J. B. Cole
Animal Improvement Programs Laboratory
Agricultural Research Service, USDA
Beltsville, MD 20705-2350, USA
john....
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (2) Cole
Overview
Why are we sequenc...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (3) Cole
Introduction
Several studie...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (4) Cole
Chromosome 18 is different
...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (5) Cole
Marker effects for dystocia...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (6) Cole
Correlations in dystocia co...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (7) Cole
The QTL also affects gestat...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (8) Cole
Overview of the dystocia co...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (9) Cole
This is a gene-rich region
...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (10) Cole
Copy number variants are p...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (11) Cole
Where did this problem com...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (12) Cole
What if we look at a diffe...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (13) Cole
We don’t have birth weight...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (14) Cole
GWAS for birth weight PTA
...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (15) Cole
Are we measuring anything ...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (16) Cole
KEGG pathways for birth we...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (17) Cole
Sequencing is becoming ver...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (18) Cole
Sequencing successes at AI...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (19) Cole
Original pedigree-based de...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (20) Cole
Modified pedigree & haplot...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (21) Cole
DNA Quality
Molecular prep...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (22) Cole
Sample preparation time is...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (23) Cole
DNA quality
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (24) Cole
Library quality
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (25) Cole
Sequencing stage
• Illumin...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (26) Cole
Anatomy of a flow cell
8 l...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (27) Cole
Sequencing by synthesis
ht...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (28) Cole
How many scientists does i...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (29) Cole
Flowcell 1: Cluster densit...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (30) Cole
Flowcell 2: Cluster densit...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (31) Cole
The Aftermath
Total Time (...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (32) Cole
Variant detection
• Alignm...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (33) Cole
Computational Logistics
De...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (34) Cole
Storage considerations
Wha...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (35) Cole
Why should you use a pipel...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (36) Cole
Galaxy server
NextGene
Cus...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (37) Cole
Challenges
Annotation
This...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (38) Cole
Conclusions
Sequencing is ...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (39) Cole
Acknowledgments
AIPL: Dere...
Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (40) Cole
Questions?
http://gigaom.c...
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Use of NGS to identify the causal variant associated with a complex phenotype

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Use of NGS to identify the causal variant associated with a complex phenotype

  1. 1. J. B. Cole Animal Improvement Programs Laboratory Agricultural Research Service, USDA Beltsville, MD 20705-2350, USA john.cole@ars.usda.gov Use of NGS to identify the causal variant associated with a complex phenotype
  2. 2. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (2) Cole Overview Why are we sequencing? How did we select the animals to sequence? What are the steps involved in the process? What do you do with the reads once you have them? Where are we now?
  3. 3. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (3) Cole Introduction Several studies (Kuhn et al., 2003; Cole et al., 2007; Seidenspinner et al., 2009) have reported QTL on BTA 18 associated with dystocia Bioinformatic analysis using SNP data has not identified the causal variant Next generation sequencing (NGS) has recently been used to find causal variants for novel recessive disorders
  4. 4. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (4) Cole Chromosome 18 is different Markers on chromosome 18 have large effects on several traits: Dystocia and stillbirth: Sire and daughter calving ease and sire stillbirth Conformation: rump width, stature, strength, and body depth Efficiency: longevity and net merit Large calves contribute to reduced lifetimes and decreased profitability
  5. 5. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (5) Cole Marker effects for dystocia complex AR-BFGL-NGS-109285 Cole et al., 2009 (J. Dairy Sci. 92:2931–2946) ARS-BFGL-NGS-109285 Cole et al., 2009 (J. Dairy Sci. 92:2931–2946)
  6. 6. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (6) Cole Correlations in dystocia complex
  7. 7. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (7) Cole The QTL also affects gestation length Maltecca et al. 2011. Animal Genetics, 42:6, 585-591.
