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The Drosophila Gene Collection

The Drosophila Gene Collection. Mark Stapleton Berkeley Drosophila Genome Project Lawrence Berkeley National Lab. Poly (A) Signal. Transcription Start. Stop codon. Start codon. Mature protein-coding transcript features. EST / cDNA Project. Generate High Quality cDNA libraries

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The Drosophila Gene Collection

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  1. TheDrosophilaGene Collection Mark Stapleton Berkeley Drosophila Genome Project Lawrence Berkeley National Lab

  2. Poly (A) Signal Transcription Start Stop codon Start codon Mature protein-coding transcript features

  3. EST / cDNA Project Generate High Quality cDNA libraries - head, 0-22hr embryo, larval/pupal, S2 cell line, testes, ovary Random sample end sequence ~ 80k 5’ ESTs (Science: ‘00) ~180k 5’ ESTs (Gen Res: ’02) Clustering, Full-length sequence and analyze - Inter Se and utilizing genome sequence (Gen Res: ’02, Gen Bio: ’02) Experiments Annotation

  4. Poly (A) Signal Transcription Start Stop codon Start codon cDNA library methodology

  5. cDNA library technologies 1) Ling’s libraries (Rubin lab) * From embryo, head, larval/pupal, S2, ovary, and testes. * “Vanilla” libraries using oligo dT primed Stratagene kit. 2) Carninci libraries (RIKEN) * From embryo and head tissues. * Cap-trapped, oligo dT primed, trehalose-stabilized RT. 3) BDGP libraries * From whole adult.

  6. Advantages/disadvantages of each method Ling’s libraries not enriched for full-length, but sampled from many tissues and exist as plasmid libraries. RIKEN libraries were Cap-trapped, but contain many SNPs due to the conditions used for 1st and 2nd strand synthesis. Only as phagemid libraries. RLM method has only one library made so far, holds great promise…. But it has the potential of RNA ligating to incompletely de-PO4 transcripts.

  7. Assessment of new Adult library compared to cap-trapped Riken Head library

  8. Rate of diminishing returns for thenormalized Riken embryonic cDNA library 1%

  9. SLIP - Self Ligation of Inverse PCR products Advantages over RT-PCR • Captures 5’ and 3’ UTRs • Captures splice variants • Extends predictions Summary • Attempts 3,829 • Recovered 2,047 • Success rate 53% Hoskins et al., (2005) NAR 33(21):e185 Wan et al., (2006) Nat Proto 1:624

  10. cDNAs Sequencing Corrects Gene Models Extends gene model at both 5’ and 3’ ends Merges three separate gene models

  11. Full-length sequenced Libraries and 5’ ESTs LD (0-22hr embryo) 35,257 GM (Ovaries) 13,570 HL (Adult head) 3,293 GH (Adult head) 21,059 LP (Mixed larval/pupal) 14,976 SD (S2 cells) 20,154 AT,UT (testes library) 23,294 RE (0-22hr embryo) 61,181 ‏ RH (Adult head) 55,816 TA (Adult) 871 Total 249,471 12,581 from random approach representing 9,423 genes. 3,064 from directed SLIP approach representing 1,813 genes. Represents ~ 75% of the 14,549 predicted genes. ~ Half of the remaining 25% are in process, which leaves ~1,500 genes.

  12. Towards completion of the DGC RACE to define the ends of ORF-short transcripts followed by RT-PCR. Generate cDNA libraries from complex tissues: total disc and total adult. Perform SLIP-directed screens on new libraries.

  13. Purpose of the DGC

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