Sequencing gene rich regions of the Medicago truncatula genome

1. Sequencing gene rich regions of the Medicagotruncatula genome Molecular Breeding of Forage and Turf Noble Foundation May 21, 2003 Dr. Doris Kupfer Advanced Center for Genome Technology Department of Chemistry and Biochemistry University of Oklahoma dkupfer@ou.edu www.genome.ou.edu

2. Why sequence the Medicago genome? • An important forage crop • A genetically tractable model legume • A relatively small (~500 Mbp) diploid genome • Active legume research community • Medicago Research Consortium • Large collection of ESTs • Excellent BAC library • Integrated physical and genetic map

3. Sequence Pipeline at the University of Oklahoma Genome Center, OU-ACGT DNA GenBank Sequencing (ABI 3700) Growing subclones (HiGroTM) Subclone isolation II (VPrepTM) DNA shearing (HydroshearTM) Data assembly and Analysis Thermocycling (ABI 9700) Subclone Isolation I (Mini-StaccatoTM) Colony Piking (QPixIITM) Closure Miscelaneous liquid handling Primer Synthesis

4. Hydroshear • GeneMachines, Inc. San Carlos, CA • Precision-drilled ruby orifice • Pump retraction speed range 0 – 40 • A 100 to 300 ul sample sheared at a retraction speed • setting of 10 produces DNA 1- 4 Kbp fragments

5. Genetix QPixII Colony Picker Digitizes colonies and picks in batches of 96 into 384-well plates Pins are sterilized after each set of 96 colonies are picked

6. Cell Growth in 384 Well Plates in a HiGro • Capacity: 48 shallow, 384 well plates or 24 deep well plates. • Cells are grown in supplemented TB medium • Cells are shaken at 520 rpm for 22 hours at 370C. • After 3.5 hours, oxygen is added @ 0.5 ft3/min for 0.5 second every 30 seconds.

7. 4 built in shakers Robotic 386 well plate loader and stacker 384 tip pipettor Zymark SciClone with Twister II

8. Subclone Isolation (Mini-StaccatoTM) • This Zymark robot has 384 cannula array, four built in shakers, three attached storage racks, built-in barcoding and a Twister II robotic arm. • This automation has allow us to perform the DNA isolation completely unattended from as many as eighty 384 well plates of bacterial cells per day.

9. Subclone Isolation (Mini-StaccatoTM) A series of three isolation solutions are added to each 384 well plates which are shaken on the four autocentering magnetic shakers on the SciClone workspace deck.

10. Subclone Isolation (Mini-StaccatoTM) • Once all three solutions have been added, the plates are transferred from the SciClone workspace deck to a storage rack by the Twister II robotic arm.

11. Subclone Isolation and Sequencing Reaction Pipetting (Velocity 11 VPrep) • Liquid handling station with 384-channel pipettor head • Four movable shelves on either side of the pipettor head • Used for subclone isolation, sequencing reaction set-up and clean-up.

12. Capillary Electrophoresis DNA Sequencing • Our present sequencing capacity with fourteen ABI 3700 capillary electrophoresis DNA sequencers is two 384-well or eight 96-well sample plates per day/machine. (>10,000 samples/day) • DNA sequencing data is transferred to the Sun computer workgroup for base calling (Phred), assembly (Phrap) and analysis (Consed).

13. Data assembly and Analysis Phred/Phrap/Consed Sun V880 server Exgap • 32 GB RAM running Solaris 8 OS and 3 TB of data stored on RAID-5 arrays with autoloader tape backup • Also: • 12 workstations each with 1 GB RAM

14. Primer synthesis (Mermade IV) for PCR-based closure and finishing • Standard phosphoramidite chemistry in an argon- filled reaction chamber. • 192 primers synthesized at 2.5 nmole scale twice each day. • 2.5 nanomole synthesis (50 cents/oligo) typically is used for either PCR or DNA sequencing primers, but can be scaled to 10 nanomole.

15. Medicago truncatula Mapped BAC Approach in collaboration with Doug Cook and DJ Kim at U.C. Davis • Focus is on gene rich euchromatic regions • Initial sequencing of 1000 BACs with known biological markers and covering regions of biological interest as supplied to us by the UC Davis group. • Once the BACs are received, we create the shotgun libraries, isolate the sequencing templates and obtain the working draft sequence followed by closure and finishing.

16. UC Davis -------- Oklahoma University

17. Pachytene FISH From D. Cook, et al (2001)

18. UC Davis BAC Tiling Path

20. May12,2003

26. Blast Homology with M.truncatula ESTs and Arabidopsis thaliana

27. Medicago GC Content for Regions Sequenced to Date

28. Exon Size Distribution (All Sequence Data) (FgenesH vs. Genscan) 12000 10000 FgenesH = 7838 genes Genscan = 6693 genes 8000 Number of Exons 6000 4000 2000 0 1-50 401-500 101-200 51-100 701-800 301-400 501-600 601-700 201-300 801-900 901-1000 Exon Size Range

29. Intron Size Distribution (All Sequence Data) (FgenesH vs. Genscan) 8000 7000 FgenesH = 7838 genes Genscan = 6693 genes 6000 5000 Numberof Introns 4000 3000 2000 1000 0 1-50 51-100 501-600 401-500 701-800 601-700 301-400 201-300 801-900 101-200 901-1000 Intron Size Range

30. Initial Intron/Exon Findings Using FELINEs The FELINEs software was developed by us and OUHSC group to examine conserved elements in introns/exons identified by comparison of ESTs to genomic data.