  8. 8. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (8) Cole Overview of the dystocia complex The key marker is ARS-BFGL-NGS-109285 at (rs109478645 ) 57,585,121 Mb on BTA18 Intronic to SIGLEC12 (sialic acid binding Ig-like lectin 12) Recent results indicate effects on gestation length (Maltecca et al., 2011) and calf birth weight (Cole et al., unpublished data)
  9. 9. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (9) Cole This is a gene-rich region http://useast.ensembl.org/Bos_taurus/Location/View?r=18%3A57583000-57587000 http://www.ncbi.nlm.nih.gov/gene?cmd=Retrieve&dopt=Graphics&list_uids=618463
  10. 10. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (10) Cole Copy number variants are present ARS-BFGL-NGS-109285 is flanked by CNV There’s a loss and a gain to the left (8 SNP region) There’s a gain to the right (10 SNP region) This can result in assembly problems Hou et al. 2011. Genomic characteristics of cattle copy number variations. BMC Genomics. 12:127.http://www.biomedcentral.com/1471-2164/12/127
  11. 11. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (11) Cole Where did this problem come from? http://aipl.arsusda.gov/CF-queries/Bull_Chromosomal_EBV/bull_chromosomal_ebv.cfm? 40,803 daughters
  12. 12. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (12) Cole What if we look at a different trait? Cole et al. (2007) proposed the following mechanism: SIGLEC12 may sequester circulating leptin This increases gestation length Calf birth weight (BW) is higher because of increased gestation length Higher BW is associated with dystocia
  13. 13. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (13) Cole We don’t have birth weight data Birth weights are not routinely recorded in the US Collaborated with Hermann Swalve’s group to develop a selection index prediction of BW PTA Performed GWAS and gene set enrichment analysis to search for interesting associations
  14. 14. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (14) Cole GWAS for birth weight PTA h Cole et al.(2013), unpublished data
  15. 15. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (15) Cole Are we measuring anything new? Identified a SNP intronic to LHX4, which is associated with cow body weight and length (Ren et al., 2010, Mol. Bio. Reprod., 37:417-422). 4 SNP in the QTL region on BTA 18 had large effects Several other SNP with large effects intronic or adjacent to genes with unknown functions
  16. 16. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (16) Cole KEGG pathways for birth weight What does regulation of the actin cytoskeleton have to do with birth weight in cattle? That is, do these results make sense? Maybe…these pathways may be involved in establishment & maintenance of pregnancy, as well as coordination of growth and development. Cole et al.(2013), unpublished data
  17. 17. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (17) Cole Sequencing is becoming very affordable
  18. 18. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (18) Cole Sequencing successes at AIPL/BFGL Simple loss-of-function mutations APAF1 – Spontaneous abortions in Holstein cattle (Adams et al., 2012) CWC15 – Early embryonic death in Jersey cattle (Sonstegard et al., 2013) Weaver syndrome – Neurological degeneration and death in Brown Swiss cattle (McClure et al., 2013)
  19. 19. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (19) Cole Original pedigree-based design Bull A (1968) AA, SCE: 8 Bull B (1962) AA, SCE: 7 MGS Bull H (1989) Aa, SCE: 14 Bull I (1994) Aa, SCE: 18 Bull E (1982) Aa, SCE: 8 Bull F (1987) Aa, SCE: 15 Bull C (1975) AA, SCE: 8 = 10δBull D (1968) ??, SCE: 7 MGS Bull E (1974) Aa, SCE: 10 MGS
  20. 20. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (20) Cole Modified pedigree & haplotype design Bull A (1968) AA, SCE: 8 Bull B (1962) AA, SCE: 7 MGS Bull H (1989) Aa, SCE: 14 Bull I (1994) Aa, SCE: 18 Bull E (1982) Aa, SCE: 8 Bull F (1987) Aa, SCE: 15 Bull C (1975) AA, SCE: 8 = 10δ Bull E (1974) Aa, SCE: 10 MGS Bull J (2002) Aa, SCE: 6 Bull K (2002) Aa, SCE: 15 Bull J (2002) aa, SCE: 15 These bulls carry the haplotype with the largest, negative effect on SCE: Bull D (1968) ??, SCE: 7 Couldn’t obtain DNA:
