California Institute of Technology
Supplemental materialsComparative Analysis of the D. melanogaster modENCODE Transcriptome AnnotationZhen-Xia Chen1*, David Sturgill1*, Jiaxin Qu2, Huaiyang Jiang2, Soo Park3, Nathan Boley4, Ana Maria Suzuki5, Anthony R. Fletcher6, David C. Plachetzki7, Peter C. FitzGerald8, Carlo G. Artieri1, Joel Atallah7, Olga Barmina7, James B. Brown4, Kerstin P. Blankenburg2, Emily Clough1, Abhijit Dasgupta9, Sai Gubbala2, Yi Han2, Joy C. Jayaseelan2, Divya Kalra2, Yoo-Ah Kim10, Christie L. Kovar2, Sandra L. Lee2, Mingmei Li2, James D. Malley6, John H. Malone1, Tittu Mathew2, Nicolas R. Mattiuzzo1, Mala Munidasa2, Donna M. Muzny2, Fiona Ongeri2, Lora Perales2, Teresa M. Przytycka10, Ling-Ling Pu2, Garrett Robinson4, Rebecca L. Thornton2, Nehad Saada2, Steven E. Scherer2, Harold E. Smith1, Charles Vinson8, Crystal B. Warner2, Kim C. Worley2, Yuan-Qing Wu2, Xiaoyan Zou2, Peter Cherbas11, Manolis Kellis12, Michael B. Eisen13, Fabio Piano14, Karin Kionte14, David H. Fitch14, Paul W. Sternberg15, Asher D. Cutter16, Michael O. Duff17, Roger A. Hoskins3, Brenton R. Graveley17, Richard A. Gibbs2, Peter J. Bickel4, Artyom Kopp7, Piero Carninci5, Susan E. Celniker3, Brian Oliver1^, and Stephen Richards21National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 50 South Drive, Bethesda MD 20892, 2Human Genome Sequencing Center, Baylor College of Medicine, One Baylor Plaza, Houston TX 77030, 3Department of Genome Dynamics, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley CA 94720, 4Department of Statistics, University of California, Berkeley CA 94720, 5Technology Development Group, RIKEN Omics Science Center, and RIKEN Center for Life Science Technologies, Division of Genomic Technologies Yokohama City, Kanagawa Japan 230-0045, 6Division of Computational Bioscience, Center For Information Technology, National Institutes of Health, 12 South Drive, Bethesda MD 20814, 7Department of Evolution and Ecology, University of California, One Shields Avenue, Davis CA 95616, 8National Cancer Institute, National Institutes of Health, Bethesda MD 20892, 9Clinical Trials and Outcomes Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda MD 20892, 10National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, 20892, 11Department of Biology, Indiana University, 1001 East 3rd Street, Bloomington IN 47405, 12Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge MA 20139, 13Molecular and Cell Biology, University of California, Berkeley CA 94720, 14Department of Biology, New York University, New York, NY 10003, 15HHMI and Division of Biology, California Institute of Technology, Pasadena, California 91125, 16Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Canada, 17Department of Genetics and Developmental Biology, Institute for Systems Genomics, University of Connecticut Health Center, 400 Farmington Avenue, Farmington, Connecticut 06030-6403. * Equal contributors ^Corresponding