New sequencing technologies:

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1 New sequencing technologies: possibili4es and limita4ons for archival DNA studies 1

2 Outline Basic concepts of NGS Overview NGS sequencing plaoorms NGS applica4ons: Whole genome Targeted- resequencing technologies 2

3 Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven

4 Next Genera4on Sequencing DNA/RNA Sequencing Library NGS 4

5 Sequencing library Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven

6 Sequencing concept Produce a non- biased source of nucleic acid material from the genome Randomly breaking genomic DNA into smaller sizes Ligate adaptors AYach template to a solid surface or support Spa4ally separate template for billions of simultaneous sequencing reac4ons Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven

7 Next Genera4on Sequencing DNA/RNA Sequencing Library NGS 7

8 Roche GS FLX 454 & Roche Junior 454 SEQUENCING 8

9 454 sequencing 9

10 454 sequencing 10

11 Illumina HiSeq2500/2000 & Illumina MiSeq ILLUMINA (SOLEXA) SEQUENCING 11

12 Illumina Sequencing 12

13 Illumina Sequencing 13

14 Life Technologies SOLiD 5500 Gene4c Analyzer SOLID SEQUENCING 14

15 SOLiD sequencing 15

16 SOLiD sequencing 16

17 Life Technologies: Ion Proton & Ion PGM ION TORRENT SEQUENCING 17

18 Ion Torrent Sequencing 18

19 Ion Torrent Sequencing 19

20 Ion Torrent Sequencing Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 20

21 Helicos BioSciences: November 15, 2012, bankrupt HELISCOPE SEQUENCING 21

22 Heliscope sequencing 22

23 Pacific Biosciences PacBio RS II SMRT SEQUENCING 23

24 SMRT sequencing 24

25 SMRT sequencing 25

26 Oxford Nanopore Technologies: GridION & MiION NANOPORE SEQUENCING 26

27 Whole genome sequencing Human genome (3Gb) Roche = 6 runs Illumina HiSeq:@100x = 1 flow cell PacBio = 16 SMRT cells Yeast (12Mb) Roche 454: 2 genomes@30x = 1 run Illumina HiSeq:150 genomes@100x = 1 flow cell PacBio RS: finished genome = 8 SMRT cells E. coli (5Mb) Roche 454: 4 genomes@30x = 1 run Illumina HiSeq:>300 genomes@100x = 1 flow cell PacBio RS: finished genome = 4 SMRT cells

28 DNA SEQUENCING TARGETED RE- SEQUENCING

29 Next Genera4on Sequencing Random genome sequencing?????? Sanger sequencing Targeted bp Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven

30 Next Genera4on Sequencing DNA/RNA Sequencing Library Target enrichment NGS 30

31 Target enrichment strategies Random genome sequencing Hybrid Capture PCR based Sanger sequencing Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven

32 Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven

33 Target enrichment strategies Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven

34 Hybrid Capture Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven

35 PCR based approaches Uniplex Mul4plex Fluidigm Raindance Mul4plicon ampliseq Raindance Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven

36 PCR based approaches: Raindance

37 PCR based approaches: Fluidigm Access Array

38 PCR based approaches: AmpliSeq

39 New developments Single Cell sequencing FFPE material Improved library prep protocols Improved enzymes 39

40 Conclusions Preferred sequencing technologies: Illumina Heliscope Protocols have been op4mized for low- input material Preferred target selec4on strategy: Hybrid capture PCR based mul4plexing 40

41 Genomics Core Website: hyp://gc.uzleuven.be/ Contact:

42 Polymerases - VeraSeq Ultra Features Features Uracil literate Superior performances Specifica4ons and Benefits Proof reading but can read through uracil Industrial leading fidelity (25x higher than Taq) High sensi4vity (<150pg input) Extreme speed (15s/kb), faster cycling, increase throughput Broad range of GC content Long range amplifica4on (up to 8kb) High yield (higher yield than VS2.0) Simplified PCR op4miza4on Unmatched purity Broad field of use Broad Mg 2+ and DMSO concentra4on range Reac4on deck 4me up to 24 hr at room temperature No BSA or other animal derived protein addi4ves Including clinical applica4ons

43 Polymerases VeraSeq ULtra and Uracil Deamina4on of cytosine to uracil in DNA leads to the GC to AT transi4on muta4on Caused by: DNA damage/oxida4on Uracil can be removed by a Uracil- DNA Glycosylase (UDG)