New sequencing technologies: possibili4es and limita4ons for archival DNA studies 1
Outline Basic concepts of NGS Overview NGS sequencing plaoorms NGS applica4ons: Whole genome Targeted- resequencing technologies 2
Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven
Next Genera4on Sequencing DNA/RNA Sequencing Library NGS 4
Sequencing library Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven
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
Next Genera4on Sequencing DNA/RNA Sequencing Library NGS 7
Roche GS FLX 454 & Roche Junior 454 SEQUENCING 8
454 sequencing 9
454 sequencing 10
Illumina HiSeq2500/2000 & Illumina MiSeq ILLUMINA (SOLEXA) SEQUENCING 11
Illumina Sequencing 12
Illumina Sequencing 13
Life Technologies SOLiD 5500 Gene4c Analyzer SOLID SEQUENCING 14
SOLiD sequencing 15
SOLiD sequencing 16
Life Technologies: Ion Proton & Ion PGM ION TORRENT SEQUENCING 17
Ion Torrent Sequencing 18
Ion Torrent Sequencing 19
Ion Torrent Sequencing Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 20
Helicos BioSciences: November 15, 2012, bankrupt HELISCOPE SEQUENCING 21
Heliscope sequencing 22
Pacific Biosciences PacBio RS II SMRT SEQUENCING 23
SMRT sequencing 24
SMRT sequencing 25
Oxford Nanopore Technologies: GridION & MiION NANOPORE SEQUENCING 26
Whole genome sequencing Human genome (3Gb) Roche 454: @1x = 6 runs Illumina HiSeq:@100x = 1 flow cell PacBio RS: @1x = 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
DNA SEQUENCING TARGETED RE- SEQUENCING
Next Genera4on Sequencing Random genome sequencing?????? Sanger sequencing Targeted 700-1000 bp Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven
Next Genera4on Sequencing DNA/RNA Sequencing Library Target enrichment NGS 30
Target enrichment strategies Random genome sequencing Hybrid Capture PCR based Sanger sequencing Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven
Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven
Target enrichment strategies Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven
Hybrid Capture Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven
PCR based approaches Uniplex Mul4plex Fluidigm Raindance Mul4plicon ampliseq Raindance Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven
PCR based approaches: Raindance
PCR based approaches: Fluidigm 48.48 Access Array
PCR based approaches: AmpliSeq
New developments Single Cell sequencing FFPE material Improved library prep protocols Improved enzymes 39
Conclusions Preferred sequencing technologies: Illumina Heliscope Protocols have been op4mized for low- input material Preferred target selec4on strategy: Hybrid capture PCR based mul4plexing 40
Genomics Core Website: hyp://gc.uzleuven.be/ Contact: gc@uzleuven.be jeroen.vanhoudt@uzleuven.be jeroen.vanhoudt@med.kuleuven.be
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
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)