author.CONTENTSThis file:Methods and referencesTable S1: Sample and assembly information for new Drosophila genomesTable S2: Matrix of patristic distancesTable S3: Summary of RNA-Seq sequencing depth Table S4: Brief view and key to: Sample identifiers and accessionsTable S5: Brief view and key to: First CDS exon RPKM for each sampleTable S6: Brief view and key to: CDS exon validation and evolution Table S7: Brief view and key to: UTR exon validation and evolution Table S8: Brief view and key to: ncRNA exon validation and evolution Table S9: Brief view and key to: Intron validation and evolutionTable S10: Brief view and key to: Intergenic validation and evolutionTable S11: DNA element validation totals and ratesTable S12: Brief view and key to: Promoter summaryTable S13: Brief view and key to: Splice junction validation and evolution Table S14: RNA element validation totals and ratesTable S15: Brief view and key to: Splicing eventsTable S16: Brief view and key to: RNA editing validation and evolutionTable S17: Expression of conserved intergenic regions.File S1-8: Brief view and key to: CAGE peaksSeparate data files:Table S4: Sample identifiers and accessionsTable S5: First CDS exon RPKM for each sampleTable S6: CDS exon validation and evolution Table S7: UTR exon validation and evolution Table S8: ncRNA exon validation and evolution Table S9: Intron validation and evolutionTable S10: Intergenic validation and evolutionTable S12: Promoter summaryTable S13: Splice junction validation and evolution Table S15: Splicing eventsTable S16: RNA editing validation and evolutionFile S1: CAGE peaks in D. melanogaster mixed-sex carcassFile S2: CAGE peaks in D. melanogaster ovaryFile S3: CAGE peaks in D. melanogaster testis, replicate 1 File S4: CAGE peaks in D. melanogaster testis, replicate 2File S5: CAGE peaks in D. pseudoobscura female carcass File S6: CAGE peaks in D. pseudoobscura male carcassFile S7: CAGE peaks in D. pseudoobscura ovaryFile S8: CAGE peaks in D. pseudoobscura testisMethodsGenomesD. biarmipes, D. eugracilis, D. ficusphila, D. takahashii, D. elegans, D. rhopaloa, D. bipectinata, and D. kikkawai were maintained on cornmeal media. Flies for these species were inbred by single pair, full-sib crosses for 10-18 generations (with the exception of D. rhopaloa, which did not tolerate inbreeding). All genome strains have been deposited in the San Diego (USA) and Ehime (Japan) Drosophila species stock centers. We prepared shotgun genomic, 3kb paired-end, and 8kb paired-end libraries for sequencing on a GS FLX Titanium Genome Sequencer (Roche, Inc. Branford, CT). 3 and 8 kb 454 mate pair libraries were prepared according to the manufacturer’s protocol with modifications. 5 ?g (15ug for 14kb) genomic DNA is sheared to 2-4 kb with a Covaris (Covaris, Inc. Woburn, MA) or to 14-18 kb by Hydroshear (Digilab INC, Holliston, MA). 