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  • 1. C. Titus Brown Assistant Professor MMG, CSE, BEACON Michigan State University May 2014 ctb@msu.edu Applying mRNAseq to non-model organisms: challenges, opportunities, and solutions
  • 2. We practice open science! Everything discussed here:  Code: github.com/ged-lab/ ; BSD license  Blog: http://ivory.idyll.org/blog (‘titus brown blog’)  Twitter: @ctitusbrown  Grants on Lab Web site: http://ged.msu.edu/research.html  Preprints available.
  • 3. Sequencing has become very inexpensive.
  • 4. Sequencing costs  Approximately $1000 of mRNAseq will yield a decent transcriptome.  Multiple samples will allow you to generate gene inventories.  For the ascidian project I will show you,  1 graduate student,  2 transcriptomes,  3 genomes…
  • 5. Mapping => quantitation Reference transcriptome required.
  • 6. Interpreting RNAseq requires gene models: http://www.hitseq.com/images/RNA-seq_AS.jpg
  • 7. The challenges of non-model transcriptomics  Missing or low quality genome reference.  Evolutionarily distant.  Most extant computational tools focus on model organisms –  Assume low polymorphism (internal variation)  Assume reference genome  Assume somewhat reliable functional annotation  More significant compute infrastructure …and cannot easily or directly be used on critters of interest.
  • 8. Outline 1. Challenges of non-model transcriptomics. 2. Lamprey: too much data, not enough genome 3. Digital normalization as a coping mechanism 4. …applied to Molgulid ascidians… 5. …and back to lamprey. 6. More transcriptome challenges 7. What’s next? Note: I also work on metagenomics, which I will not discuss t
  • 9. Sea lamprey in the Great Lakes  Non-native  Parasite of medium to large fishes  Caused populations of host fishes to crash Li Lab / Y-W C-D
  • 10. The problem of lamprey:  Diverged at base of vertebrates; evolutionarily distant from model organisms.  Large, complicated genome (~2 GB)  Relatively little existing sequence.  We sequenced the liver genome…
  • 11. Lamprey has incomplete genomic sequence J. Smith et al., PNAS 2009 Evidence of somatic recombination; 100s of mb of sequence eliminated from genome during development. More recent evidence (unpub, J. Smith et al.) suggests that this loss is developmentally regulated, results in changes in gene expression (due to loss of genes!), and is tissue specific. Liver genome is not the entire genome.
  • 12. Lamprey tissues for which we have mRNAseq embryo stages (late blastula, gastrula, neurula, 22b, neural- crest migration, 24c1,24c2) metamorphosis 3 (intestine, kidney) ovulatory female head skin adult intestine metamorphosis 4 (intestine, kidney) preovulatory female eye adult kidney metamorphosis 5 (liver, intestine, kidney) preovulatory female tail skin brain paired metamorphosis 6 (intestine, kidney) prespermiating male gill freshwater (gill, intestine, kidney) metamorphosis 7 (intestine, kidney) mature adult male rope tissue larval (gill, kidney, liver, intestine) monocytes spermiating male gill juvenile (intestine, liver, kidney) brain (0,3,21 dpi) spermiating male head skin lips spinal cord (0.3.21 dpi) supraneural tissue metamorphosis 1 (intestine, kidney) spermiating male muscle small parasite distal intestine, kidney, proximal intestine metamorphosis 2 (liver, intestine, salt water (gill, intestine)
  • 13. Assembly It was the best of times, it was the wor , it was the worst of times, it was the isdom, it was the age of foolishness mes, it was the age of wisdom, it was th It was the best of times, it was the worst of times, it was the age of wisdom, it was the age of foolishness …but for lots and lots of fragments!
  • 14. Shared low-level transcripts may not reach the threshold for assembly.
  • 15. Main problem (4 years ago): We have a massive amount of data that challenges existing computers when we try to assemble it all together.
