Metagenomika – NGS (454, Illumina, IonTorrent) Petra Vídeňská, Ph.D. 1 https://nanohub.org/site/resources/2014/01/20215/slides/020.01.jpg Next Generation Sequencing • https://upload.wikimedia.org/wikipedia/commons/2/2e/Mapping_Reads.png 2 history-of-genome-sequencing_FINAL[1].pdf - Adobe Reader • The total number of genes is estimated at around 30,000--much lower than previous estimates of 80,000 to 140,000. • Almost all (99.9%) nucleotide bases are exactly the same in all people. • The functions are unknown for over 50% of discovered genes. Využití next generation sekvenování • http://media.wiley.com/mrw_images/els/articles/a0022508/image_n/nfgz001.gif 3 picture of Completely Sequenced Genomes Počet kompletně osekvenovaných genomů Statistics - Maxthon 2.5.14 www.genomesonline.org 4 Náklady na sekvenování genomu 5 Sekvenování nové (druhé) generace •= masivní paralelní sekvenování •Umožňuje najednou sekvenaci miliónů různých fragmentů DNA (cDNA, i RNA) o délce cca 30-1000 bp (dle zvolené platformy a sekvenačního kitu) •Dochází k zmnožení fragmentu (emulzní PCR, můstková amplifikace) – větší signál při inkorporaci nukleotidů během sekvenace, umožňující detekci Sekvenování 3. generace •Nevyužívá amplifikace za účelem zvýšení signálu (měla by být vyšší přesnost –accurancy) •Produkuje dlouhá čtení •Dobrá prosekvenovanost GC bohatých oblastí •Epigenetika •Zatím dvě dostupné technologie – PacBio a Nanopore (MinION) •I Illumina chystá nový systém • http://www.medgadget.com/wp-content/uploads/2014/01/HiSeq-X.jpg Dostupné platformy •454 (Roche) •SOLiD (Life Technologies) •Illumina (Illumina) •Ion Torrent (Life Technologies) •PACBIO, Sequel System (Pacific BioSciences) •MiniON (Oxford Nanopore Technologies) •BGISEQ-500 (BGI) – – • http://www.nucleomics.be/wp-content/uploads/roche454-sequencing.png Solid PI System Pacific Biosystems Sequel product20150910_7921_PacBio_Sequel.CR2 https://www.genomeweb.com/sites/default/files/public/images/articles/bgiseq-500_2.jpg 2 vs 3 generace 8 Sekvenování nové generace My Clips by videnska - Mozilla Firefox 10 https://www.researchgate.net/profile/Ioana_Berindan-Neagoe/publication/282061980/figure/fig1/AS:298 534453694466@1448187568133/Figure-1-Overview-of-the-main-steps-in-Next-Generation-Sequencing-workfl ow.png Sekvenační workflow 454 •Roche •454 GS Junior (35 MB) x 454 GS FLX (700 MB) • • • • •Příprava templátu: EM PCR na kuličkách •Sekvenace syntézou •Detekce chemiluminiscenční - pyrosekvenování • 11 454 Shotgun – příprava knihovny 12 Nebulization DNA End Repair 3’ 5’ 3’ 5’ Adaptor Ligation (A&B) 3’ 5’ 3’ 5’ B A DNA End Repair 3’ 5’ 3’ 5’ B A Library Quantification, read lenght check http://cache1.bioon.com.cn/picture/200712/2007129233929711.jpg 454 • 13 454 Technology to Detect Low-level Drug-resistant HIV Variants - Mozilla Firefox https://www.youtube.com/watch?v=nFfgWGFe0aA 14 GS Junior Titanium PicoTiterPlate Kit - Mozilla Firefox 454 sekvenace by videnska - Mozilla Firefox sekvenace by videnska - Mozilla Firefox sekvenace by videnska - Mozilla Firefox 15 454 ⚡Presentation "1 Ultra-High Throughput DNA Sequencing on the 454/Roche GS-FLX Methods, Automation, Applications Graham Wiley Roe Lab." - Mozilla Firefox 16 ⚡Presentation "1 Ultra-High Throughput DNA Sequencing on the 454/Roche GS-FLX Methods, Automation, Applications Graham Wiley Roe Lab." - Mozilla Firefox 17 454 – podrobný workflow •http://cfgbc.mf.uni-lj.si/people/damjana/teaching/fg-fkkt/4-GS-JuniorTechnology.pdf 