High Throughput Profiling of Prokaryotic Species

,Klik om de stijl te bewerken,Klik om de modelstijlen te bewerken,Tweede niveau,Derde niveau,Vierde niveau,Vijfde niveau,*,*,Klik om de stijl te bewerken,Klik om de modelstijlen te bewerken,Tweede niveau,Derde niveau,Vierde niveau,Vijfde niveau,*,*,Klik om de stijl te bewerken,Klik om de modelstijlen te bewerken,Tweede niveau,Derde niveau,Vierde niveau,Vijfde niveau,*,*,Klik om de stijl te bewerken,Klik om de modelstijlen te bewerken,Tweede niveau,Derde niveau,Vierde niveau,Vijfde niveau,*,*,Klik om de stijl te bewerken,Klik om de modelstijlen te bewerken,Tweede niveau,Derde niveau,Vierde niveau,Vijfde niveau,*,*,Klik om de stijl te bewerken,Klik om de modelstijlen te bewerken,Tweede niveau,Derde niveau,Vierde niveau,Vijfde niveau,*,*,Klik om de stijl te bewerken,Klik om de modelstijlen te bewerken,Tweede niveau,Derde niveau,Vierde niveau,Vijfde niveau,*,*,High Throughput Profiling of Prokaryotic Species,Joachim De Schrijver,Vakgroep Wiskundige Modellering, Statistiek en Bio-informatica,1,Overview,Sequencing technology,Roche/454 GS-FLX (454),Illumina,Prokaryotic profiling,De novo genome sequencing,Metagenomics,SNP profiling,Species quantification,Viral profiling,De novo genome sequencing,2,Sequencing technology,Classic chain-terminator sequencing,Dye chain-terminator sequencing,Next-generation sequencing,3,Sequencing technology,Next-gen sequencing principle,Massive parallel,Add ACTGs,Catch a signal,4,Sequencing technology,Roche/454 GS-FLX+ (454),Pyrosequencing,problems with homopolymers (e.g. AAAAAA),Long-read sequencing: 500-1000 bp,Variable sequencing length,1 million reads/run,1Gb/run,Sequencing speed: 1 day/run,Next-next generation: IonTorrent PGM/Proton,5,Sequencing technology,Illumina,Sequence by synthesis,Short-read sequencing: 36, 72, , 150bp,Fixed sequencing length,1 billion reads/run,100Gb/run (= 33 x human genome!),Sequencing speed: 3 day 10 days length,Solid,Short-read sequencing (similar to Illumina),6,Sequencing technology,454,Illumina,7,Sequencing technology,Price per run: $10000/run,Supporting IT hardware,Peripheral devices such as fragmentation instrument, PCR equipment ,Negotiating power,Use service centers!,Nxtgnt (BE), GATC(EU), Baseclear(NL), BGI ,No overhead cost, no maintenance etc.,Cheaper,8,Sequencing technology,Next-generation sequencing has become,2,nd,generation sequencing,Next-next-generation sequencing is almost there: 3,rd,generation sequencing,Helicos: True Single Molecule Sequencing,IonTorrent/Life: Cheap and fast,Nanopore: Unlimited read size,9,Sequencing technology,Evolution sequencing technology goes hand in hand with evolution of,IT infrastructure/hardware,Analysis software,Hardware,1 Illumina run 100Gb text-file 5million page book,Processing power/storage are an issue!,Software,Mapping to a human genome: couple of hours,10,Overview,Sequencing technology,Roche/454 GS-FLX (454),Illumina,Prokaryotic profiling,De novo genome sequencing,Metagenomics,SNP profiling,Species quantification,Viral profiling,De novo genome sequencing,11,Prokaryotic profiling,Prokaryotic genomics 101,Prokaryotes = bacterias + archaea,Prokaryotic genomes,Large circular genome (0.5 10 Mb) chromosome,Small plasmids (1-1000 kb) (virulence factors, antibiotics resistance ),(Almost) no introns,Easy ORF annotation,12,Overview,Sequencing technology,Roche/454 GS-FLX (454),Illumina,Prokaryotic profiling,De novo genome sequencing,Metagenomics,SNP profiling,Species quantification,Viral profiling,De novo genome sequencing,13,Prokaryotic profiling: de novo genome sequencing,1953: Watson/Crick discover DNA helix,1977: First complete genome,bacteriophage,X174,1995: First genome of free-living organism,H. influenza,2001: First draft of the human genome,2006: 200 complete bacterial genomes,2012: An uncountable number of bacterial genomes have been sequenced using next-gen sequencing,14,Prokaryotic profiling: de novo genome sequencing,Complete bacterial genomes used to be,Expensive,Difficult to obtain,Nature or Science work,Remained complex until the invention of next-generation sequencing,15,Prokaryotic profiling:de novo genome sequencing,Using next-generation sequencing, de novo sequencing has become,Relatively easy,Relatively cheap,Routine research,Already 10 complete bacterial genomes published in 2012,More than just an assembly!,16,Prokaryotic profiling: de novo genome sequencing,Practical,Get some DNA from an,isolated species,of interest,Sequence: long or short reads (1-10 days),Obtain your sequences,Assemble (1h),Pure de novo assembly,Guided assembly,Annotate the genome (days-weeks),17,Prokaryotic profiling: de novo genome sequencing,Assembly:,Multiple short reads,1 long sequence,Existing software,Velvet,SSAKE,Newbler,SSAKE,Source: Nature 2009, MacLean et al.,18,Prokaryotic profiling: de novo genome sequencing,Relatively cheap,Sequencing cost: depending on coverage,Illumina, 30x, 5Gb genome: $10-$100,454, 30x, 5Gb genome: $1000-$5000,Equipment,IT infrastructure, sequencing equipment, people ,Relatively easy,Need for IT support,No out-of-the-box standard solution for everything,Several different software packages for assembly,19,Overview,Sequencing technology,Roche/454 GS-FLX (454),Illumina,Prokaryotic profiling,De novo genome sequencing,Metagenomics,SNP profiling,Species quantification,Viral profiling,De