14 kb mate pair fragments were further size selected on a 0.7% agarose gel. The DNA fragments were end-repaired (NEBNext End-Repair Module; Cat. No. E6050L), and LoxP adaptor ligated (NEBNext Quick Ligation Module Cat. No. E6056L). Nicked DNA was repaired by strand displacement with the Bst DNA Polymerase and the DNA fragments were quantitated. 100 ng (300 ng for 8 kb) size-selected fragments were circularized by Cre Recombinase (NEB, Cat No. M0298L), and any remaining linear molecules were removed by DNase/Exonuclease digestion. The circularized DNA fragments were sheared again by the Covaris (Covaris, Inc. Woburn, MA) to an average fragment length of 500 bp. After end repair, fragments containing the biotinylated junction linker from the circularized size-selected fragments were purified using streptavidin-coated magnetic beads (Invitrogen, Carlsbad, CA). These purified fragments were adapter ligated and PCR enriched. The library was size-selected using AMPure size exclusion beads (Beckman Coulter Genomics, Inc.; Cat. No. A63882). These dsDNA amplified molecules were immobilized once more on streptavidin-coated magnetic beads, and single-stranded Paired End DNA library was released by alkaline treatment, then neutralized and cleaned using MinElute PCR purification columns (Qiagen, Valencia, CA). All libraries were checked for quality on an Agilent 2100 Bioanalyzer (Santa Clara, CA) using an RNA Pico 6000 Lab Chip. Library concentrations were determined using a Quant-iT RiboGreen RNA Assay Kit (Life Technologies) and each library diluted to 108 molecules prior to sequencing.A single-stranded 454 sequencing library was used as template for single-molecule emulsion PCR on 28-mm diameter beads. The amplified template beads were recovered after emulsion breaking and selective enrichment. The sequencing primer was annealed to the template and the beads were incubated with Bst DNA polymerase, apyrase and single-stranded binding protein. A slurry of the template beads, enzyme beads (required for signal transduction) and packing beads (for Bst DNA polymerase retention) was loaded into the wells of a picotiter plate, inserted in the flow cell and subjected to pyro-sequencing on the Genome Sequencer XLR Titanium instrument (Roche, Inc. Branford, CT). The XLR/Titanium Genome Sequencer flows 400 cycles of four solutions containing either dTTP, aSdATP, dCTP and dGTP reagents, in that order, over the cell. Each dNTP flow was imaged by a CCD (charge-coupled device) camera on the sequencer, and images were processed in real time to identify template-containing wells and to compute associated signal intensities. The images were further processed for chemical and optical cross-talk, phase errors and read quality before base calling was performed for each template bead. We utilized Illumina technology to correct for any 454 homopolymer errors that may have otherwise been incorporated in the reference genome sequences. High molecular weight double strand genomic DNA samples were constructed