA) moleculen
referentiegenoom
= Sequentie van DNA in stukjes
bepaald en terug in volgorde
gelegd
Humaan Genoom project, nieuwe Verschillen tussen individuen In welke mate zijn ze aanwezig?
organismen (bv. Sars-CoV-2) (SNPs)
Mutaties (ziekten)
Construct verificatie
Klinische toepassingen: diagnose,
farmacogenetica, NIPT (niet
invasieve prenatale test), …
Gedeeltelijk of volledig (zie RNAseq, ChIPseq,…)
WGS = whole-genome sequencing
WES = whole-exome sequencing= coderende sequentie= 1.5% van genoom
Targeted sequencing= specifieke gewenste regio’s
Transcriptoom: RNA → cDNA
3
, 3.2.1.1 SANGER SEQUENCING = 1ST GENERATION
= nieuwsynthese met dideoxy-keten terminatie
1. Mengsel van:
- Template/matrijs (= clone of PCR amplicon bv van genomisch DNA-fragment)
- Polymerase + primer + mengsel van 4 dNTPs + ddNTPs (elke ddNTP met verschillende fluorescente label)
2. Polymerase zorgt voor synthese van DNA (juiste verhouding dNTPs/ddNTPs)
- Inbouwen dNTP = verdere synthese
- Inbouwen ddNTP = STOP synthese
3. Scheiding strengen op gel of capillair → elektroferogram
Kijken welke lengte overeen komt met welke eindbase
MAAR:
- Beperkt tot ~500 nt, moeite met herhalingen
- Voor betrouwbaarheid: best twee aparte reacties
(FW of RV primer) om beide richtingen af te lezen
ELEKTROFEROGRAM AFLEZEN
➔ Les nog bekijken
Gebruiken voor
mutatiedefectie
1ste 30 bp niet afleesbaar meestal 500-700 bp goed afleesbaar (max 1000)
Phred score: software om aft e lezen met welke kwaliteitsscores per base: Q = -10*log10(P)
→ P = probabiliteit van foutieve base call
→ base calling met Phred-score zitten verwerkt in FASTQ file (bioinformatica)
Om zeker te zijn dat het geen seq-fout
is een fwd en rv reactie uitvoeren
4
3.2.1 DNA SEQUENTIEBEPALING ........................................................................................................................... 3
EVOLUTIE IN METHODEN ................................................................................................................................................. 3
BREDE TOEPASSINGEN ................................................................................................................................................... 3
3.2.1.1 SANGER SEQUENCING = 1ST GENERATION ................................................................................................................ 4
Elektroferogram aflezen ......................................................................................................................................... 4
Verwezenlijkingen .................................................................................................................................................. 5
Gevolgen: .................................................................................................................................................................................. 5
Nieuwe uitdagingen: .................................................................................................................................................................. 5
3..2.1.2 SHORT-READ NEXT-GENERATION SEQUENCING= 2ND GENERATIE SEQUENTIEBEPALING ........................................................ 5
3 pijlers: ................................................................................................................................................................ 5
4 stappen .............................................................................................................................................................. 6
1. Aanmaak DNA library = verzameling van DNA templates .................................................................................................. 6
2. Klonale in vitro amplificatie ............................................................................................................................................. 7
3. Sequencing/ sequentiebepaling ...................................................................................................................................... 8
4. Data analyse ................................................................................................................................................................. 10
Commerciële platformen ..................................................................................................................................... 10
Illumina ................................................................................................................................................................................... 10
Ion torrent ............................................................................................................................................................................... 13
Ultima genomics ..................................................................................................................................................................... 15
Complete genomics ................................................................................................................................................................ 16
Element biosciences ............................................................................................................................................................... 16
Mogelijkheden en beperkingen van 2nd generation sequencing .............................................................................. 17
3.2.1.3 LONG READ DNA SEQUENCING= 3DE GENERATION SEQUENCING ............................................................................. 18
Helicos tSMS ....................................................................................................................................................... 18
PacBio SMRT ....................................................................................................................................................... 19
Nanopore seuencing ............................................................................................................................................ 20
stratos genomics (Roche) ..................................................................................................................................... 20
3.2.1.4 EPIGENETICA SEQUENTIEBEPALING ....................................................................................................................... 21
Methoden............................................................................................................................................................ 22
