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A decade into Next Generation Sequencing on marine non-model organisms: current state and developments
- 1. A decade intoNext Generation Sequencing on marine non-model organisms: Current state and developments Alexander Jueterbock 2017-03-01 @AJueterbock Next Generation Sequencing 2017-03-01 1 / 43
- 2. NGS history Nextgeneration sequencing History of sequencing 1953 Watson and Crick: Double helix structure 1977 First generation Sanger sequencing 1983 Mullis:PCR 1997 Next Generation Sequencing 2003 Human genome - after 1 decade 454 Pyrosequencing2006 Illumina NGS Numerous NGS technologies @AJueterbock Next Generation Sequencing 2017-03-01 2 / 43
- 3. NGS history Nextgeneration sequencing Next generation sequencing - low costs and high throughput from www.sciencenews.org @AJueterbock Next Generation Sequencing 2017-03-01 3 / 43
- 4. NGS history Nextgeneration sequencing NGS platforms Next Generation First Generation @AJueterbock Next Generation Sequencing 2017-03-01 4 / 43
- 5. NGS history Nextgeneration sequencing Typical NGS library preparation workow Van Dijk et al., 2014, Trends in Genetics @AJueterbock Next Generation Sequencing 2017-03-01 5 / 43
- 6. NGS history Nextgeneration sequencing NGS platforms SOLiD 454 Roche PyrosequencingIon Torrent Illumina market leader @AJueterbock Next Generation Sequencing 2017-03-01 6 / 43
- 7. Genomics Omics overview Genomics DNA Epigenomics Methylation, Histonemodication, Non-coding RNA Transcriptomics mRNA Metagenomics Genomes of microorganisms Stability,heritability eectonphenotype Complexityandexibility responsetoenvironment adjusted from Kellermayer, 2010, American Journal of Medical Genetics, Part A @AJueterbock Next Generation Sequencing 2017-03-01 7 / 43
- 8. Genomics NGS omics applications Seq-MethodsApplications Genomics WGS Reduced representation gDNA targeted DNA cpDNA mtDNA Assembly Markers Variations Epigenomics Bis-seq MethylRAD MeDIP ChIP-seq gDNA ncRNA Histone modication Methylation Expression Transcriptomics RNA-seq mRNA smallRNA Assembly Expression Variations Characterization Metagenomics WGS Amplicon gDNA 16S rRNA Function Variation Phylogenetics Stability,heritability Complexityandexibility adjusted from Kellermayer, 2010, American Journal of Medical Genetics, Part A @AJueterbock Next Generation Sequencing 2017-03-01 8 / 43
- 9. Genomics Reference genome Genomesizes Phaeodactylum tricornutum (Diatom) Ectocarpus siliculosus (Brown alga) Eurytemora anis (Copepod) Patiria miniata (Bat star) Crassostrea gigas (Pacic oyster) Salmo salar (Atlantic salmon) Orcinus orca Mb Gb wikipedia @AJueterbock Next Generation Sequencing 2017-03-01 9 / 43
- 10. Genomics Reference genome Genomesizes Phaeodactylum tricornutum (Diatom) Ectocarpus siliculosus (Brown alga) Eurytemora anis (Copepod) Patiria miniata (Bat star) Crassostrea gigas (Pacic oyster) Salmo salar (Atlantic salmon) Orcinus orca Zostera marina (Seagrass) Olsen et al., 2016, Nature Mb Gb wikipedia @AJueterbock Next Generation Sequencing 2017-03-01 9 / 43
- 11. Genomics Reference genome Assemblybased on Illumina fragment and mate pair libraries Genome (203 Mb, N50: 79,958) Reads Contigs (12,588) Mate-pair Scaold (2,200) @AJueterbock Next Generation Sequencing 2017-03-01 10 / 43
- 12. Genomics Reference genome Zostera- adaptation to the marine evironment Abundance of genes and Transposable El- ements, TEs (63%) in 10 largest scaolds TEs associated with gained genes Olsen et al., 2016, Nature @AJueterbock Next Generation Sequencing 2017-03-01 11 / 43
- 13. Genomics Reference genome Zostera- adaptation to the marine evironment Abundance of genes and Transposable El- ements, TEs (63%) in 10 largest scaolds TEs associated with gained genes Olsen et al., 2016, Nature @AJueterbock Next Generation Sequencing 2017-03-01 11 / 43
- 14. Genomics RADseq NGS omicsapplications Seq-Methods Applications Genomics WGS Reduced representation gDNA targeted DNA cpDNA mtDNA Assembly Markers Variations Epigenomics Bis-seq MethylRAD MeDIP ChIP-seq gDNA ncRNA Histone modication Methylation Expression Transcriptomics RNA-seq mRNA smallRNA Assembly Expression Variations Characterization Metagenomics WGS Amplicon gDNA 16S rRNA Function Variation Phylogenetics Stability,heritability Complexityandexibility adjusted from Kellermayer, 2010, American Journal of Medical Genetics, Part A @AJueterbock Next Generation Sequencing 2017-03-01 12 / 43