into Illumina paired-end libraries according to the manufacturer’s protocol (Illumina Inc., San Diego, CA). Briefly, between 1 and 5 ?g of genomic DNA in 100ul volume was sheared into fragments of approximately 300 base pairs with the Covaris S2 or E210 system (Covaris, Inc. Woburn, MA). The setting was 10% Duty cycle, Intensity of 4,200 Cycles per Burst, for 120 seconds. Fragments were processed through DNA End-Repair in 100 ?l containing sheared DNA, 10 ?l 10X buffer, 5?l End?Repair Enzyme Mix and H2O (NEBNext End-Repair Module; Cat. No. E6050L) at 20°C for 30 minutes; A-tailing was performed in 50 ?l containing End-Repaired DNA, 5 ?l 10X buffer, 3 ?l Klenow Fragment (NEBNext dA-Tailing Module; Cat. No. E6053L) at 37°C for 30 minutes, each step followed by purification using a QIAquick PCR purification kit (Cat. No. 28106). Resulting fragments were ligated with Illumina PE adapters and the NEBNext Quick Ligation Module (Cat. No. E6056L). After ligation, size selection was carried out by using 2% low-melt agarose gel running in 1X TBE. Gel slices were excised from 290bp to 320bp and the size-selected DNA was purified using a Qiagen MinElute gel extraction kit and eluted in 30 ?l EB buffer. PCR with Illumina PE 1.0 and 2.0 primers was performed in 25-μl reactions containing 12.5 ?l of 2x Phusion High-Fidelity PCR master mix, 2.5 ?l size-selected fragment DNA, 0.3 ?l each primer and H2O. The standard thermocycling for PCR was 30 s at 98°C for the initial denaturation followed by 10 cycles of 10 s at 98°C, 30 s at 65°C and 30 s at 72°C and a final extension of 5 min. at 72°C. Agencourt? XP? Beads (Beckman Coulter Genomics, Inc.; Cat. No. A63882) were used to purify the PCR products. After Bead purification, PCR products were quantified using PicoGreen (Cat. No. P7589) and their size distribution analyzed using the Agilent Bioanalyzer 2100 DNA Chip 7500 (Cat. No. 5067-1506). We sequenced 15 ?l of 10 nM final library on Illumina’s Genome Analyzer IIx system according to the manufacturer’s specifications. Briefly, cluster generations were performed on an Illumina cluster station. 36-76 cycles of sequencing were carried out with each library in a separate, single flow cell lane on the Illumina GA II. Sequencing analysis was first done with Illumina analysis pipeline. Sequencing image files were processed to generate base calls and phred-like base quality scores and to remove low-quality reads. We took a 454 based approach to assembling genome sequence for the newly reported species, generating approximately 20X fragment sequence, and 30X “clone” or mate pair coverage in 3 kb and 8 kb mate pairs. These data were assembled with the Celera CABOG assembler (version 6.1, 2010/03/22). Genomic sequence generated on the Illumina platform was then aligned to the draft genome assemblies [release 1 versions of new genomes [e.g. D. biarmipes?