  • 16. Solution: Digital normalization (a computational version of library normalization) Suppose you have a dilution factor of A (10) to B(1). To get 10x of B you need to get 100x of A! Overkill!! This 100x will consume disk space and, because of errors, memory. We can discard it for you…
  • 17. Digital normalization
  • 18. Digital normalization
  • 19. Digital normalization
  • 20. Digital normalization
  • 21. Digital normalization
  • 22. Digital normalization
  • 23. Digital normalization approach A digital analog to cDNA library normalization, diginorm:  Is single pass: looks at each read only once;  Does not “collect” the majority of errors;  Keeps all low-coverage reads;  Smooths out coverage of sequencing. => Enables analyses that are otherwise completely impossible.
  • 24. Evaluating diginorm – how?  Can’t assemble lamprey w/o diginorm; are results any good & how would we know?  Need comparative data set  …ascidians!
  • 25. Looking at the Molgula… Putnam et al., 2008, Nature.Modified from Swalla 2001
  • 26. Sea squirts! Molgula oculata Molgula occulta Molgula oculata Ciona intestinalis Elijah Lowe; collaboration w/Billie Swalla
  • 27. Challenging organisms to work on --  Only spawn ~1 month out of the year  Located off the northern coast of France (Roscoff)  Hybrids not found outside of lab conditions  Species cannot be cultured (yet)  Wet lab techniques are not fully developed for species
  • 28. Tail loss and notochord genes a) M. oculata b) hybrid (occulta egg x oculata sperm) c) M. occulta Notochord cells in orange Swalla, B. et al. Science, Vol 274, Issue 5290, 1205-1208 , 15 November 1996
  • 29. Diginorm applied to Molgula embryonic mRNAseq
  • 30. Substantial time savings (3-5x) << RAM Elijah Lowe
  • 31. Question: does it matter what assembly pipeline you use? (No) 3 70 25 1 36 13563 35 13 7 4 23 8 1 6 5 Diginorm V/O Raw V/O Diginorm trinity Raw trinity Numbers are putative orthologs (reciprocal best hits) w/Ciona intestinalis, calculated for each assembly. Elijah Lowe
  • 32. How complete are these transcriptomes? Elijah Lowe
  • 33. Shift in differentially expressed genes from gastrulation to neurulation M. ocu vs. M. occ gastrula M. ocu vs. M. occ neurula Differentially expressed during neurulation in M. ocu vs M. occ
  • 34. Notochord gene expression similar to tailed species -10 -5 0 5 10 15 -10-5051015 Expression difference Hybrid vs Parent species log2(hybrid)-log2(oculata) log2(hybrid)-log2(occulta)
  • 35. M. occulta transgenic NoTrlc Alberto Stolfi & Lionel Christiaen
  • 36. Lionel Christaen Claudia Racioppi NYU Statione Zoologica Napoli
  • 37. Enabling Molgula research…  Develop candidate genes to generate hypotheses about gene network evolution;  Rapid development of genomic resources => reporter constructs. Doesn’t answer any biological questions directly, but enables us to go looking for things much faster!
  • 38. Transcriptome assembly thoughts  We can (now) assemble really big data sets, and get pretty good results.  We have lots of evidence (some presented here :) that some assemblies are not strongly affected by digital normalization. (Note: normalization algorithm is now standard part of Trinity mRNAseq pipeline.)
  • 39. Transcriptome results - lamprey  Started with 5.1 billion reads from 50 different tissues. (4 years of computational research, and about 1 month of compute time, GO HERE) Ended with:
  • 40. Lamprey transcriptome basic stats  616,000 transcripts (!)  263,000 transcript families (!) (This seems like a lot.)
  • 41. Lamprey transcriptome basic stats  616,000 transcripts  263,000 transcript families  Only 20436 transcript families have transcripts > 1kb (compare with mouse: 17331 of 29769 genes are > 1kb) So, estimation by thumb ~ not that off, for long transcripts.