18 Illumina • • • • •Příprava templátu: hybridizace na sklíčku, tvorba klastrů •Sekvenace syntézou •Detekce fluorescence odštěpené značky z reverzního terminátoru (nukleotidu) • • HiSeq 2500 available now. http://www.illumina.com/images/products/miseq.jpg 19 20 Sequencing Platforms | Compare NGS platforms (benchtop, production-scale) - Mozilla Firefox Přístroje Illumina 21 Sequencing Platforms | Compare NGS platforms (benchtop, production-scale) - Mozilla Firefox Přístroje Illumina ILLUMINA_LOGO_RGB_new G:\Photos\Renders_MiSeq\Legacy\MG\PPT_Request\2A_Front_view_closed_LED_on_Transparent.png MiSeq specifications READ LENGTH (BP) TOTAL TIME* OUTPUT 1 × 36 (V2) ~4 hrs 540-610 Mb 2 × 25 (V2) ~5.5 hrs 750-850 Mb 2 × 150 (V2) ~24 hrs 4.5-5.1 Gb 2 × 250 (V2) ~39 hrs 7.5-8.5 Gb 2 × 75 (V3) ~20hrs 3.3-3.8 Gb 2 × 300 (V3) ~ 55hrs 13.2-15 Gb RUN TYPE READS PASSING FILTER V2 V3 Single Reads 12-15 M 22-25 M Paired-End Reads 24-30 M 44-50 M Roche (13-3-2013) http://www.gsjunior.com/instrument-workflow.php ILLUMINA_LOGO_RGB_new NextSeq specifications ILLUMINA_LOGO_RGB_new http://bioinformative.net/wp-content/uploads/2013/09/illumina-hiseq-2500.jpeg HiSeq 2500 specifications Illumina • 25 Příprava knihovny Fragmentation Enzymatic Mechanical Nextera TruSeq Amplification The aim of the sample prep step is to obtain nucleic acid fragments with adapters attached on both ends Same general template architecture regardless of application (gDNA, RNA, miRNA, ChIP-seq, exon pull-down, etc.) Sekvenační technologie Online Courses: Sequencing - Google Chrome Agrigenomics - Microsoft PowerPoint 27 Single vs Pair End Ready •Single reads –Small RNA • • •Pair-End reads –DeNovo assembling Agrigenomics - Microsoft PowerPoint 28 ILLUMINA_LOGO_RGB_new The aim of the sample prep step is to obtain nucleic acid fragments with adapters attached on both ends Sample Prep Dual Index Library shown Same general template architecture regardless of application (gDNA, RNA, miRNA, ChIP-seq, exon pull-down, etc.) Cluster Generation Bind single DNA molecules to surface Amplify on surface ~1000 molecules per ~ 1 µm cluster [USEMAP] Skip Overview Script: A flow cell is a glass slide sandwich with 1 channel. The channel is coated with oligos that are complementary to the adapters. Flow cell hybridization and cluster generation is automated on the MiSeq. First, single DNA molecules (the prepared samples) bind to the flow cell surface. Then, the samples are amplified to form clusters. Approximately 1,000 molecules are included in a cluster. The last step is the hybridization of the sequencing primer. Hybridize Fragment & Extend Adapter sequence 3’ extension Surface of flow cell coated with a lawn of oligo pairs Single DNA libraries are hybridized to primer lawn Bound libraries then extended by polymerases Denatured Library Temp. Ramp: 96-40 °C Wash Buffer HT2 Schematic representation of cluster generation process Newly synthesized strand Original template discard Original template washed away Newly synthesized strand is covalently attached to flow cell surface AMP Premix AMP1 Phusion HFE 90 sec Temp. Ramp: 20°C Denature Double-Stranded DNA Cluster formn 3 NOTE: Single molecules bind to flow cell in a random pattern Hybridize Fragment & Extend NaOH Wash Wash Buffet HT2 33 Cluster formn 3 Hybridize Fragment & Extend AMP Manifold Ramp to 60°C Formamide AT1 34 ILLUMINA_LOGO_RGB_new Bridge Amplification Cluster formn 4 Single-stranded