(Dbia_1.0)], and used those reads to correct 454 homopolymer errors in the final references [release 2 versions of new genomes [e.g. D. biarmipes?(Dbia_2.0)]. The average contig N50 of the eight assemblies was 206 kb and scaffold N50 of 1.1 Mb. Of the eight assemblies, D. rhopaloa in particular had the worst assembly statistics. This was likely due to the inability of this species to be inbred, and the large number of individuals used for DNA isolation must have resulted in considerable heterozygosity, a known problem in genome assembly PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Kb25lczwvQXV0aG9yPjxZZWFyPjIwMDQ8L1llYXI+PFJl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==
ADDIN EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Kb25lczwvQXV0aG9yPjxZZWFyPjIwMDQ8L1llYXI+PFJl
Y051bT4xMzA8L1JlY051bT48RGlzcGxheVRleHQ+KEpvbmVzIGV0IGFsLiAyMDA0KTwvRGlzcGxh
eVRleHQ+PHJlY29yZD48cmVjLW51bWJlcj4xMzA8L3JlYy1udW1iZXI+PGZvcmVpZ24ta2V5cz48
a2V5IGFwcD0iRU4iIGRiLWlkPSJ3cHBhMHIydjBzZGR6NmU1cHA1djIwMGplcjB0cmFmMng5cGUi
PjEzMDwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYtdHlwZSBuYW1lPSJKb3VybmFsIEFydGljbGUi
PjE3PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxhdXRob3JzPjxhdXRob3I+Sm9uZXMsIFQuPC9h
dXRob3I+PGF1dGhvcj5GZWRlcnNwaWVsLCBOLiBBLjwvYXV0aG9yPjxhdXRob3I+Q2hpYmFuYSwg
SC48L2F1dGhvcj48YXV0aG9yPkR1bmdhbiwgSi48L2F1dGhvcj48YXV0aG9yPkthbG1hbiwgUy48
L2F1dGhvcj48YXV0aG9yPk1hZ2VlLCBCLiBCLjwvYXV0aG9yPjxhdXRob3I+TmV3cG9ydCwgRy48
L2F1dGhvcj48YXV0aG9yPlRob3JzdGVuc29uLCBZLiBSLjwvYXV0aG9yPjxhdXRob3I+QWdhYmlh
biwgTi48L2F1dGhvcj48YXV0aG9yPk1hZ2VlLCBQLiBULjwvYXV0aG9yPjxhdXRob3I+RGF2aXMs
IFIuIFcuPC9hdXRob3I+PGF1dGhvcj5TY2hlcmVyLCBTLjwvYXV0aG9yPjwvYXV0aG9ycz48L2Nv
bnRyaWJ1dG9ycz48YXV0aC1hZGRyZXNzPlN0YW5mb3JkIEdlbm9tZSBUZWNobm9sb2d5IENlbnRl
ciwgUGFsbyBBbHRvLCBDQSA5NDMwNCwgVVNBLjwvYXV0aC1hZGRyZXNzPjx0aXRsZXM+PHRpdGxl
PlRoZSBkaXBsb2lkIGdlbm9tZSBzZXF1ZW5jZSBvZiBDYW5kaWRhIGFsYmljYW5zPC90aXRsZT48
c2Vjb25kYXJ5LXRpdGxlPlByb2MgTmF0bCBBY2FkIFNjaSBVIFMgQTwvc2Vjb25kYXJ5LXRpdGxl
PjxhbHQtdGl0bGU+UHJvY2VlZGluZ3Mgb2YgdGhlIE5hdGlvbmFsIEFjYWRlbXkgb2YgU2NpZW5j
ZXMgb2YgdGhlIFVuaXRlZCBTdGF0ZXMgb2YgQW1lcmljYTwvYWx0LXRpdGxlPjwvdGl0bGVzPjxw
ZXJpb2RpY2FsPjxmdWxsLXRpdGxlPlByb2MgTmF0bCBBY2FkIFNjaSBVIFMgQTwvZnVsbC10aXRs
ZT48YWJici0xPlByb2NlZWRpbmdzIG9mIHRoZSBOYXRpb25hbCBBY2FkZW15IG9mIFNjaWVuY2Vz
IG9mIHRoZSBVbml0ZWQgU3RhdGVzIG9mIEFtZXJpY2E8L2FiYnItMT48L3BlcmlvZGljYWw+PGFs
dC1wZXJpb2RpY2FsPjxmdWxsLXRpdGxlPlByb2MgTmF0bCBBY2FkIFNjaSBVIFMgQTwvZnVsbC10
aXRsZT48YWJici0xPlByb2NlZWRpbmdzIG9mIHRoZSBOYXRpb25hbCBBY2FkZW15IG9mIFNjaWVu
Y2VzIG9mIHRoZSBVbml0ZWQgU3RhdGVzIG9mIEFtZXJpY2E8L2FiYnItMT48L2FsdC1wZXJpb2Rp
Y2FsPjxwYWdlcz43MzI5LTM0PC9wYWdlcz48dm9sdW1lPjEwMTwvdm9sdW1lPjxudW1iZXI+MTk8
L251bWJlcj48ZWRpdGlvbj4yMDA0LzA1LzA1PC9lZGl0aW9uPjxrZXl3b3Jkcz48a2V5d29yZD5D
YW5kaWRhIGFsYmljYW5zLypnZW5ldGljczwva2V5d29yZD48a2V5d29yZD4qRGlwbG9pZHk8L2tl
eXdvcmQ+PGtleXdvcmQ+Kkdlbm9tZSwgRnVuZ2FsPC9rZXl3b3JkPjxrZXl3b3JkPkhldGVyb3p5
Z290ZTwva2V5d29yZD48L2tleXdvcmRzPjxkYXRlcz48eWVhcj4yMDA0PC95ZWFyPjxwdWItZGF0
