Next generation DNA sequencing technologies. theory & prac-ce

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1 Next generation DNA sequencing technologies theory & prac-ce

2 Outline Next- Genera-on sequencing (NGS) technologies overview NGS applica-ons NGS workflow: data collec-on and processing the exome sequencing pipeline

3 PART I: NGS technologies Next- Genera-on sequencing (NGS) technologies overview

4 Landmarks in DNA sequencing 1953 Discovery of DNA double helix structure 1977 A Maxam and W Gilbert "DNA seq by chemical degrada-on" F Sanger"DNA sequencing with chain- termina-ng 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 drah sequence of the human genome 2003 human genome completed Life Sciences markets first NGS machine

5

6 Massive parallel sequencing 11/10/13

7 DNA Sequencing the next generation Commercially available technologies Roche 454 GSFLX -tanium Junior Illumina HiSeq2000/2500 MiSeq Life SOLiD 5500 Ion torrent/proton (Helicos BioSciences HeliScope) Pacific Biosciences PacBio RS 11/10/13

8 DNA Sequencing the next generation The newer technologies cons-tute various strategies that rely on a combina-on of Library/template prepara-on Parallel sequencing

9 Template preparation: STEP1

10 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 anach or immobilize the template to a solid surface or support the spa-ally separated template sites allows thousands to billions of sequencing reac-ons to be performed simultaneously

11 Template preparation Clonal amplifica-on Roche 454 Illumina HiSeq Life SOLiD Life Ion Torrent Single molecule sequencing Helicos BioSciences HeliScope Pacific Biosciences PacBio RS

12 Template preparation: Clonal amplification In solu-on emulsion PCR (empcr) Roche 454 Life SOLiD Solid phase Bridge PCR Illumina HiSeq Life SOLiD - wildfire 11/10/13

13 Template preparation: Clonal amplification empcr 11/10/13

14 Template preparation: SOLiD 454 Ion Torrent Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven 11/10/13

15 Template preparation: Clonal amplification Bridge PCR 11/10/13

16 Template preparation: Single molecule templates Heliscope PacBio 11/10/13

17 Sequencing Sequencing By Synthesis (SBS) Roche 454 Illumina HiSeq Life Ion Torrent (label- free) Helicos BioSciences HeliScope Pacific Biosciences PacBio RS Sequecing By Liga-on Life SOLiD 11/10/13

18 454 - Pyrosequencing Pico-tre plate Pyrosequencing 11/10/13

19 454 - Pyrosequencing 11/10/13

20 Ion torrent label free sequencing 11/10/13

21 HiSeq Heliscope

22 Sequencing PacBio single molecule 11/10/13

23 Sequencing by ligation 11/10/13

24 DNA Sequencing 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 scien-fic approaches in basic, applied and clinical research NGS allows to study different aspects of the gene-c architecture at the whole genome scale 11/10/13

25 Whole-Genome SEQUENCING DNA SEQUENCING

26 Whole-Genome SEQUENCING

27 WGS - Copy number variation analysis

28 WGS - Structural variation analysis

29 Whole-Genome Sequencing (WGS) Copy number varia-on analysis Sequencing a genome at x Sequencing a genome at 1-3x Structural varia-on analysis Sequencing a genome at 5-10x Whole genome re- sequencing Sequencing a genome at >30x yeast, fruit fly, bacterial genomes, human

30 Targeted re-sequencing DNA SEQUENCING

31 The beginning Random genome sequencing?????? Sanger sequencing Targeted bp

32 DNA Sequencing the next generation Library/template prepara-on Library enrichment for target Sequencing and imaging

33 Target enrichment strategies Random genome sequencing Hybrid Capture PCR based Sanger sequencing

34 Target enrichment strategies

35 Target enrichment strategies

36 Target enrichment strategies

37 Hybrid Capture In solu-on Agilent Nimblegen... Solid phase Agilent Nimblegen Febit...

38 Hybrid Capture In solu-on Rela-vely cheap High throughput is possible Small amounts of DNA sufficient Solid phase Straighworward method Flexible Higher amounts of DNA

39 PCR based approaches Uniplex Mul-plex Fluidigm Raindance Mul-plicon Longrange PCR products Raindance

40 RNA Sequencing Rapid expression profiling, transcriptome sequencing and small RNA s

41 RNA-seq

42 RNAseq: Gene Expression through sequencing Supports discovery, screening, and profiling Does not require prior gene knowledge or annota-on Unique combina-on of Qualita-ve and quan-ta-ve measurement Digital counts vs analog intensi-es Increased dynamic range and sensi-vity No probes or primers Any species - Even when reference genome not available Analyze gene expression

43 RNAseq: summary Coun-ng or Profiling 10 million total reads of 35 bp length from poly- A selected RNA will give performance bener than any microarray Studying Alterna-ve Splicing or quan-fying csnps for most transcripts Deeper profiling of 50 to 100 million reads, with read lengths of 50 to 100 bps, from poly- A selected RNA using mrna- Seq assay Complete Annota-on of an en-rely New Transcriptome ~500 Million reads of 100 bp read length from mul-ple -ssues Normalized stranded mrna- Seq & ncrnas Small RNA- Seq for micrornas

44 PART III: NGS workflow data collec-on and processing the exome sequencing pipeline

45 Whole Exome Sequencing The human genome Genome = 3Gb Exome = 30Mb exons Protein coding genes cons-tute only approximately 1% of the human genome It is es-mated that 85% of the muta-ons 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 exome capture Seq - 2.5Gbases total cost Jeroen Van Houdt - 1/01/2010 Genomics Core - UZ Leuven- KU Leuven 1/08/2010 1/01/2011

48 Exome sequencing capacity HiSeq specifica-ons: 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 Exome sequencing

50 Data processing workflow Data forma ng & QC Mapping & QC Variant calling Variant annota-on Variant filtering/comparison

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

52 DATA GENERATION Prepare sample library Perfom exome capture Perform sequencing

53 DATA GENERATION Prepare sample library Perfom exome capture Perform sequencing

54 DATA GENERATION Prepare sample library Perfom exome capture Perform sequencing

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

56

57 NGS data processing: overview 1 Mapping Duplicate marking Local realignment Base quality recalibra-on Analysis- ready mapped reads

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

59 DATA PROCESSING QC NGS Mapping QC HC

60 DATA PROCESSING QC NGS Mapping QC HC

61 DATA GENERATION DATA PROCESSING DATA STORAGE Image processing Base calling Sequence Data Gb / exome QC sequencing Mapping sequences QC capture exp Mapping results 5 Gb / exome Variant Calling Variant Annota-on

62

63

64

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

66 SNPs vs Indels INDEL SNP

67 exonic vs non-exonic stopgain SNV nonsynonymous SNV nonframeshih inser-on nonframeshih dele-on non- coding frameshih inser-on frameshih dele-on

68 Exonic synonymous SNV stoploss SNV stopgain SNV nonsynonymous SNV nonframeshih inser-on nonframeshih dele-on frameshih inser-on frameshih dele-on

69 Exonic stoploss SNV stopgain SNV nonframeshih inser-on nonframeshih dele-on frameshih inser-on frameshih dele-on

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

71

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

73 DNA Sequencing the next generation 11/10/13

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