molecule flips over and forms a bridge by hybridizing to adjacent, complementary primer Hybridized primer is extended by polymerases AMP Premix AMP1 ILLUMINA_LOGO_RGB_new Bridge Amplification Cluster formn 5 AMP Mix AMX1 Contains BST pol & nucleotides ILLUMINA_LOGO_RGB_new Denature Double-Stranded Bridge Cluster formn 6 Double-stranded bridge is denatured - 1st cycle denaturation Result: Two copies of covalently bound single-stranded templates Formamide AT1 ILLUMINA_LOGO_RGB_new Bridge Amplification Cluster formn 7 Single-stranded molecules flip over to hybridize to adjacent primers Hybridized primer is extended by polymerase AMP Premix APM1 ILLUMINA_LOGO_RGB_new Bridge Amplification Cluster formn 8 Bridge amplification cycle repeated until multiple bridges are formed AMP Mix AMX1 Contains BST pol & nucleotides ILLUMINA_LOGO_RGB_new Linearization dsDNA bridges are denatured PE Linearization LMX1 Ramp 37.9 °C, 30 min Temp Ramp: 20 °C ILLUMINA_LOGO_RGB_new Reverse Strand Cleavage Reverse strands cleaved and washed away, leaving a cluster with forward strands only Wash Buffer HT2 ILLUMINA_LOGO_RGB_new Blocking Free 3’ ends are blocked to prevent unwanted DNA priming Blocking Mix BMX 38 °C, 30 min 60 °C, 15 min 20°C, HT2, HT1 Washes Read 1 Primer Hybridization Sequencing primer Sequencing primer is hybridized to adapter sequence 0.1 NAOH Seq. Primer 60 °C, 5 min 20 °C, HT2, HT1 Washes Agrigenomics - Microsoft PowerPoint Single molecule array Sequencing by sythesis (SBS) Tvorba klastrů (3x106 – 3x109) Flow cell hand 5’ 3’ T G C T A n=100 DNA (1 ng – 1 µg) Příprava knihovny 1. Sample prep – paired ends, mate pairs and multiplexing. 2. Clusters – use of Phusion 3. SBS – improved reagents  greater accuracy by reducing background noise, improved polymerase reduces time and increases accuracy, increase read length. 4. Data density – improvements in cluster analysis  much higher densities and higher accuracy. Sequencing A image C image T image G image After imaging is complete for one section (tile), the flow cell is moved to the next tile and the process is repeated Clusters are images using LED and filter combinations specific for each fluorescently-labeled nucleotide Script: After Cluster Generation is complete, the clusters with the fluorescently labeled incorporated nucleotides are imaged using LEDs. The MiSeq flow cell includes 12 tiles, arranged in a single lane. Imaging is performed 4 times for a single tile at a time. (This is a called a cycle.) The process repeats until all tiles in the lane have been imaged. Online Courses: Sequencing - Google Chrome Pair - End Sequencing – Dual Indexed Online Courses: Sequencing - Google Chrome Online Courses: Sequencing - Google Chrome Online Courses: Sequencing - Google Chrome Online Courses: Sequencing - Google Chrome Online Courses: Sequencing - Google Chrome Online Courses: Sequencing - Google Chrome Online Courses: Sequencing - Google Chrome Online Courses: Sequencing - Google Chrome Online Courses: Sequencing - Google Chrome Sekvenační technologie Online Courses: Sequencing - Google Chrome Sekvenační technologie Online Courses: Sequencing - Google Chrome Images Generated on the Instrument •https://accounts.illumina.com/ • BaseSpace * Is a powerful website computing platform * for storing my genomics data on a cloud * for analyzing my sequences * for sharing my genetic data Ion Torrent •Ion PGM x Ion Proton • • • •The chip is the machine •Příprava