ZXM+PGRhdGU+TWF5IDExPC9kYXRlPjwvcHViLWRhdGVzPjwvZGF0ZXM+PGlzYm4+MDAyNy04NDI0
IChQcmludCkmI3hEOzAwMjctODQyNCAoTGlua2luZyk8L2lzYm4+PGFjY2Vzc2lvbi1udW0+MTUx
MjM4MTA8L2FjY2Vzc2lvbi1udW0+PHdvcmstdHlwZT5SZXNlYXJjaCBTdXBwb3J0LCBVLlMuIEdv
diZhcG9zO3QsIFAuSC5TLjwvd29yay10eXBlPjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD5odHRw
Oi8vd3d3Lm5jYmkubmxtLm5paC5nb3YvcHVibWVkLzE1MTIzODEwPC91cmw+PC9yZWxhdGVkLXVy
bHM+PC91cmxzPjxjdXN0b20yPjQwOTkxODwvY3VzdG9tMj48ZWxlY3Ryb25pYy1yZXNvdXJjZS1u
dW0+MTAuMTA3My9wbmFzLjA0MDE2NDgxMDE8L2VsZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjxsYW5n
dWFnZT5lbmc8L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+AG==
ADDIN EN.CITE.DATA (Jones et al. 2004). These assemblies are draft genomes and may contain errors, so users should exercise caution when using these data. Typical errors in draft genome sequences include mis-assemblies of repeated sequences, collapses of repeated regions, and unmerged overlaps (e.g. due to polymorphisms) creating artificial duplications. However base accuracy in contigs (contiguous blocks of sequence) is usually very high with most errors near the ends of contigs. Phylogenetic analysisWe conducted phylogenetic analysis of the twelve Drosophila species with previously published genome sequences PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5DbGFyazwvQXV0aG9yPjxZZWFyPjIwMDc8L1llYXI+PFJl
Y051bT43PC9SZWNOdW0+PERpc3BsYXlUZXh0PihDbGFyayBldCBhbC4gMjAwNyk8L0Rpc3BsYXlU
ZXh0PjxyZWNvcmQ+PHJlYy1udW1iZXI+NzwvcmVjLW51bWJlcj48Zm9yZWlnbi1rZXlzPjxrZXkg
YXBwPSJFTiIgZGItaWQ9IndwcGEwcjJ2MHNkZHo2ZTVwcDV2MjAwamVyMHRyYWYyeDlwZSI+Nzwv
a2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYtdHlwZSBuYW1lPSJKb3VybmFsIEFydGljbGUiPjE3PC9y
ZWYtdHlwZT48Y29udHJpYnV0b3JzPjxhdXRob3JzPjxhdXRob3I+Q2xhcmssIEEuIEcuPC9hdXRo
b3I+PGF1dGhvcj5FaXNlbiwgTS4gQi48L2F1dGhvcj48YXV0aG9yPlNtaXRoLCBELiBSLjwvYXV0
aG9yPjxhdXRob3I+QmVyZ21hbiwgQy4gTS48L2F1dGhvcj48YXV0aG9yPk9saXZlciwgQi48L2F1
dGhvcj48YXV0aG9yPk1hcmtvdywgVC4gQS48L2F1dGhvcj48YXV0aG9yPkthdWZtYW4sIFQuIEMu
PC9hdXRob3I+PGF1dGhvcj5LZWxsaXMsIE0uPC9hdXRob3I+PGF1dGhvcj5HZWxiYXJ0LCBXLjwv
YXV0aG9yPjxhdXRob3I+SXllciwgVi4gTi48L2F1dGhvcj48YXV0aG9yPlBvbGxhcmQsIEQuIEEu
PC9hdXRob3I+PGF1dGhvcj5TYWNrdG9uLCBULiBCLjwvYXV0aG9yPjxhdXRob3I+TGFycmFjdWVu
dGUsIEEuIE0uPC9hdXRob3I+PGF1dGhvcj5TaW5naCwgTi4gRC48L2F1dGhvcj48YXV0aG9yPkFi
YWQsIEouIFAuPC9hdXRob3I+PGF1dGhvcj5BYnQsIEQuIE4uPC9hdXRob3I+PGF1dGhvcj5BZHJ5
YW4sIEIuPC9hdXRob3I+PGF1dGhvcj5BZ3VhZGUsIE0uPC9hdXRob3I+PGF1dGhvcj5Ba2FzaGks
IEguPC9hdXRob3I+PGF1dGhvcj5BbmRlcnNvbiwgVy4gVy48L2F1dGhvcj48YXV0aG9yPkFxdWFk
cm8sIEMuIEYuPC9hdXRob3I+PGF1dGhvcj5BcmRlbGwsIEQuIEguPC9hdXRob3I+PGF1dGhvcj5B
cmd1ZWxsbywgUi48L2F1dGhvcj48YXV0aG9yPkFydGllcmksIEMuIEcuPC9hdXRob3I+PGF1dGhv
cj5CYXJiYXNoLCBELiBBLjwvYXV0aG9yPjxhdXRob3I+QmFya2VyLCBELjwvYXV0aG9yPjxhdXRo
b3I+QmFyc2FudGksIFAuPC9hdXRob3I+PGF1dGhvcj5CYXR0ZXJoYW0sIFAuPC9hdXRob3I+PGF1
dGhvcj5CYXR6b2dsb3UsIFMuPC9hdXRob3I+PGF1dGhvcj5CZWd1biwgRC48L2F1dGhvcj48YXV0
