Download presentation
Presentation is loading. Please wait.
1
Next–generation DNA sequencing technologies – theory & practice
2
Outline Next-Generation sequencing (NGS) technologies – overview
NGS targeted re-sequencing – fishing out the regions of interest NGS workflow: data collection and processing – the exome sequencing pipeline
3
PART I: NGS technologies
Next-Generation sequencing (NGS) technologies – overview
4
DNA Sequencing – the next generation
The automated Sanger method is considered as a ‘first- generation’ technology, and newer methods are referred to as next- generation sequencing (NGS).
5
Landmarks in DNA sequencing
1953 Discovery of DNA double helix structure 1977 A Maxam and W Gilbert "DNA seq by chemical degradation" F Sanger"DNA sequencing with chain-terminating inhibitors" 1984 DNA sequence of the Epstein-Barr virus, 170 kb 1987 Applied Biosystems - first automated sequencer 1991 Sequencing of human genome in Venter's lab 1996 P. Nyrén and M Ronaghi - pyrosequencing 2001 A draft sequence of the human genome 2003 human genome completed Life Sciences markets first NGS machine
6
2005
7
DNA Sequencing – the next generation
Random genome sequencing 25 Mb 300k reads 110bp Sanger sequencing Targeted bp
8
DNA Sequencing – the next generation
The newer technologies constitute various strategies that rely on a combination of Library/template preparation Sequencing and imaging
9
DNA Sequencing – the next generation
Commercially available technologies Roche – 454 GSFLX titanium Junior Illumina HiSeq2000 MySeq Life – SOLiD 5500xl Ion torrent Helicos BioSciences – HeliScope Pacific Biosciences – PacBio RS
10
DNA Sequencing – the next generation
11
Template preparation: STEP1
Produce a non-biased source of nucleic acid material from the genome
12
Template preparation: STEP1
Produce a non-biased source of nucleic acid material from the genome
13
Template preparation Produce a non-biased source of nucleic acid material from the genome Current methods: randomly breaking genomic DNA into smaller sizes Ligate adaptors attach or immobilize the template to a solid surface or support the spatially separated template sites allows thousands to billions of sequencing reactions to be performed simultaneously
14
Template preparation Clonal amplification Single molecule sequencing
Roche – 454 Illumina – HiSeq Life – SOLiD Single molecule sequencing Helicos BioSciences – HeliScope Pacific Biosciences – PacBio RS
15
Template preparation: Clonal amplification
In solution – emulsion PCR (emPCR) Roche – 454 Life – SOLiD Solid phase – Bridge PCR Illumina – HiSeq
16
Template preparation: Clonal amplification - emPCR
17
Sequencing SOLiD 454
18
Pyrosequencing Picotitre plate Pyrosequencing
19
Pyrosequencing
20
Sequencing by ligation
21
Sequencing by ligation
22
Sequencing by ligation
23
Template preparation: Clonal amplification – Bridge PCR
24
Template preparation: Single molecule templates
Heliscope BioPac
25
HiSeq Heliscope
26
DNA Sequencing – the next generation
The major advance offered by NGS is the ability to cheaply produce an enormous volume of data The arrival of NGS technologies in the marketplace has changed the way we think about scientific approaches in basic, applied and clinical research
27
PART II: NGS targeted resequencing
fishing out the regions of interest
28
Random genome sequencing
The beginning Random genome sequencing ??? Sanger sequencing Targeted bp
29
DNA Sequencing – the next generation
Library/template preparation Library enrichment for target Sequencing and imaging
30
Target enrichment strategies
Random genome sequencing Hybrid Capture PCR based Sanger sequencing
31
Target enrichment strategies
32
Target enrichment strategies
33
Target enrichment strategies
34
Target enrichment strategies: MIP
35
Hybrid Capture In solution Agilent Nimblegen ... Solid phase Febit
36
Hybrid Capture In solution Relatively cheap
High throughput is possible Small amounts of DNA sufficient Solid phase Straightforward method Flexible Higher amounts of DNA
37
Target enrichment strategies
38
PCR based approaches Uniplex Multiplex Fluidigm Raindance Multiplicon
Longrange PCR products
39
PCR based approaches: Raindance
40
PCR based approaches: Fluidigm
48.48 Access Array
41
PCR based approaches: Fluidigm
48.48 Access Array
42
PCR based approaches: Fluidigm
48.48 Access Array
43
Target enrichment strategies
44
PART III: NGS workflow data collection and processing – the exome sequencing pipeline
45
Whole Exome Sequencing
The human genome Genome = 3Gb Exome = 30Mb exons Protein coding genes constitute only approximately 1% of the human genome It is estimated that 85% of the mutations with large effects on disease-related traits can be found in exons or splice sites
46
Exome sequencing gDNA 3 Gb Exome 38Mb NGS
47
The past, present & future
48
Exome sequencing capacity
HiSeq specifications: 2 flow cells 16 lanes (8 per flow cell) Gbases per flow cell 10 days for a single run Exome throughput 96 @ 60x coverage per run 60x coverage per year
49
Data processing workflow
Data formatting & QC Mapping & QC Variant calling Variant annotation Variant filtering/comparison
50
Data processing
52
DATA GENERATION DATA PROCESSING DATA STORAGE INTERPRETATION RESULTS REPORTING & VALIDATION
53
Prepare sample library
DATA GENERATION Prepare sample library Perfom exome capture Perform sequencing
54
Prepare sample library
DATA GENERATION Prepare sample library Perfom exome capture Perform sequencing
55
Prepare sample library
DATA GENERATION Prepare sample library Perfom exome capture Perform sequencing
56
DATA GENERATION DATA PROCESSING DATA STORAGE Image processing Base calling Sequence Data 10-15 Gb / exome
58
NGS data processing: overview
1 Mapping 2 Duplicate marking 3 Local realignment 4 Base quality recalibration 5 Analysis-ready mapped reads
59
DATA GENERATION DATA PROCESSING DATA STORAGE Image processing Base calling Sequence Data 10-15 Gb / exome QC sequencing Mapping sequences QC capture exp
60
DATA PROCESSING QC NGS Mapping QC HC
61
DATA PROCESSING QC NGS Mapping QC HC
62
DATA GENERATION DATA PROCESSING DATA STORAGE Image processing Base calling Sequence Data 10-15 Gb / exome QC sequencing Mapping sequences QC capture exp Mapping results 5 Gb / exome Variant Calling Variant Annotation
66
DATA GENERATION DATA PROCESSING DATA STORAGE Image processing Base calling Sequence Data 10-15 Gb / exome QC sequencing Mapping sequences QC capture exp Mapping results 5 Gb / exome Variant Calling Variant Annotation Variant Calls 100Mb / exome
67
SNPs vs Indels
68
exonic vs non-exonic
69
Exonic
70
Exonic
71
Variants Public & Private
DATA GENERATION DATA PROCESSING DATA STORAGE Image processing Base calling Sequence Data 10-15 Gb / exome QC sequencing Mapping sequences QC capture exp Mapping results 5 Gb / exome Variant Calling Variant Annotation Variant Calls 100Mb / exome Variant Filtering Database known Variants Public & Private
73
Validated variants in candidate genes
DATA GENERATION DATA PROCESSING DATA STORAGE Image processing Base calling Sequence Data 10-15 Gb / exome QC sequencing Mapping sequences QC capture exp Mapping results 5 Gb / exome INTERPRETATION RESULTS Variant Calling Variant Annotation Variant Calls 100Mb / exome Validated variants in candidate genes Variant Filtering Database known Variants Public & Private REPORTING & VALIDATION
Similar presentations
