1. Genetic Map and Forward Genetics Tools for C. briggsae Presented by Dan Koboldt Ray Miller’s Group

2. Outline I. Physical Map of C. briggsae II. Constructing the Initial Genetic Map III. Polymorphism Discovery IV. Assay Development V. The Genetic Map Web Site VI. Resources for C. briggsae as a model organism

3. The Physical Map (cb25) Shotgun sequencing of AF16 strain Shotgun sequencing of AF16 strain 5,341 supercontigs 5,341 supercontigs 578 fingerprint contigs 578 fingerprint contigs Assembly not organized by chromosomes Assembly not organized by chromosomes

4. The Initial SNP Map 1. S. Baird made 2 sets of ~100 Recombinant Inbred lines Cross 1: AF16 X HK104 Cross 2: AF16 X VT847 2. SNPs discovered by shotgun sequencing (GSC). 3. Selected 267 SNPs from the largest contigs in the physical map 4. Genotyped the RILs 5. Assembled the map using data from 248 SNPs.

5. The Draft Genetic Map Genetic Map of Cb4 (R. Miller) The Genetic Map and the Physical Map Coverage as of v3.0: 117 ultra-contigs (~71.5%) of the C. briggsae genome. Several changes made to cb25 genome assembly (R. Waterston and L. Hillier – pers. comm). Homology with C. elegans (Ibid) briggsaeelegans Cb1I Cb2II Cb3III Cb4IV Cb5V CbXX

6. SNP Discovery in HK104 In 13,632 HK104 sequence traces: 7,669 SNPs found by all methods 15,438 SNPs found by two methods Reasons for Disagreement Quality scoring & trimming Repetitive regions

7. HK104 SNPs

8. Improving SNP Discovery Data set may be used to test the next version of Polybayes (G. Marth, pers. comm). Data set may be used to test the next version of Polybayes (G. Marth, pers. comm). New software: Polyphred, Ssaha-SNP, novoSNP New software: Polyphred, Ssaha-SNP, novoSNP High-confidence SNPs will be submitted to Wormbase High-confidence SNPs will be submitted to Wormbase Quality scores and “hits”  quantify the SNP quality Quality scores and “hits”  quantify the SNP quality

9. Structural Polymorphisms SSAHA-DIP for Deletion-Insertion Polys SSAHA found 2,842 indels of >=2 bp 627 long (>= 7bp) HK104 indels identified Two-Step Algorithm for Structural Variants 1. 1. Blast step to identify break-point reads 2. 2. Pair-wise alignment to characterize variant(s).

10. Two-step Applied to 800 Cb reads Breakpoint ReadPredicted VariantCharacterized Variant(s) pjn91h04.g127 bp insertion pjn90e12.b128 bp insertion27 bp insertion pjn90h04.b144 bp insertion45 bp insertion pjn88f02.b170 bp insertion67 bp insertion pjn88e06.g188 bp insertion86 bp insertion pjn89d12.g1101 bp insertion93 bp insertion pjn89f10.b1151 bp insertion pjn91b04.g1247 bp insertion241 bp insertion pjn89c08.b1271 bp insertion pjn89f09.b145 bp deletion48 bp deletion pjn89h09.g151 bp deletion61 bp deletion and 12bp insertion pjn88d09.g1653 bp deletion651 bp deletion pjn91b04.b1953 bp deletion956 bp deletion pjn89d08.b11.3 kb deletion1.267 kb deletion pjn91d10.b1277 bp deletionpossible segmental duplication

11. Forward Genetics Tools SNPs and Indels FP-TDI genotyping assays FP-TDI genotyping assays RFLP assays (T. Harris) RFLP assays (T. Harris) PCR fragment length (PLP) assays PCR fragment length (PLP) assays Array-based technology (S. Baird) Array-based technology (S. Baird) Insertional Mutagenesis Mos1 insertions (M-A. Felix) Mos1 insertions (M-A. Felix)

12. Web Site at http://snp.wustl.edu

14. Genetic Map Online

15. Browse Polymorphisms click!

17. Future Directions Improving/integrating the genetic map More genotyping to improve/resolve coverage More genotyping to improve/resolve coverage Additional HK104 SNP discovery (454 run?) Additional HK104 SNP discovery (454 run?) Developing and sharing resources Identification of snip-SNPs Identification of snip-SNPs Insertion-deletion validation / array design Insertion-deletion validation / array design Detecting structural variants in C. elegans Detecting structural variants in C. elegans

18. Take-Home Messages C. briggsae now has powerful tools to support it as a model organism. C. briggsae now has powerful tools to support it as a model organism. Comparative studies of C. elegans and C. briggsae are becoming feasible and should be considered. Comparative studies of C. elegans and C. briggsae are becoming feasible and should be considered.

19. Acknowledgements C. briggsae Genetic Map Advisory Committee Scott Baird, Helen Chamberlin, Bhagwati Gupta, Eric Haag, and Ray Miller Scott Baird, Helen Chamberlin, Bhagwati Gupta, Eric Haag, and Ray Miller Other Collaborators Marie-Anne Felix, Todd Harris, LaDeana Hillier, Gabor Marth, Bob Waterston Marie-Anne Felix, Todd Harris, LaDeana Hillier, Gabor Marth, Bob Waterston Funding Support NIH NIH

20. Thank You EMBO & I.G.C* Beautiful Country Free Food *Gulbenkian, not Gularenkian