- 15. Genomics RADseq RAD sequencing RAD mutatedcut site cut site PCR duplicate Restriction/Shearing PCR Sequencing Analysis Inated homozygosity Baird et al., 2008, PLoS ONE; Schweyen et al., 2014, Biological Bulletin @AJueterbock Next Generation Sequencing 2017-03-01 13 / 43
- 16. Genomics RADseq forpopulation genomics ddRAD case studies Population genomics Natural samples from North-Atlantic Fisheries-induced selection 3-year size selection on Guppy ddRAD pool-sequencing (16 ind.) ddRAD pool-sequencing (20 ind.) Alignment to genome Variant calling (total 1,800 SNPs, 340 common) Alignment to genome Variant calling (total 51,338 SNPs) Estimate genetic dierentiation Talk of Marvin Choquet at 12:15 Test for putatively adaptive SNPs Talk of Irina Smolina at 12:00 @AJueterbock Next Generation Sequencing 2017-03-01 14 / 43
- 17. Epigenomics NGS omics applications Seq-MethodsApplications Genomics WGS Reduced representation gDNA targeted DNA cpDNA mtDNA Assembly Markers Variations Epigenomics Bis-seq MethylRAD MeDIP ChIP-seq gDNA ncRNA Histone modication Methylation Expression Transcriptomics RNA-seq mRNA smallRNA Assembly Expression Variations Characterization Metagenomics WGS Amplicon gDNA 16S rRNA Function Variation Phylogenetics Stability,heritability Complexityandexibility adjusted from Kellermayer, 2010, American Journal of Medical Genetics, Part A @AJueterbock Next Generation Sequencing 2017-03-01 15 / 43
- 18. Epigenomics Epigenetic variation addsa level of variation to the genome Allis et al., 2015 @AJueterbock Next Generation Sequencing 2017-03-01 16 / 43
- 19. Epigenomics NGS omics applications Seq-MethodsApplications Genomics WGS Reduced representation gDNA targeted DNA cpDNA mtDNA Assembly Markers Variations Epigenomics Bis-seq MethylRAD MeDIP ChIP-seq gDNA ncRNA Histone modication Methylation Expression Transcriptomics RNA-seq mRNA smallRNA Assembly Expression Variations Characterization Metagenomics WGS Amplicon gDNA 16S rRNA Function Variation Phylogenetics Stability,heritability Complexityandexibility adjusted from Kellermayer, 2010, American Journal of Medical Genetics, Part A @AJueterbock Next Generation Sequencing 2017-03-01 17 / 43
- 20. Epigenomics DNA methylation Whatis the methylome? The set of DNA methylation modications in an organism's genome Zakhari, 2013, Alcohol research : current reviews @AJueterbock Next Generation Sequencing 2017-03-01 18 / 43
- 21. Epigenomics DNA methylation Eco-evolutionaryimportance of DNA-methylation Speciation Heritable phenotypic variation Adaptation independant of genotype @AJueterbock Next Generation Sequencing 2017-03-01 19 / 43
- 22. Epigenomics DNA methylation Bisulteconversion allows to detect methylcytosine www.atdbio.com @AJueterbock Next Generation Sequencing 2017-03-01 20 / 43
- 23. Epigenomics DNA methylation MethylRAD Wanget al., 2015, Open Biology @AJueterbock Next Generation Sequencing 2017-03-01 21 / 43
- 24. Epigenomics The methylomeof seagrass Epigenetic variation in seagrass clones Epigenetic variation in a clonal meadow on the Åland Islands? @AJueterbock Next Generation Sequencing 2017-03-01 22 / 43
- 25. Epigenomics The methylomeof seagrass Methylation response to heat stress in seagrass New grown shoots @AJueterbock Next Generation Sequencing 2017-03-01 23 / 43
- 26. Transcriptomics NGS omics applications Seq-MethodsApplications Genomics WGS Reduced representation gDNA targeted DNA cpDNA mtDNA Assembly Markers Variations Epigenomics Bis-seq MethylRAD MeDIP ChIP-seq gDNA ncRNA Histone modication Methylation Expression Transcriptomics RNA-seq mRNA smallRNA Assembly Expression Variations Characterization Metagenomics WGS Amplicon gDNA 16S rRNA Function Variation Phylogenetics Stability,heritability Complexityandexibility adjusted from Kellermayer, 2010, American Journal of Medical Genetics, Part A @AJueterbock Next Generation Sequencing 2017-03-01 24 / 43
