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C.Titus Brown
Assistant Professor
MMG, CSE, BEACON
Michigan State University
May 2014
ctb@msu.edu
Large-scale transcriptom...
We practice open science!
Everything discussed here:
 Code: github.com/ged-lab/ ; BSD license
 Blog: http://ivory.idyll....
We practice open science!
Everything discussed here:
 Code: github.com/ged-lab/ ; BSD license
 Blog: http://ivory.idyll....
The challenges of non-model
transcriptomics
 Missing or low quality genome reference.
 Evolutionarily distant.
 Most ex...
Outline
1. Challenges of non-model transcriptomics.
2. Lamprey: too much data, not enough genome
3. Digital normalization ...
Sea lamprey in the Great Lakes
 Non-native
 Parasite of
medium to large
fishes
 Caused
populations of
host fishes to
cr...
The problem of lamprey:
 Diverged at base of vertebrates; evolutionarily
distant from model organisms.
 Large, complicat...
Lamprey has incomplete genomic sequence
J. Smith et al., PNAS 2009
Evidence of somatic recombination; 100s of
mb of sequen...
Lamprey tissues for which we have mRNAseq
embryo stages (late blastula,
gastrula, neurula, 22b, neural-
crest migration, 2...
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 foolish...
Shared low-level
transcripts may not
reach the threshold
for assembly.
Main problem (4 years ago):
We have a massive amount of data that
challenges existing computers when we try to
assemble it...
Solution: Digital normalization
(a computational version of library normalization)
Suppose you have a dilution
factor ofA ...
Digital normalization
Digital normalization
Digital normalization
Digital normalization
Digital normalization
Digital normalization
Digital normalization approach
A digital analog to cDNA library normalization, diginorm:
 Is single pass: looks at each r...
Evaluating diginorm – how?
 Can’t assemble lamprey w/o diginorm; are
results any good & how would we know?
 Need compara...
Looking at the Molgula…
Putnam et al., 2008,
Nature.Modified from Swalla 2001
Sea squirts!
Molgula oculata
Molgula occulta
Molgula oculata Ciona intestinalis
Elijah Lowe; collaboration w/Billie Swalla
Tail loss and notochord genes
a) M. oculata b) hybrid (occulta egg x oculata sperm) c) M. occulta
Notochord cells in orang...
Diginorm applied to Molgula embryonic
mRNAseq
Substantial time
savings (3-5x) << RAM
Elijah Lowe
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...
Why Trinity vs Oases?
Trinity is slightly better at picking out isoforms.
Elijah Lowe
How complete are these
transcriptomes?
Elijah Lowe
Transcriptome assembly thoughts
 We can (now) assemble really big data sets, and
get pretty good results.
 We have lots ...
Transcriptome results - lamprey
 Started with 5.1 billion reads from 50 different tissues.
(4 years of computational rese...
Lamprey transcriptome basic stats
 616,000 transcripts (!)
 263,000 transcript families (!)
(This seems like a lot.)
Lamprey transcriptome basic stats
 616,000 transcripts
 263,000 transcript families
 Only 20436 transcript families hav...
Common vs rare genes
#transcripts
# samples
Camille Scott
Can look at transcripts by tissue --
Camille Scott
Too… many… samples…
Camille Scott
Presence/absence clustering
Expression-based clustering
Some known biology recapitulated; and… ???
Camille Scott
Next challenges
OK, we can deal with volume of data, make pretty
pictures, and ... Now what?
Contamination!
Both experimental or “real” contaminants are big probems.
Camille Scott
Pathway predictions vary dramatically
depending on data set, annotation
Likit Preeyanon
KEGG pathway
comparison
across sev...
The problem of lopsided gene characterization is
pervasive: e.g., the brain "ignorome"
"...ignorome genes do not differ fr...
Practical implications of diginorm
 Data is (essentially) free;
 For some problems, analysis is now cheaper
than data ga...
1. khmer-protocols
 Effort to provide standard “cheap” assembly
protocols for the cloud.
 Entirely copy/paste; ~2-6 days...
CC0; BSD; on github; in reStructuredText.
A few thoughts on our approach…
 Explicitly a “protocol” – explicit steps, copy-paste,
customizable.
 No requirement for...
Can we crowdsource bioinformatics?
We already are! Bioinformatics is already a tremendously open and
collaborative endeavo...
2. Data availability is important for
annotating distant sequences
Anything else Mollusc Cephalopod
no similarity
Can we incentivize data sharing?
 ~$100-$150/transcriptome in the cloud
 Offer to analyze people’s existing data for fre...
First results: Loligo
genomic/transcriptome resources
Putting other people’s sequences where my mouth is:
w/Josh Rosenthal...
“Research singularity”
The data a researchers generates in their lab constitutes
an increasingly small component of the da...
Acknowledgements
Lab members involved Collaborators
 Adina Howe (w/Tiedje)
 Jason Pell
 Arend Hintze
 Qingpeng Zhang
...