31. Consensus Logogram of the 5’GU vs the 5’AU Class of Introns in Medicago truncatula determined by FELINES Analysis AU intron consensus GU intron consensus Based on 8862 Introns

32. PrintrepeatAnalysis of M. truncatula BAC AC121240 vs. A. thaliana Chr.2 Expansion, Duplication, Repeat Elements

33. PIP of M. truncatula BAC AC121240 vs. A. thaliana Chr.2

34. Medicago truncatula Summary and Conclusions • At present we have received six (6) sets of 96 well characterized BACs from the UC Davis group. • Of these, all 576 BACs have been isolated, have shotgun libraries constructed, and are being sequenced. • Data for almost all of the first five 96 sets and several of the sixth 96 set of BACs (>61 million bp) have been submitted to GenBank and assigned accession numbers as of May 14, 2003 . • We have scaled up our sequencing and are close to our goal of obtaining a working draft sequence (5-6 fold coverage) of 96 BACs/month.

35. Medicago truncatula Summary and Conclusions • Average Gene Density of 140 to 160 genes per million bp in the euchromatic, gene rich regions of the 8 Medicago truncatula chromosomes based on 55 finished (phase 3) BACs covering ~6.5 Mbp. • Very close to Doug Cook’s 1 gene per 6.5 Kbp. • Genome characteristics such as %GC, intron/exon size and conserved cis sequences reveal Medicago characteristics • The sequence of the Medicago truncatula genome shows homology to the sequenced Arabidopsis thalianagenome but expansion, rearrangements and duplications are evident.

36. Data Release and Preliminary Annotation • All our sequence data is available through links on our web site to GenBank and on our ftp site at URL: ftp.genome.ou.edu/medicago • keyword and blast searches can be done on our web site at URL: http://www.genome.ou.edu/medicago.html • Additional annotation via Genome Browser database are available on our web site at URL: http://www.genome.ou.edu/medicago_table.html • E-mail suggestions for additional annotation to Bruce Roe at: broe@ou.edu

37. Future Plans • Complete working draft sequence of ~1000 mapped Medicago truncatula BACs over the next 6 months with funding from the Noble Foundation • Finish a significant number of these BACs with additional • funding from the DOE • Pending NSF application to: • complete the genomic sequence of unfinished BACs to fewer than one uncertain base in 10,000-The Bermuda rules • and • sequence to working draft and finish an additional ~750 mapped BACs • Obtain the contiguous sequenceof the Gene Rich regions of four of the 8 Medicago truncatula genome at OU, with the remaining four being completed by our international partners at TIGR, Sanger, and Genoscope.

41. Laboratory Organization Bruce Roe, PI Support Teams Reagents & Equip. Maint. Informatics Production DNA Synthesis Administration Jim White Steve Kenton Hongshing Lai Sean Qian Rose Morales-Diaz* Mounir Elharam* Yonas Tesfai Steve Shaull** Doug White Work-study Undergraduates** Phoebe Loh* Sulan Qi Bart Ford* Mounir Elharam* Doug White Kay Lynn Hale Dixie Wishnuck Tami Womack Mary Catherine Williams Research Teams Limei Yang Angie Prescott* Audra Wendt** Mandi Aycock** Doris Kupfer Julia Kim* Sun So Graham Wiley** Lauren Ritterhouse** Axin Hua Weihong Xu Fares Najar Chunmei Qu Keqin Wang Carson Qu Shuling Li ShaoPing Lin Honggui Jia Hongming Wu Baifang Qin Peng Zhang Ziyun Yao Steve Shaull* Youngju Yoon Jami Milam Sara Downard** Trang Do Anh Do Lily Fu Yang Ye James Yu Tessa Manning** Stephan Deschamps Shelly Oommen Christopher Lau Yanhong Li Fu Ying Liping Zhou Ruihua Shi Junjie Wu Pheobe Loh * Sulan Qi Bart Ford* Lin Song Ying Ni Huarong Jiang Funding from the Noble Foundation, DOE, (pending NSF) Collaborators at UC Davis and the Noble Foundation * Previous undergraduate research student ** Present undergraduate research student

42. TheACGTTeam

44. Medicago truncatula Summary and Conclusions • Average Gene Density of 140 to 160 genes per million bp in the euchromatic, gene rich regions of the 8 Medicago truncatula chromosomes based on 55 finished (phase 3) BACs covering ~6.5 Mbp. • Close to Doug’s 1 gene per 6.5 Kbp. • We have observed numerous unique repeated sequences in heterochromatic and euchromatic regions of the Medicago truncatula genome. • The sequence of the Medicago truncatula chloroplast genome is complete and shows a high degree of homology to the sequenced Arabidopsis thalianachloroplast genome.

45. Medicago truncatula chloroplast genome

46. Arabidopsis thalianachloroplast Medicago truncatula chloroplast