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Affymetrix Probeset Mapping and Medicago Genome Annotation (Mt4.0 RC1). Xinbin Dai, Ph. D . Agenda. About Affymetrix Medicago GeneChip Mapping Algorithm and Tool Bioinformatics Resources for Medicago Truncatula. Affymetrix GeneChip Probes. mRNA. EXON-I. EXON-II. EXON-III.
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AffymetrixProbeset Mapping and Medicago Genome Annotation (Mt4.0 RC1) Xinbin Dai, Ph. D.
Agenda • About AffymetrixMedicagoGeneChip • Mapping Algorithm and Tool • Bioinformatics Resources for Medicago Truncatula
Affymetrix GeneChip Probes mRNA EXON-I EXON-II EXON-III 3’ UTR 5’ UTR Target Transcript Probeset:11 Probes 25-mer Perfect match - PM 1 5 10 15 20 25 Mismatch - MM 1 5 10 15 20 25
Probeset Types • 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.
About MedicagoGeneChip Reference sequences: early version of IMGAG, DFCI GeneIndex and alfalfa EST
Mapping Algorithm and Tool • 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 [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] 1 5 10 15 20 25 Originated from Affymetrix, Inc.
AffyProbeMapping: An Online AffymetrixProbeset Mapping Tool http://bioinfo3.noble.org/affymap/ • Input sequence: • Transcript • cDNA • EST/Unigene • CDS
Output of AffyProbeMapping: AffyProbeMapping also supports Affymetrix chips for other species: Lotus Japonica, Arabidopsis thaliana, rice, soybean, maize, populus, cotton and tomato
Bioinformatics & Data Resources for Medicago Truncatula Data Sources: • Mt3.5v4(2011, version for Nature paper): optical mapping44,124 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 illuminacontigsonto pseudochromosomes.84,993 gene loci (BAC+illumina). Chr sequences frozen; some of gene models might be removed. • DFCI Gene index Release 11294k ESTs/ETs 68,814 Unigenes Originated from Affymetrix, Inc.
Statistics on Mt3.5v4 vs. Probesets Mapping Results usingAffyProbeMapping
Statistics on Mt4RC1 vs. Probesets Mapping Results usingAffyProbeMapping
Statistics on GeneIndex R11 vs. Probesets Mapping Results usingAffyProbeMapping
Mapping between Medicago genome vs. AffyMedicago Chip http://bioinfo3.noble.org/affymap/Dataset.gy
Bioinformatics Tools For Medicago • Sequence Search and Annotation • DOBLAST --- http://bioinfo3.noble.org/doblast/ , a parallel computing accelerated BLAST search tool • Features: • Preload many Medicagodata resource • Capable of handling big dataset • “Tab-delimited bioparseroutput format” works friendly with Excel
Bioinformatics Tools For Medicago • Sequence Download and Cut by Coordinates. • “Sequence Download” page of DOBLAST --- batch download sequences or cut sequences by Coordinates • Preload many Medicago data resources • Batch download • Get a fragment of sequence by coordinates
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 • http://plantgrn.noble.org/LegumeIP/ • We are updating to Version 2