- 27. Transcriptomics NGS omics applications Seq-MethodsApplications Genomics WGS Reduced representation gDNA targeted DNA cpDNA mtDNA Assembly Markers Variations Epigenomics Bis-seq MethylRAD MeDIP ChIP-seq gDNA ncRNA Histone modication Methylation Expression Transcriptomics RNA-seq mRNA smallRNA Assembly Expression Variations Characterization Metagenomics WGS Amplicon gDNA 16S rRNA Function Variation Phylogenetics Stability,heritability Complexityandexibility adjusted from Kellermayer, 2010, American Journal of Medical Genetics, Part A @AJueterbock Next Generation Sequencing 2017-03-01 25 / 43
- 28. Transcriptomics RNAseq andtemperature adaptation RNAseq to identify transcriptomic adaptation in seagrass Sampling sites Summer sea surface temperatures Jueterbock et al., 2016, Molecular Ecology @AJueterbock Next Generation Sequencing 2017-03-01 26 / 43
- 29. Transcriptomics RNAseq andtemperature adaptation RNAseq libraries of heatstressed samples Jueterbock et al., 2016, Molecular Ecology @AJueterbock Next Generation Sequencing 2017-03-01 27 / 43
- 30. Transcriptomics RNAseq andtemperature adaptation Dierential expression analysis Gene 1 Gene 2 Population 1 or control Population 2 or stress 5,000 out of 13,000 uniquely mapped genes were heat-responsive @AJueterbock Next Generation Sequencing 2017-03-01 28 / 43
- 31. Transcriptomics RNAseq andtemperature adaptation Separating neutral from adaptive dierentiation Gene 1 Gene 2 Population 1 Population 2 SNP 140,000 SNPs in total Neutral dierentiation Mediterranean @AJueterbock Next Generation Sequencing 2017-03-01 29 / 43
- 32. Transcriptomics RNAseq andtemperature adaptation Putatively adaptive dierentiation 21 genes were likely involved in parallel adaptation to warm temperatures 21 genes adaptively dierentiated Jueterbock et al., 2016, Molecular Ecology @AJueterbock Next Generation Sequencing 2017-03-01 30 / 43
- 33. Metagenomics NGS omics applications Seq-MethodsApplications Genomics WGS Reduced representation gDNA targeted DNA cpDNA mtDNA Assembly Markers Variations Epigenomics Bis-seq MethylRAD MeDIP ChIP-seq gDNA ncRNA Histone modication Methylation Expression Transcriptomics RNA-seq mRNA smallRNA Assembly Expression Variations Characterization Metagenomics WGS Amplicon gDNA 16S rRNA Function Variation Phylogenetics Stability,heritability Complexityandexibility adjusted from Kellermayer, 2010, American Journal of Medical Genetics, Part A @AJueterbock Next Generation Sequencing 2017-03-01 31 / 43
- 34. Metagenomics Metagenomics history Metagenomicstimeline Escobar-Zepeda et al., 2015, Frontiers in Genetics @AJueterbock Next Generation Sequencing 2017-03-01 32 / 43
- 35. Metagenomics Metagenomics history 16SrRNA metaproling vs WGS metagenomics www.gatc-biotech.com @AJueterbock Next Generation Sequencing 2017-03-01 33 / 43
- 36. Metagenomics Metagenomics history NGSOmics applications Seq-Methods Applications Genomics WGS Reduced representation gDNA targeted DNA cpDNA mtDNA Assembly Markers Variations Epigenomics Bis-seq MethylRAD MeDIP ChIP-seq gDNA ncRNA Histone modication Methylation Expression Transcriptomics RNA-seq mRNA smallRNA Assembly Expression Variations Characterization Metagenomics WGS Amplicon gDNA 16S rRNA Function Variation Phylogenetics Stability,heritability Complexityandexibility adjusted from Kellermayer, 2010, American Journal of Medical Genetics, Part A @AJueterbock Next Generation Sequencing 2017-03-01 34 / 43
- 37. Metagenomics The seagrassmicrobiome Local variation in seagrass microbiome Chloe Marechal, poster 192, Session 016, 03/03/2017, 11:00 - 12:00 @AJueterbock Next Generation Sequencing 2017-03-01 35 / 43
- 38. Bottlenecks and perspectivesHuge data Big data genalice.com @AJueterbock Next Generation Sequencing 2017-03-01 36 / 43
- 39. Bottlenecks and perspectivesHuge data Bioinformatics data File size: several Gb Number of lines: 1,000,000 @AJueterbock Next Generation Sequencing 2017-03-01 37 / 43
- 40. Bottlenecks and perspectivesHuge data Bioinformatics data analysis Computational infrastructure needed Pipelines often have to be re-established for each non-model species Open source software increases reproducibility as compared with commercial software Knowledge of both biology and informatics One month analysis contracts are often too short Lacking standards for metagenomics data analysis @AJueterbock Next Generation Sequencing 2017-03-01 38 / 43