Voorbeeld ........................................................................................................................................................... 23
Oplossen met .......................................................................................................................................................................... 23
Toepassingen ...................................................................................................................................................... 27
3.2.1.5 SINGLE-CELL SEQUENTIEBEPALING ....................................................................................................................... 28
Methoden voor WGA: ........................................................................................................................................... 28
PCR ......................................................................................................................................................................................... 28
MDA ........................................................................................................................................................................................ 28
MALBAC .................................................................................................................................................................................. 29
Combinatorial indexering ......................................................................................................................................................... 29
3.2.2 DNA GENOTYPEREN ............................................................................................................................... 31
SHORT TANDEM REPEAT GENOTYPERING ........................................................................................................................... 32
TAQMAN GENOTYPERING .............................................................................................................................................. 33
MICROARRAYS ........................................................................................................................................................... 34
HapMap project ................................................................................................................................................... 34
Ilumina= marktleider ............................................................................................................................................ 34
Alfymetrix ............................................................................................................................................................ 35
2 verschillende manieren om SNP uit te lezen .......................................................................................................................... 35
Toepassingen: ..................................................................................................................................................... 36
GWAS ...................................................................................................................................................................................... 36
Imputatie................................................................................................................................................................................. 36
Manhattan plot: ....................................................................................................................................................................... 37
1
, PRS ......................................................................................................................................................................................... 37
CNV ........................................................................................................................................................................................ 37
3.2.3 DNA HYBRIDISEREN ............................................................................................................................................ 38
2
,3.2 DNA
3.2.1 DNA SEQUENTIEBEPALING
EVOLUTIE IN METHODEN
1ste generation 2nd generation 3rd generation
Sanger sequencing Next generation Next-next generation
Cloneren, elektroferese Massieve parallel sequencing van Sequencing van individuele DNA
clonaal in vitro geamplificieerd DNA moleculen (geen amplificatie)
Human genome project Short read NGS Long read NGS
Voor kleine projecten Illumina, ion torrent Nanpore, pacific biosciences
30-100 bp 50 000-1mlj bp
$3 billion $5-10 000 $800 -1000
10 jaar 2 weken uren
BREDE TOEPASSINGEN
De novo genoom sequentie Resequencing Sequentiebepaling als teller
bepaling
Ongekende genomen Individuen tov Aantallen DNA (of RNA) moleculen
referentiegenoom
= Sequentie van DNA in stukjes
bepaald en terug in volgorde
gelegd
Humaan Genoom project, nieuwe Verschillen tussen individuen In welke mate zijn ze aanwezig?
organismen (bv. Sars-CoV-2) (SNPs)
Mutaties (ziekten)
Construct verificatie
Klinische toepassingen: diagnose,
farmacogenetica, NIPT (niet
invasieve prenatale test), …
Gedeeltelijk of volledig (zie RNAseq, ChIPseq,…)
WGS = whole-genome sequencing
WES = whole-exome sequencing= coderende sequentie= 1.5% van genoom
Targeted sequencing= specifieke gewenste regio’s
Transcriptoom: RNA → cDNA
3
, 3.2.1.1 SANGER SEQUENCING = 1ST GENERATION
= nieuwsynthese met dideoxy-keten terminatie
1. Mengsel van:
- Template/matrijs (= clone of PCR amplicon bv van genomisch DNA-fragment)
- Polymerase + primer + mengsel van 4 dNTPs + ddNTPs (elke ddNTP met verschillende fluorescente label)
2. Polymerase zorgt voor synthese van DNA (juiste verhouding dNTPs/ddNTPs)
- Inbouwen dNTP = verdere synthese
- Inbouwen ddNTP = STOP synthese
3. Scheiding strengen op gel of capillair → elektroferogram
Kijken welke lengte overeen komt met welke eindbase
MAAR:
- Beperkt tot ~500 nt, moeite met herhalingen
- Voor betrouwbaarheid: best twee aparte reacties
(FW of RV primer) om beide richtingen af te lezen
ELEKTROFEROGRAM AFLEZEN
➔ Les nog bekijken
Gebruiken voor
mutatiedefectie
1ste 30 bp niet afleesbaar meestal 500-700 bp goed afleesbaar (max 1000)
Phred score: software om aft e lezen met welke kwaliteitsscores per base: Q = -10*log10(P)
→ P = probabiliteit van foutieve base call
→ base calling met Phred-score zitten verwerkt in FASTQ file (bioinformatica)
Om zeker te zijn dat het geen seq-fout
is een fwd en rv reactie uitvoeren
4
