ULTRASEQUENCING. Next Generation Sequencing: methods and applications. Genòmica i Proteòmica Pablo Lammers Curs 12/13 NIU 1323099

Sanger sequencing Since 1975. Frederick Sanger Human Genome Project New necessities 1 sample -> 1 read -> 3 to 9€ 1 read -> 1 kbp (max.) 1 run -> 16/48/96 samples

Next Generation Sequencing Libraries preparation DNA Sonication Physical methods Fragmentation Amplification Chemical methods Adapters ligation Sequencing reaction 1 run -> 1000 € 1 read -> 100 to 400 bp 1 run -> >100 M reads 1 run -> 24 small genomes

NGS platforms 454 Roche – GS Junior Applied Biosystems: SOLiD Illumina - MiSeq Invitrogen – Ion Torrent Pacific Biosciences – PacBio RS II

454 (Roche) One fragment = One bead emPCR: Emulsion PCR amplification Sequencing: One bead = One read Pyrosequencing 1 M reads/run Read lenght: 250-500 bp DNA captured bead containing millions of copies of a single clonally amplified fragment Library construction emPCR PTP loading

Applied Biosystems: SOLiD Amplification by emPCR Hybridization to beads. Beads covalently attached to glass slide. Ligation Based Sequencing with Di-Base probes (fluorescently labeled with 4 dyes) Image capture (fluorophore) 100-500 M reads/run Read lenght: 50-100 bp

Illumina 1. Library preparation 2. Clusters generation 3. Sequencing Sequencing by synthesis 1. Library preparation 2. Clusters generation 3. Sequencing DNA fragmentation Adapter oligos ligated Isothermal bridge amplification Purification 100 M reads/run Read lenght: 80-250 bp

Ion Torrent Nucleotide incorporated to a single DNA strain wells -> chemical info from DNA seq -> into digital info (basecalls) DNA fragmented Attached to beads Each bead in a well one of the 4 nucleotides Nucleotide incorporated to a single DNA strain ion H released pH chemichal changes -> into voltage each 15 sec -> wash and repeat (different nucleotide) 10 M – 1 G reads/run Read lenght: 200-400 bp

Pacific Biosystems (PACBIO) Amplification not required SMRT: Single Molecule Real Time seq ZMW: Zero-mode waveguide DNA template-polymerase complex -> immobilized at the bottom of the ZMW Each nucleotide labeled with a different colored fluorosphore Read lenght: 4 kbp Maximum: 23 kbp Phospholinked nucleotides -> introduced into the ZMW chamber Base held -> light pulse produced

Applications Cancer research Population genomics studies Metagenomics RNA-seq Comparative genomics Disease association studies Species clasification Forensics