The Drosophila Gene Collection Mark Stapleton Berkeley Drosophila Genome Project Lawrence Berkeley National Lab

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

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) EST / cDNA Project Annotation Experiments

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

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.

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 1 st and 2 nd 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.

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

Rate of diminishing returns for the normalized Riken embryonic cDNA library 1%

SLIP - Self Ligation of Inverse PCR products Summary Attempts3,829 Recovered2,047 Success rate 53% Advantages over RT-PCR Captures 5’ and 3’ UTRs Captures splice variants Extends predictions Hoskins et al., (2005) NAR 33(21):e185 Wan et al., (2006) Nat Proto 1:624

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

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 Total249,471 Libraries and 5’ ESTs Full-length sequenced 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.

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.

Purpose of the DGC