Efficient online
counting of k-mers
Trimming reads
on abundance
Efficient De
Bruijn graph
representations
Read
abundance
nor...
2014 ucl
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  1. 1. C.Titus Brown Assistant Professor MMG, CSE, BEACON Michigan State University May 2014 ctb@msu.edu Large-scale transcriptome sequencing of non-model organisms: coping mechanisms
  2. 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 LabWeb site: http://ged.msu.edu/research.html  Preprints available. Everything is > 80% reproducible.
  3. 3. 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 LabWeb site: http://ged.msu.edu/research.html  Preprints available. Everything is > 80% reproducible by you.
  4. 4. 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.
  5. 5. 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? (Implications of free data + free data analysis.)
  6. 6. 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
  7. 7. 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…
  8. 8. 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.
  9. 9. 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)
  10. 10. 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!
  11. 11. Shared low-level transcripts may not reach the threshold for assembly.
  12. 12. Main problem (4 years ago): We have a massive amount of data that challenges existing computers when we try to assemble it all together.
  13. 13. Solution: Digital normalization (a computational version of library normalization) Suppose you have a dilution factor ofA (10) to B(1). To get 10x of B you need to get 100x ofA! Overkill!! This 100x will consume disk space and, because of errors, memory. We can discard it for you…
  14. 14. Digital normalization
  15. 15. Digital normalization
  16. 16. Digital normalization
  17. 17. Digital normalization
  18. 18. Digital normalization
  19. 19. Digital normalization
  20. 20. 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.
  21. 21. Evaluating diginorm – how?  Can’t assemble lamprey w/o diginorm; are results any good & how would we know?  Need comparative data set  …ascidians!
  22. 22. Looking at the Molgula… Putnam et al., 2008, Nature.Modified from Swalla 2001
  23. 23. Sea squirts! Molgula oculata Molgula occulta Molgula oculata Ciona intestinalis Elijah Lowe; collaboration w/Billie Swalla
  24. 24. 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
  25. 25. Diginorm applied to Molgula embryonic mRNAseq
  26. 26. Substantial time savings (3-5x) << RAM Elijah Lowe
  27. 27. 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
  28. 28. Why Trinity vs Oases? Trinity is slightly better at picking out isoforms. Elijah Lowe
  29. 29. How complete are these transcriptomes? Elijah Lowe
  30. 30. 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 ofTrinity mRNAseq pipeline.)
  31. 31. 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:
  32. 32. Lamprey transcriptome basic stats  616,000 transcripts (!)  263,000 transcript families (!) (This seems like a lot.)
  33. 33. 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.
  34. 34. Common vs rare genes #transcripts # samples Camille Scott
  35. 35. Can look at transcripts by tissue -- Camille Scott
  36. 36. Too… many… samples… Camille Scott Presence/absence clustering
  37. 37. Expression-based clustering Some known biology recapitulated; and… ??? Camille Scott
  38. 38. Next challenges OK, we can deal with volume of data, make pretty pictures, and ... Now what?
  39. 39. Contamination! Both experimental or “real” contaminants are big probems. Camille Scott
  40. 40. Pathway predictions vary dramatically depending on data set, annotation Likit Preeyanon KEGG pathway comparison across several different gene annotation sets for chicken
  41. 41. 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.Slide courtesy Erich Schwarz
  42. 42. 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).
  43. 43. 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
  44. 44. CC0; BSD; on github; in reStructuredText.
  45. 45. 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.
  46. 46. Can we crowdsource bioinformatics? We already are! Bioinformatics is already a tremendously open and collaborative endeavor. (Let’s take advantage of it!) “It’s as if somewhere, out there, is a collection of totally free software that can do a far better job than ours can, with open, published methods, great support networks and fantastic tutorials. But that’s madness – who on Earth would create such an amazing resource?” - http://thescienceweb.wordpress.com/2014/02/21/bioinformatics -software-companies-have-no-clue-why-no-one-buys-their- products/
  47. 47. 2. Data availability is important for annotating distant sequences Anything else Mollusc Cephalopod no similarity
  48. 48. 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 MarineTranscriptome Project” blog post;
  49. 49. First results: Loligo genomic/transcriptome resources Putting other people’s sequences where my mouth is: w/Josh Rosenthal and Benton Gravely
  50. 50. “Research singularity” The data a researchers generates in their lab constitutes an increasingly small component of the data used to reach a conclusion. Corollary:The true value of the data an individual investigator generates should be considered in the context of aggregate data. Even if we overcome the social barriers and incentivize sharing, we are, needless to say, not remotely prepared for sharing all the data.
  51. 51. 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.
  52. 52. Efficient online counting of k-mers Trimming reads on abundance Efficient De Bruijn graph representations Read abundance normalization Streaming algorithms for assembly, variant calling, and error correction Cloud assembly protocols Efficient graph labeling & exploration Data set partitioning approaches Assembly-free comparison of data sets HMM-guided assembly Efficient search for target genes Currentresearch (khmer software)
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