  • 42. Common vs rare genes #transcripts # samples Camille Scott
  • 43. Can look at transcripts by tissue - - Camille Scott
  • 44. Too… many… samples… Camille Scott Presence/absence clustering
  • 45. Expression-based clustering Some known biology recapitulated; and… ??? Camille Scott
  • 46. Next steps with lamprey  Far more complete transcriptome than the one generated from the genome!  (…but suffering from contamination, oversensitivity to unprocessed transcripts, …?)  Enabling studies in –  Basal vertebrate phylogeny  Biliary atresia  Evolutionary origin of brown fat (previously thought to be mammalian only!)  Pheromonal response in adults  Spinal cord regeneration
  • 47. Next challenges OK, we can deal with volume of data, make pretty pictures, and ... Now what?
  • 48. Contamination! Both experimental or “real” contaminants are big pro Camille Scott
  • 49. Pathway predictions vary dramatically depending on data set, annotation Likit Preeyanon KEGG pathway comparison across several different gene annotation sets for chicken
  • 50. The problem of lopsided gene characterization is pervasive: e.g., the brain "ignorome" "...ignorome genes do not differ from well-studied genes in terms of connectivity in coexpression networks. Nor do they differ with respect to numbers of orthologs, paralogs, or protein domains. The major distinguishing characteristic between these sets of genes is date of discovery, early discovery being associated with greater research momentum—a genomic bandwagon effect." Ref.: Pandey et al. (2014), PLoS One 11, e88889.lide courtesy Erich Schwarz
  • 51. Practical implications of diginorm  Data is (essentially) free;  For some problems, analysis is now cheaper than data gathering (i.e. essentially free);  …plus, we can run most of our approaches in the cloud (per-hour rental compute resources – e.g. Amazon Web Services).
  • 52. 1. khmer-protocols  Effort to provide standard “cheap” assembly protocols for the cloud.  Entirely copy/paste; ~2-6 days from raw reads to assembly, annotations, and differential expression analysis.  Open, versioned, forkable, citable. (“Don’t bother me unless it doesn’t work.”) Read cleaning Diginorm Assembly Annotation RSEM differential expression
  • 53. CC0; BSD; on github; in reStructuredText.
  • 54. A few thoughts on our approach…  Explicitly a “protocol” – explicit steps, copy-paste, customizable.  No requirement for computational expertise or significant computational hardware.  ~1-5 days to teach a bench biologist to use.  $100-150 of rental compute (“cloud computing”)…  …for $1000 data set.  Adding in quality control and internal validation steps.
  • 55. 2. Data availability is important for annotating distant sequences Anything else Mollusc Cephalopod no similarity
  • 56. Can we incentivize data sharing?  ~$100-$150/transcriptome in the cloud  Offer to analyze people’s existing data for free, IFF they open it up within a year. See: • CephSeq white paper. • “Dead Sea Scrolls & Open Marine Transcriptome Project” blog post; Note: data sets can now be cited.
  • 57. First results: Loligo genomic/transcriptome resources Putting other people’s sequences where my mouth is: w/Josh Rosenthal and Benton Grav
  • 58. Acknowledgements Lab members involved Collaborators  Adina Howe (w/Tiedje)  Jason Pell  Arend Hintze  Qingpeng Zhang  Elijah Lowe  Likit Preeyanon  Jiarong Guo  Tim Brom  Kanchan Pavangadkar  Eric McDonald  Camille Scott  Jordan Fish  Michael Crusoe  Leigh Sheneman  Billie Swalla (UW)  Josh Rosenthal (UPR)  Weiming Li, MSU  Ona Bloom (Feinstein), Jen Morgan (MBL), Joe Buxbaum (MSSM) Funding USDA NIFA; NSF IOS; NIH; BEACON.
  • 59. Elijah Lowe MSU
  • 60. C. Titus Brown Billie J. Swalla MSU UW
  • 61. Thanks!