novo genome sequencing,20,Prokaryotic profiling:Metagenomics,De novo genome assembly,Study of 1 single species,Need for species isolation,Metagenomics analysis,Study of a community of species,No need for isolation (culturing bias!),Study the collective,gene pool and function,of the community/ecology,No need for individual functions,21,Prokaryotic profiling:Metagenomics,Practical,Get bacterial DNA or RNA from a sample,Soil,Gut/Fecal,Ocean water (e.g. Craig Venter),Sequence: long or short reads (1-10 days),Obtain your sequences,Map on a database of known genes (1 day),Annotate/analyse the community (weeks),22,Prokaryotic profiling:Metagenomics,23,Prokaryotic profiling:Metagenomics,2010:,Giant Panda genome (2,nd,carnivore),No umami taster receptor - no meat affinity,The panda is more a dog than a bear,The panda is a carnivore eating bamboo!,24,Prokaryotic profiling:Metagenomics,Still 2010 !: Panda microbiome,Gut microbiome of the panda reveals the presence of bamboo/cellulose degrading pathways,25,Prokaryotic profiling:Metagenomics,26,Prokaryotic profiling:Metagenomics,A clinical example: gut microbiome can predict diabetes and malnourishment,Plos One (2011), Brown et al.,Plos One (2010), Valladares et al.,Gut Pathology (2011),Gupta et al.,27,Overview,Sequencing technology,Roche/454 GS-FLX (454),Illumina,Prokaryotic profiling,De novo genome sequencing,Metagenomics,SNP profiling,Species quantification,Viral profiling,De novo genome sequencing,28,Prokaryotic profiling:SNP profiling,Classical SNP analysis - practical,Design PCR primers,Generate amplicons,Re-sequence using long read sequencing,Conserve SNP blocks,Detect SNPs,Correlate SNPs to drug resistance, severity of symptoms ,29,Prokaryotic profiling:SNP profiling,Amplicon resequencing is the same for human, prokaryotic, viral analyses,Many standardized out-of-the-box solutions available,Very simple analysis,Watch out for the overkill,Dont use a bazooka to kill a fly!,Throughput can be too high,30,Prokaryotic profiling:SNP profiling,31,Prokaryotic/Viral profiling:SNP profiling,Profile the coding region of hepatitis C,Lauck et al. 2012,32,Prokaryotic profiling:SNP profiling,Use next-generation sequencing to predict the optimal HIV therapy,Thielen et al. 2012,33,Overview,Sequencing technology,Roche/454 GS-FLX (454),Illumina,Prokaryotic profiling,De novo genome sequencing,Metagenomics,SNP profiling,Species quantification,Viral profiling,De novo genome sequencing,34,Prokaryotic profiling:Species quantification,Imagine the following research questions,Which (known) species/groups are present in a certain sample,Does this composition alter given a certain treatment, change of conditions, patients etc.,No need for de novo genome sequencing,No metagenomics: species instead of functions,35,Prokaryotic profiling:Species quantification,Prokaryotes have the gene 16S rDNA, coding for ribosomal RNA,The 16S rDNA region is 1.5 kb long,16S rDNA is specific for each species/strain,Theoretical: 4,1,500,= 10,903,possibilities,In practice: 16S rDNA sequence known for millions of species,36,Prokaryotic profiling:Species quantification,16S rDNA can be isolated in different species using universal PCR primers,Isolate/amplify different regions using the same primers,Compare the isolated sequences against a database of known sequences,37,Prokaryotic profiling:Species quantification,Practical procedure,Sample an environment and isolate DNA,Do a universal PCR amplification,Sequence using long read sequencing: the longer the better!,Obtain sequences,Map sequences against a reference database,Annotate the data,38,Prokaryotic profiling:Species quantification,Example: The Antarctica project,Which parameters determine the composition of bacterial communities in antarctical lakes?,20 different samples/lakes,Sequence 16S rDNA genes,1 x 454 run (1 million 500bp sequences),Map all sequences back to the RDP database,39,Prokaryotic profiling:Species quantification,Analyse the data using computing power,Compare different locations,Is species A present in location1, location2,Assess the distribution in a single location,How dominant is the most dominant species in location 1,How many species are in location 1,Visualize !,40,Prokaryotic profiling:Species quantification,Analyse different samples on different taxonomic levels,Include taxonomic tree of life of bacterias,Use a taxonomy browser,41,Prokaryotic profiling:Species quantification,Analyse a single location,42,Prokaryotic profiling:Species quantification,Compare different locations,43,Overview,Analysis,Lab work difficulty,Analysis difficulty,De novo genome,+ (isolate),+,Metagenomics,+,+ (pathways etc.),SNP,+ (design primers),+ (correlate),Species quantification,+ (universal primers),+,44,Overview,Sequencing technology,Roche/454 GS-FLX (454),Illumina,Prokaryotic profiling,De novo genome sequencing,Metagenomics,SNP profiling,Species quantification,Viral profiling,De novo genome sequencing,45,Viral profiling,Viral profiling,Viral profiling = prokaryotic profiling, but,Cheaper,Faster,Easier,De novo genome sequencing = OK,Dont spend $10.000 on a 100kb genome!,Multiplexing/pooling capacity is limited!,46,Viral profiling,Watch out for the overkill,An illumina run can be split into 8 lanes,20 samples per lane can be combined,Still 100Mb per sample,47,Thanks for your attention !,48,Questions,49,