templátu: Em PCR •Sekvenace syntézou •Detekce uvolněných protonů – změna pH • http://24.media.tumblr.com/tumblr_le6jqjRtHU1qft3eko1_500.jpg http://www.popsci.com/files/imagecache/bown_article_image_550w/articles/thespeediestdnasequencer.jp g http://images.businessweek.com/ss/10/09/0902_techpioneers_2011/image/11_iontorrent.jpg https://tools.invitrogen.com/Content/SFS/ProdImages/high/Ion%20PI%20Chip%20Sequencing.jpg 59 Ion Torrent • An external file that holds a picture, illustration, etc. Object name is btt212f1p.jpg 60 Product Name SKU # Product Size Number of Wells Platform List Price (CZK) Ion 314™ Chip Kit v2 4482261 1 kit 1 million wells per chip Ion Personal Genome Machine® (PGM™) System 15.808,00 Ion 316™ Chip Kit 4466616 4 pack 6 million wells per chip Ion Personal Genome Machine® (PGM™) System 28.616,00 Ion 316™ Chip Kit 4469496 8 pack 6 million wells per chip Ion Personal Genome Machine® (PGM™) System 57.232,00 Ion 316™ Chip Kit v2 4483188 4 chips 6 million wells per chip Ion Personal Genome Machine® (PGM™) System 28.616,00 Ion 316™ Chip Kit v2 4483324 8 chips 6 million wells per chip Ion Personal Genome Machine® (PGM™) System 57.232,00 Ion 318™ Chip Kit (4 pack) 4466617 4 pack 11 million wells per chip Ion Personal Genome Machine® (PGM™) System 49.280,00 Ion 318™ Chip Kit (8 pack) 4469497 8 pack 11 million wells per chip Ion Personal Genome Machine® (PGM™) System 98.560,00 Ion 318™ Chip Kit v2 4484354 4 pack 11 million wells per chip Ion Personal Genome Machine® (PGM™) System 49.280,00 Ion 318™ Chip Kit v2 4484355 8 pack 11 million wells per chip Ion Personal Genome Machine® (PGM™) System 98.560,00 Ion PI™ Chip Kit v2 4482321 8 chips 165 million wells per chip Ion Proton™ System 129.130,00 61 Ion Torrent • https://www.neb.com/~/media/NebUs/Page%20Images/NEBNext%20New%20Workflow%20images/DNA/DNAIonTorrent _Workflow2.jpg 62 http://image.slidesharecdn.com/annelieshaegeman-ngs-basicprinciplesandsequencingplatforms-141219072 130-conversion-gate02/95/ngs-basic-principles-and-sequencing-platforms-17-638.jpg?cb=1418974002 Ion Torrent 63 https://www.thermofisher.com https://www.thermofisher.com/content/dam/LifeTech/migration/images/sequencing/semiconductor-sequenc ing/ion-torrent.par.54658.image.660.400.1.step-1.gif https://www.thermofisher.com/content/dam/LifeTech/migration/images/sequencing/semiconductor-sequenc ing/ion-torrent.par.52461.image.660.400.1.step-5.gif https://www.thermofisher.com/content/dam/LifeTech/migration/images/sequencing/semiconductor-sequenc ing/ion-torrent.par.32085.image.660.400.1.step-3.gif https://www.thermofisher.com/content/dam/LifeTech/migration/images/sequencing/semiconductor-sequenc ing/ion-torrent.par.83208.image.660.400.1.step-4.gif Ion Torrent https://www.youtube.com/watch?v=WYBzbxIfuKs 64 Ion Proton, Genotypic technology, Ion Torrent CSP, Ion Torrent Certified service provider, Exome sequencing, Targeted re-sequencing, Microbial sequencing, Mitochondrial sequencing, Amplicon sequencing, ChIP sequencing, RNA sequencing, Whole transcriptome Ion Torrent 65 Příprava knihovny – celogenomové sekvenování (shot gun) 66 Příprava knihovny FRAGMENTACE DNA Sonikace /enzymaticky 67 END- REPAIR ↓ LIGACE ADAPTORŮ + NICK REPAIR ↓ SIZE SELECTION - E-gel 2 % https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcRZkuD2Do-MjRhmgt9xSy4mxPcc6v18idni31PxWCF98bS RSPLLHw 4 5 50bp ladder https://tools.lifetechnologies.com/content/sfs/gallery/high/50bpDNAladder.jpg Příprava knihovny 68 AMPLIFIKACE KNIHOVNY (?) ↓ KVANTIFIKACE KNIHOVNY - qPCR / Agilent Příprava knihovny 69 PŘÍPRAVA TEMPLÁTU - Emulzní PCR ↓ ENRICHMENT ↓ SEKVENACE http://cdn.pressebox.de/a/c9b95caa7010926e/attachments/0498331.attachment/filename/imgm_laboratorie s_ion_torrent_pgm_314_316_318_chips.jpg Příprava knihovny 70 cena chipu??? 