  21. 21. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (21) Cole DNA Quality Molecular prep Sample Collection DNA Extraction Library Construction Library Quality Control
  22. 22. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (22) Cole Sample preparation time is substantial DNA Extraction: ~12 hours (30 mins) DNA QC: ~1-2 hours (1-2 hours) Library Construction: 48 hours (12 hours) Library QC: ~2-4 hours (1 hour) Total: 3-4 days (15.5 hours)
  23. 23. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (23) Cole DNA quality
  24. 24. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (24) Cole Library quality
  25. 25. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (25) Cole Sequencing stage • Illumina cBot: • Preps DNA for sequencing • Takes 4-5 hours • Must be done 48 hours before • Illumina HiSeq 2000: • Does the sequencing • Takes ~10-14 days for 100 x 100 • Minimal hands-on time
  26. 26. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (26) Cole Anatomy of a flow cell 8 lanes per flow cell 3 columns per lane − 96 tiles per column Each tile imaged 8 times 1 from upper surface, 1 from lower Approximately 300Gb of sequence per flow cell http://www.qbi.uq.edu.au/images/genomics/genomics1.jpg
  27. 27. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (27) Cole Sequencing by synthesis https://www.broadinstitute.org/files/shared/illuminavids/sequencingSlides.pdf
  28. 28. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (28) Cole How many scientists does it take…
  29. 29. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (29) Cole Flowcell 1: Cluster densities uster densities from current HiSeq run finished 30 April 2013 (unpublished data):
  30. 30. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (30) Cole Flowcell 2: Cluster densities uster densities from current HiSeq run started 22 May 2013 (unpublished data):
  31. 31. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (31) Cole The Aftermath Total Time (sample to sequence): 3 weeks That’s assuming nothing went wrong! More realistic: months Resulting Data Large text files ~300 gigabytes compressed Analysis Often underestimated Can take months as well
  32. 32. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (32) Cole Variant detection • Alignment against a reference genome • Analysis is very disk I/O-intensive. Variant DetectionRaw Sequencer Output Alignment to the Genome
  33. 33. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (33) Cole Computational Logistics Desktop computers Viable for single lanes Long computation time Servers are better >100GB RAM and >16 processor cores Cloud Amazon Web Services iAnimal/iPlant
  34. 34. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (34) Cole Storage considerations What to save? Raw data? Processed results? How much workspace? Suggestions: Workspace 10x compressed files Save alignments Backup REGULARLY!!!
  35. 35. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (35) Cole Why should you use a pipeline? • Automates analysis • Maximizes resource consumption • Because post-docs aren’t cheap
  36. 36. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (36) Cole Galaxy server NextGene Custom pipeline Scripting languages Open-source tools Many options for analysis pipelines
  37. 37. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (37) Cole Challenges Annotation This is a mess in the cow The reference assembly may not be representative of all taurine cows Validation Doing functional genomics with large mammals is expensive – who pays? When have we proven something?
  38. 38. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (38) Cole Conclusions Sequencing is powerful, but presents many challenges Computational requirements are substantial We’re learning how much we don’t know about functional genomics in the cow Validation remains a problem
  39. 39. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (39) Cole Acknowledgments AIPL: Derek Bickhart, Dan Null, Paul VanRaden BFGL: Reuben Anderson, Steve Schroeder, Tad Sonstegard, Curt Van Tassell
  40. 40. Animal Sciences Group, Wageningen UR Livestock Research, The Netherlands, 29 May 2013 (40) Cole Questions? http://gigaom.com/2012/05/31/t-mobile-pits-its-math-against-verizons-the-loser-common-sense/shutterstock_76826245/

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