Genome sequencing GenBank 1982 Los Alamos Sequence Database Walter Goad Frederick Sanger 1958 – Nobel prize – insuline structure 1975 - Dideoxy sequencing method 1977 – Φ-X174 (5,368 bp) sequence 1980 – second Nobel prize λ phage sequence shotgun method (48,502 bp) Genome sequencing • 1986 Leroy Hood: automatic sequencing machine • 1986 Human Genome Initiative Leroy Hood Genome sequencing • 1995 John Craig Venter first bacterial genome John Craig Venter Craig Venter Global Ocean Sampling Expedition Synthetic genomics Human Longevity Inc http://www.youtube.com/watch?v=J0rDFbr hjtI 2010 Human super genome reference 2010 Human super genome reference Complexity reduction Hi-Cot selection Complexity reduction Methylation filtration (MF) E. coli McrBC (5mC restriction) Laser microdissection Sheath fluid Deflection plates Excitation light Waste Right collector Left collector Laser Scattered light Fluorescence emission Relative fluorescence intensity Chromosomes in suspension Flow sorted chromosomes Flow karyotype Flow chamber Numberofevents Flow sorted chromosomes Flow sorting Why chromosome flow sorting ? Barley (~5,000 Mbp) Bread wheat (~17,000 Mbp) Durum wheat (~13,000 Mbp) Rye (~8,000 Mbp)  Most of the genomes that will be sequenced in the near future are smaller than 500 Mbp  But many genomes are much larger (and polyploid)! 500 Mbp http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Human (~3,400 Mbp) x 103 Mbp The case of bread wheat  Grown on more areas of land than any other crop plant  A staple food of 40 % of the world’s population  Provides for 20 % of the calories consumed The development of wheat as a food source is deeply linked to the history of modern civilization … The fertile soil of the Fertile Crescent enabled humans to invent farming, cities, trade, and writing … http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Gill et al., Genetics 168:1087, 2004 Nuclear genome of bread wheat is complex  Allohexaploid species (2n = 6x = 42, AABBDD genome)  Resulted from two independent hybridization events ~17,000 Mbp (1C) ~1.2% genes http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Gill et al., Genetics 168:1087, 2004 O.5 MYA 8 TYA Divide and sequence  Chromosomes: 605 - 995 Mbp (3.6 – 5.9% of the genome) Three genomes of hexaploid wheat Triticum aestivum (2n = 6x = 42) 1C ~ 17,000 Mbp AA BB DD  Chromosome arms: 225 - 585 Mbp (1.3 – 3.4% of the genome) http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html D B ; A  Preparation of aqueous suspensions of mitotic chromosomes  Preservation of chromosome integrity and intact DNA  Discrimination of individual chromosome types  Sorting chromosomes in large quantities Integrating flow cytogenetics and genomics Critical requirements http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Chromosome sample for flow cytometry The procedure Cell cycle synchronisation in root-tip meristems Accumulation of mitotic cells in metaphase Extraction of chromosomes Fluorescent staining of chromosomal DNA Flow cytometric analysis Chromosome sorting A sample must be in a form of liquid suspension of single particles http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Doležel et al., Planta 188: 93, 1992 Suspension  Mild fixation of synchronized root tips in formaldehyde  Release of chromosomes by mechanical homogenization  Removal of large debris and tissue fragments by filtration Root tip Chromosome http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Isolation of intact chromosomes Doležel et al., Planta 188: 93, 1992 Development of flow cytogenetics at IEB Species list:  Field bean  Garden pea  Chickpea  Garden vetch  Barley  Bread wheat  Durum wheat  Rye  Oats  Norway spruce  White campion BD FACSVantage SE high-speed flow sorter • Sample rate: 500 – 1000 chromosomes / sec • Sort rate: 15 - 30 chromosomes / sec http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html The uses of flow-sorted chromosomes BAC libraries Cot fractionation HAPPY mapping Optical mapping http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html Cytogenetic mapping Immunolocalization of proteins Mapping on DNA arrays Generation of DNA markers Shotgun sequencing Linear amplification Short-insert DNA libraries Generation of DNA markers DOP - PCRPhysical mapping using PCR Chromosome sorting 102 copies 102-3 copies 103 copies 104 copies 106-7 copies Chromosome-specific BAC libraries Ligation into a dephosphorylated BAC vector E. coli transformation Size selection by PFGE Chromosome analysis and sorting I II III 3B ‘Chinese Spring’ l 2 3 S1 kbp - 2200 - 825 - 225 Colonies Ordering into 384-well plates 2 x 106 - 107 flowsorted chromosomes (~8 weeks of sorting) Partial digestion Major challenges: • Quantity of DNA (micrograms DNA) • Quality of DNA (HMW) • Cloning efficacy • Insert size http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html BAC-BY-BAC SEQUENCING BAC clones  Physical map is composed of contigs of overlapping BAC clones  BAC contigs are landed on the chromosome through markers comprised in the contigs • http://www.454.com Genome Sequencer 20 System 454 pyrosequencing (2005) DNA library preparation Fragmentace DNA Ligace adaptoru Vychytání DNA molekul denaturace emPCR Vznik emulze (olej) emPCR emPCR Vychytání kuliček Vychytání kuliček denaturace Sekvenační primer Disperze na sklíčko Disperze na sklíčko Parametry mikroreaktorů Parametry mikroreaktorů sekvenace sekvenace sekvenace sekvenace sekvenace sekvenace sekvenace sekvenace SOLID (Sequencing by Oligonucleotide Ligation and Detection) 2-base encoding sequencing (2007) Solexa (2007) HELICOS (2008) True Single Molecule Sequencing (tSMS) Single Molecule Real-Time (SMRT) Pacific Biosciences 20 zeptolitrů Ion Torrent Oxford nanopore Další technologie • Mikroelektroforéza • Sekvenování na bázi microarray