314 – kolem 2000 Kč 316 – přes 7000 318 – vice jak 12 500Kč Ion Torrent http://tools.thermofisher.com/content/sfs/prodImages/high/Ion%20OneTouch%202%20System.jpg 71 (a) Schematic of a well on an ion sequencing chip. Clonal DNA immobilized on a bead is extended by polymerase in the presence of a pure solution of one nucleotide (here 'T'). Nucleotide incorporation releases a pyrophosphate (PPi) and a hydrogen ion. The change in pH caused by release of the hydrogen ion alters the surface potential of the ion-sensitive metal oxide layer. This is converted to a voltage signal by transistors. The wells are washed and exposed sequentially to pure solutions of other nucleotides. For comparison, in high-throughput pyrosequencing, the pyrophosphate is converted to chemiluminescence by an enzymatic cascade and optically imaged. The size of the well relative to the bead has been exaggerated, although each well contains a single bead. (b) Evolution of ion sequencing chips. Increases in sensors per chip can be achieved by increasing the physical area of the sensor array, reducing the number of transistors per chip, arranging the sensors in a hexagonal rather than rectilinear geometry and reducing the well and bead size. Sensors are drawn to scale, and gray indicates sensor area not accessible to fluid. The 13-million (M) sensor design was used by Rothberg et al.1 to sequence DNA from both Escherichia coli and human. Data for a fixed ('key') DNA sequence was shown for the 165-million sensor design. The 1,100-million sensor design was proposed but its feasibility was not shown. Ion Torrent 72 https://www.thermofisher.com/cn/en/home/life-science/sequencing/dna-sequencing/microbial-sequencing /microbial-identification-ion-torrent-next-generation-sequencing/ion-16s-metagenomics-solution/_jcr _content/MainParsys/well_8185/uipar/textimage_4b52/image.img.jpg/1442958400168.jpg Ion S5 Workflow Video 02 New Ion S5 „simplicity/speed/scalability/small sample input/service & support“ https://www.thermofisher.com/kr/en/home/life-science/sequencing/dna-sequencing/targeted-sequencing/ targeted-sequencing-ion-torrent-next-generation-sequencing/_jcr_content/MainParsys/image_48e1.img.j pg/1446592021414.jpg Ion Torrent https://www.youtube.com/watch?v=jFCD8Q6qSTM&ebc=ANyPxKrMLmAe4Nmia2N3RFr_1QbGUsOzceI2sMqnIJ5gS09XPCo fTb-0cUvJdbzQhD_gKRKTL-XBahDEvoV6uOnm_78yvaG-eA 73 74 O platformách • http://dnasequencing.yolasite.com/next-generation-sequencing.php 75 Srovnání: • 454 (Junior/FLX) Illumina (MiSeq/HiSeq) Ion Torrent (PGM/ Počet čtení/run 100 tis/1 mil 35-50 milionů PE/ 8 miliard PE 5,5 mil/ 60-80 mil Průměrná délka čtení [bp] 450/700 2x300/ 2x250 400 Doba běhu 6/24 hodin 1/10 dní 7/4 hodiny Výhody délka čtení, přesnost, rychlost, snadná příprava, velké množství sekvencí, nejnižší cena rychlost, relativně nízké náklady, různé čipy (outputy) Nevýhody pracnost, cena, chybovost v polymorfismech, nízké outputy - techynologie je tak drahá, že již není více konkurenceschopná nižší přesnost na konci readů, interference u nízkodiverzních knihoven chybovost v homopolymerech, EM PCR Shotgun knihovny Amplikony 76