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 Next-Generation sequencing (NGS) technologies – overview  NGS targeted re-sequencing – fishing out the regions of interest  NGS workflow: data collection.

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Presentation on theme: " Next-Generation sequencing (NGS) technologies – overview  NGS targeted re-sequencing – fishing out the regions of interest  NGS workflow: data collection."— Presentation transcript:

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2  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 Next-Generation sequencing (NGS) technologies – overview

4  The automated Sanger method is considered as a ‘first- generation’ technology, and newer methods are referred to as next- generation sequencing (NGS).

5  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

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7 Random genome sequencing 25 Mb 300k reads 110bp Sanger sequencing Targeted bp

8  The newer technologies constitute various strategies that rely on a combination of ◦ Library/template preparation ◦ Sequencing and imaging

9  Commercially available technologies ◦ Roche – 454  GSFLX titanium  Junior ◦ Illumina  HiSeq2000  MySeq ◦ Life – SOLiD  5500xl  Ion torrent ◦ Helicos BioSciences – HeliScope ◦ Pacific Biosciences – PacBio RS

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11  Produce a non-biased source of nucleic acid material from the genome

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13  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  Clonal amplification ◦ Roche – 454 ◦ Illumina – HiSeq ◦ Life – SOLiD  Single molecule sequencing ◦ Helicos BioSciences – HeliScope ◦ Pacific Biosciences – PacBio RS

15  In solution – emulsion PCR (emPCR) ◦ Roche – 454 ◦ Life – SOLiD  Solid phase – Bridge PCR ◦ Illumina – HiSeq

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17 SOLiD454

18 Picotitre platePyrosequencing

19 Pyrosequencing

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24 HeliscopeBioPac

25 HiSeqHeliscope

26  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 fishing out the regions of interest

28 Random genome sequencing ??? Sanger sequencing Targeted bp

29  Library/template preparation  Library enrichment for target  Sequencing and imaging

30 Random genome sequencing Hybrid Capture PCR basedSanger sequencing

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35 In solution Agilent Nimblegen... Solid phase Agilent Nimblegen Febit...

36 In solution Relatively cheap High throughput is possible Small amounts of DNA sufficient Solid phase Straightforward method Flexible Higher amounts of DNA

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38 Uniplex Multiplex Fluidigm Raindance Multiplicon Longrange PCR products Raindance

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40 48.48 Access Array

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44 data collection and processing – the exome sequencing pipeline

45  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 gDNA 3 Gb Exome 38Mb NGS

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48  HiSeq specifications: ◦ 2 flow cells ◦ 16 lanes (8 per flow cell) ◦ Gbases per flow cell ◦ 10 days for a single run  Exome throughput ◦ 60x coverage per run ◦ 60x coverage per year

49 Data formatting & QCMapping & QCVariant callingVariant annotationVariant filtering/comparison

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52 DATA STORAGE DATA GENERATION DATA PROCESSING REPORTING & VALIDATION RESULTS INTERPRETATION

53 Prepare sample library Perfom exome capture Perform sequencing

54 Prepare sample library Perfom exome capture Perform sequencing

55 Prepare sample library Perfom exome capture Perform sequencing

56 Sequence Data Gb / exome Sequence Data Gb / exome DATA STORAGE DATA GENERATION DATA PROCESSING Image processing Base calling Image processing Base calling

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58 1 Mapping 2 Duplicate marking 3 Local realignment 4 Base quality recalibration 5 Analysis-ready mapped reads

59 Sequence Data Gb / exome Sequence Data Gb / exome DATA STORAGE DATA GENERATION DATA PROCESSING Image processing Base calling Image processing Base calling QC sequencing Mapping sequences QC capture exp QC sequencing Mapping sequences QC capture exp

60 QC NGSMappingQC HC

61 QC NGSMappingQC HC

62 Sequence Data Gb / exome Sequence Data Gb / exome DATA STORAGE Mapping results 5 Gb / exome Mapping results 5 Gb / exome DATA GENERATION DATA PROCESSING Image processing Base calling Image processing Base calling QC sequencing Mapping sequences QC capture exp QC sequencing Mapping sequences QC capture exp Variant Calling Variant Annotation Variant Calling Variant Annotation

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66 Sequence Data Gb / exome Sequence Data Gb / exome DATA STORAGE Mapping results 5 Gb / exome Mapping results 5 Gb / exome Variant Calls 100Mb / exome Variant Calls 100Mb / exome DATA GENERATION DATA PROCESSING Image processing Base calling Image processing Base calling QC sequencing Mapping sequences QC capture exp QC sequencing Mapping sequences QC capture exp Variant Calling Variant Annotation Variant Calling Variant Annotation

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71 Sequence Data Gb / exome Sequence Data Gb / exome DATA STORAGE Mapping results 5 Gb / exome Mapping results 5 Gb / exome Variant Calls 100Mb / exome Variant Calls 100Mb / exome DATA GENERATION DATA PROCESSING Image processing Base calling Image processing Base calling QC sequencing Mapping sequences QC capture exp QC sequencing Mapping sequences QC capture exp Variant Calling Variant Annotation Variant Calling Variant Annotation Database known Variants Public & Private Database known Variants Public & Private Variant Filtering

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73 Sequence Data Gb / exome Sequence Data Gb / exome DATA STORAGE Mapping results 5 Gb / exome Mapping results 5 Gb / exome Variant Calls 100Mb / exome Variant Calls 100Mb / exome DATA GENERATION DATA PROCESSING Image processing Base calling Image processing Base calling QC sequencing Mapping sequences QC capture exp QC sequencing Mapping sequences QC capture exp Variant Calling Variant Annotation Variant Calling Variant Annotation Database known Variants Public & Private Database known Variants Public & Private Variant Filtering REPORTING & VALIDATION RESULTS Validated variants in candidate genes INTERPRETATION


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