- 41. Bottlenecks and perspectivesPerspectives Perspectives Microsatellites largely replaced by SNPs for population genetics Third Generation Sequencers open up opportunities for genomics, epigenomics, and metagenomics CRISPR genome editing, a breakthrough also for non-model organisms? @AJueterbock Next Generation Sequencing 2017-03-01 39 / 43
- 42. Bottlenecks and perspectivesPerspectives Perspectives Microsatellites largely replaced by SNPs for population genetics Third Generation Sequencers open up opportunities for genomics, epigenomics, and metagenomics CRISPR genome editing, a breakthrough also for non-model organisms? @AJueterbock Next Generation Sequencing 2017-03-01 39 / 43
- 43. Bottlenecks and perspectivesThird generation sequencing Third generation sequencing platforms NGS 3rd Generation First Generation single molecule sequencing no PCR bias characterization of DNA modications @AJueterbock Next Generation Sequencing 2017-03-01 40 / 43
- 44. Bottlenecks and perspectivesThird generation sequencing Third generation sequencing platforms NGS 3rd Generation First Generation Pacic Biociences high cost input DNA 10µg raw error 10-15% circular consensus sequence error correction to 0.01% @AJueterbock Next Generation Sequencing 2017-03-01 40 / 43
- 45. Bottlenecks and perspectivesThird generation sequencing Third generation sequencing platforms NGS 3rd Generation First Generation Pacic Biociences Oxford Nanopore up to 200kbp read length 5-30% raw error size of USB stick @AJueterbock Next Generation Sequencing 2017-03-01 40 / 43
- 46. Bottlenecks and perspectivesThird generation sequencing Perspectives Microsatellites largely replaced by SNPs for population genetics Third Generation Sequencers open up opportunities for genomics, epigenomics, and metagenomics CRISPR genome editing, a breakthrough also for non-model organisms? @AJueterbock Next Generation Sequencing 2017-03-01 41 / 43
- 47. References References I Allis, CD,ML Caparros, T Jenuwein, and D Reinberg (2015). Epigenetics. P. 984. Baird, NA, PD Etter, TS Atwood, MC Currey, AL Shiver, ZA Lewis, et al. (2008). Rapid SNP discovery and genetic mapping using sequenced RAD markers. In: PLoS ONE 3.10. Escobar-Zepeda, A, AVP De Le??n, and A Sanchez-Flores (2015). The road to metagenomics: From microbiology to DNA sequencing technologies and bioinformatics. In: Frontiers in Genetics 6.DEC, pp. 115. Haas, BJ and MC Zody (2010). Advancing RNA-Seq analysis. In: Nature Biotechnology 28.5, pp. 421423. Jueterbock, A, SU Franssen, N Bergmann, J Gu, JA Coyer, TBH Reusch, et al. (2016). Phylogeographic dierentiation versus transcriptomic adaptation to warm temperatures in Zostera marina, a globally important seagrass. In: Molecular Ecology 25.21, pp. 53965411. @AJueterbock Next Generation Sequencing 2017-03-01 41 / 43
- 48. References References II Kellermayer, R(2010). Omics as the ltering gateway between environment and phenotype: The inammatory bowel diseases example. In: American Journal of Medical Genetics, Part A 152 A.12, pp. 30223025. Olsen, JL, P Rouzé, B Verhelst, Yc Lin, T Bayer, J Collen, et al. (2016). The genome of the seagrass Zostera marina reveals angiosperm adaptation to the sea. In: Nature 530.7590, pp. 331335. Schweyen, H, A Rozenberg, and F Leese (2014). Detection and removal of PCR duplicates in population genomic ddRAD studies by addition of a degenerate base region (DBR) in sequencing adapters. In: Biological Bulletin 227.2, pp. 146160. Van Dijk, EL, H Auger, Y Jaszczyszyn, and C Thermes (2014). Ten years of next-generation sequencing technology. In: Trends in Genetics 30.9, pp. 41826. @AJueterbock Next Generation Sequencing 2017-03-01 42 / 43
- 49. References References III Wang, S,J Lv, L Zhang, J Dou, Y Sun, X Li, et al. (2015). MethylRAD: a simple and scalable method for genome-wide DNA methylation proling using methylation-dependent restriction enzymes. In: Open Biology 5.11, p. 150130. Zakhari, S (2013). Alcohol metabolism and epigenetics changes. In: Alcohol research : current reviews 35.1, pp. 616. @AJueterbock Next Generation Sequencing 2017-03-01 43 / 43