1. Xinbin Dai, Ph. D. Affymetrix Probeset Mapping and Medicago Genome Annotation (Mt4.0 RC1)

2. About Affymetrix Medicago GeneChip Mapping Algorithm and Tool Bioinformatics Resources for Medicago Truncatula Agenda

3. Affymetrix GeneChip Probes 5’ UTR EXON-I EXON-IIEXON-III3’ UTR mRNA Probeset: 11 Probes Target Transcript 25-mer 1255101520 1255101520 Perfect match - PM Mismatch - MM

4. id_at: Designates probe sets that uniquely recognize target transcripts id_a_at: Designates probe sets that recognize alternative transcripts from the same gene. id_s_at: Designates probe sets with common probes among multiple transcripts from different genes. id_x_at: Designates probe sets where it was not possible to select either a unique probe set or a probe set with identical probes among multiple transcripts. Rules for cross-hybridization were dropped in order to design the _x probe sets. These probe sets share some probes identically with two or more sequences and, therefore, these probe sets may cross-hybridize in an unpredictable manner. GeneChip® Expression Analysis Data Analysis Fundamentals. Probeset Types

5. About Medicago GeneChip TypeNum of probe sets Percent in the Mtr. set Notes Unique probe sets: e.g. Mtr.10097.1.S1_at 4418286.80 Unique to one gene Alternative (_a_), e.g.: Mtr.10267.1.S1_a_at 1162.28Alternative probe sets to one gene Shared (_s_), e.g. Mtr.10146.1.S1_s_at 47939.42Common to multiple genes Others (_x_), e.g.: Mtr.10093.1.S1_x_at 18093.55Other probe sets with complicated mapping Total50900100 Reference sequences: early version of IMGAG, DFCI GeneIndex and alfalfa EST

6. Gene transcripts were matched to corresponding Affymetrix probe sets using a position-weighted scoring index in which mismatches near the middle of a probe were most heavily penalized as follows: A perfect match for a probe set yields a score of 45 Matches were declared when at least 8 of 11 probes had scores of 43 or higher. Cutoff for matching: 43x8=344 Mapping Algorithm and Tool 1255101520 [1,1,1,1,1,2,2,2,2,2,3,3,3,3,3,2,2,2,2,2,1,1,1,1,1] Originated from Affymetrix, Inc.

7. AffyProbeMapping: An Online Affymetrix Probeset Mapping Tool http://bioinfo3.noble.org/affymap/ Input sequence: Transcript cDNA EST/Unigene CDS

8. Output of AffyProbeMapping: AffyProbeMapping also supports Affymetrix chips for other species: Lotus Japonica, Arabidopsis thaliana, rice, soybean, maize, populus, cotton and tomato

9. Bioinformatics & Data Resources for Medicago Truncatula Originated from Affymetrix, Inc. Data Sources: Mt3.5v4(2011, version for Nature paper): optical mapping 44,124 BAC-based gene loci + 18,264 illumina (nr) gene model Mt3.5v5(2012, minor changes): 45,859 BAC-based gene loci + 18,264 illumina gene model Mt4 RC1(2013, PAG 2013 conference): anchored illumina contigs onto pseudochromosomes. 84,993 gene loci (BAC+illumina). Chr sequences frozen; some of gene models might be removed. DFCI Gene index Release 11 294k ESTs/ETs  68,814 Unigenes

10. Statistics on Mt3.5v4 vs. Probesets Mapping Results using AffyProbeMapping Num of cDNAMatching probe_setPercent 37,385059.92 18,354129.42 6,649>=210.66 62,388Total100

11. Statistics on Mt4RC1 vs. Probesets Mapping Results using AffyProbeMapping Num of cDNAMatching probe_setPercent 58,660069.02 20,257123.83 6,076>=27.15 84,993Total100

12. Statistics on GeneIndex R11 vs. Probesets Mapping Results using AffyProbeMapping Num of cDNAMatching probe_setPercent 29,722043.2 32,848147.7 6,244>=29.1 68,814Total100

13. Mapping between Medicago genome vs. AffyMedicago Chip http://bioinfo3.noble.org/affymap/Dataset.gy

14. Bioinformatics Tools For Medicago Sequence Search and Annotation –DOBLAST --- http://bioinfo3.noble.org/doblast/, a parallel computing accelerated BLAST search tool Features: o Preload many Medicago data resource o Capable of handling big dataset o “Tab-delimited bioparser output format” works friendly with Excel

15. Bioinformatics Tools For Medicago Sequence Download and Cut by Coordinates. –“Sequence Download” page of DOBLAST --- batch download sequences or cut sequences by Coordinates o Preload many Medicago data resources o Batch download o Get a fragment of sequence by coordinates

16. DOBLAST sequence download page

17. Bioinformatics Tools For Medicago LegumeIP: An Integrative Platform to Study Gene Function and Genome Evolution in Legumes. Features: –Synteny analysis among model legumes –Phylogenetic analysis for gene family –Gene to gene association analysis –Gbrowser o http://plantgrn.noble.org/LegumeIP/ o We are updating to Version 2

18. LegumeIP: Synteny analysis for Medicago genome

19. LegumeIP: Phylogenetic analysis for Medicago gene family

20. LegumeIP: Gene association network analysis for Medicago gene