aG9yPkJodXRrYXIsIEEuPC9hdXRob3I+PGF1dGhvcj5CbGFuY28sIEUuPC9hdXRob3I+PGF1dGhv
cj5Cb3NhaywgUy4gQS48L2F1dGhvcj48YXV0aG9yPkJyYWRsZXksIFIuIEsuPC9hdXRob3I+PGF1
dGhvcj5CcmFuZCwgQS4gRC48L2F1dGhvcj48YXV0aG9yPkJyZW50LCBNLiBSLjwvYXV0aG9yPjxh
dXRob3I+QnJvb2tzLCBBLiBOLjwvYXV0aG9yPjxhdXRob3I+QnJvd24sIFIuIEguPC9hdXRob3I+
PGF1dGhvcj5CdXRsaW4sIFIuIEsuPC9hdXRob3I+PGF1dGhvcj5DYWdnZXNlLCBDLjwvYXV0aG9y
PjxhdXRob3I+Q2FsdmksIEIuIFIuPC9hdXRob3I+PGF1dGhvcj5CZXJuYXJkbyBkZSBDYXJ2YWxo
bywgQS48L2F1dGhvcj48YXV0aG9yPkNhc3BpLCBBLjwvYXV0aG9yPjxhdXRob3I+Q2FzdHJlemFu
YSwgUy48L2F1dGhvcj48YXV0aG9yPkNlbG5pa2VyLCBTLiBFLjwvYXV0aG9yPjxhdXRob3I+Q2hh
bmcsIEouIEwuPC9hdXRob3I+PGF1dGhvcj5DaGFwcGxlLCBDLjwvYXV0aG9yPjxhdXRob3I+Q2hh
dHRlcmppLCBTLjwvYXV0aG9yPjxhdXRob3I+Q2hpbndhbGxhLCBBLjwvYXV0aG9yPjxhdXRob3I+
Q2l2ZXR0YSwgQS48L2F1dGhvcj48YXV0aG9yPkNsaWZ0b24sIFMuIFcuPC9hdXRob3I+PGF1dGhv
cj5Db21lcm9uLCBKLiBNLjwvYXV0aG9yPjxhdXRob3I+Q29zdGVsbG8sIEouIEMuPC9hdXRob3I+
PGF1dGhvcj5Db3luZSwgSi4gQS48L2F1dGhvcj48YXV0aG9yPkRhdWIsIEouPC9hdXRob3I+PGF1
dGhvcj5EYXZpZCwgUi4gRy48L2F1dGhvcj48YXV0aG9yPkRlbGNoZXIsIEEuIEwuPC9hdXRob3I+
PGF1dGhvcj5EZWxlaGF1bnR5LCBLLjwvYXV0aG9yPjxhdXRob3I+RG8sIEMuIEIuPC9hdXRob3I+
PGF1dGhvcj5FYmxpbmcsIEguPC9hdXRob3I+PGF1dGhvcj5FZHdhcmRzLCBLLjwvYXV0aG9yPjxh
dXRob3I+RWlja2J1c2gsIFQuPC9hdXRob3I+PGF1dGhvcj5FdmFucywgSi4gRC48L2F1dGhvcj48
YXV0aG9yPkZpbGlwc2tpLCBBLjwvYXV0aG9yPjxhdXRob3I+RmluZGVpc3MsIFMuPC9hdXRob3I+
PGF1dGhvcj5GcmV5aHVsdCwgRS48L2F1dGhvcj48YXV0aG9yPkZ1bHRvbiwgTC48L2F1dGhvcj48
YXV0aG9yPkZ1bHRvbiwgUi48L2F1dGhvcj48YXV0aG9yPkdhcmNpYSwgQS4gQy48L2F1dGhvcj48
YXV0aG9yPkdhcmRpbmVyLCBBLjwvYXV0aG9yPjxhdXRob3I+R2FyZmllbGQsIEQuIEEuPC9hdXRo
b3I+PGF1dGhvcj5HYXJ2aW4sIEIuIEUuPC9hdXRob3I+PGF1dGhvcj5HaWJzb24sIEcuPC9hdXRo
b3I+PGF1dGhvcj5HaWxiZXJ0LCBELjwvYXV0aG9yPjxhdXRob3I+R25lcnJlLCBTLjwvYXV0aG9y
PjxhdXRob3I+R29kZnJleSwgSi48L2F1dGhvcj48YXV0aG9yPkdvb2QsIFIuPC9hdXRob3I+PGF1
dGhvcj5Hb3RlYSwgVi48L2F1dGhvcj48YXV0aG9yPkdyYXZlbHksIEIuPC9hdXRob3I+PGF1dGhv
cj5HcmVlbmJlcmcsIEEuIEouPC9hdXRob3I+PGF1dGhvcj5HcmlmZml0aHMtSm9uZXMsIFMuPC9h
dXRob3I+PGF1dGhvcj5Hcm9zcywgUy48L2F1dGhvcj48YXV0aG9yPkd1aWdvLCBSLjwvYXV0aG9y
PjxhdXRob3I+R3VzdGFmc29uLCBFLiBBLjwvYXV0aG9yPjxhdXRob3I+SGFlcnR5LCBXLjwvYXV0
aG9yPjxhdXRob3I+SGFobiwgTS4gVy48L2F1dGhvcj48YXV0aG9yPkhhbGxpZ2FuLCBELiBMLjwv
YXV0aG9yPjxhdXRob3I+SGFscGVybiwgQS4gTC48L2F1dGhvcj48YXV0aG9yPkhhbHRlciwgRy4g
TS48L2F1dGhvcj48YXV0aG9yPkhhbiwgTS4gVi48L2F1dGhvcj48YXV0aG9yPkhlZ2VyLCBBLjwv
YXV0aG9yPjxhdXRob3I+SGlsbGllciwgTC48L2F1dGhvcj48YXV0aG9yPkhpbnJpY2hzLCBBLiBT
LjwvYXV0aG9yPjxhdXRob3I+SG9sbWVzLCBJLjwvYXV0aG9yPjxhdXRob3I+SG9za2lucywgUi4g
QS48L2F1dGhvcj48YXV0aG9yPkh1YmlzeiwgTS4gSi48L2F1dGhvcj48YXV0aG9yPkh1bHRtYXJr
LCBELjwvYXV0aG9yPjxhdXRob3I+SHVudGxleSwgTS4gQS48L2F1dGhvcj48YXV0aG9yPkphZmZl
LCBELiBCLjwvYXV0aG9yPjxhdXRob3I+SmFnYWRlZXNoYW4sIFMuPC9hdXRob3I+PGF1dGhvcj5K
ZWNrLCBXLiBSLjwvYXV0aG9yPjxhdXRob3I+Sm9obnNvbiwgSi48L2F1dGhvcj48YXV0aG9yPkpv
bmVzLCBDLiBELjwvYXV0aG9yPjxhdXRob3I+Sm9yZGFuLCBXLiBDLjwvYXV0aG9yPjxhdXRob3I+
S2FycGVuLCBHLiBILjwvYXV0aG9yPjxhdXRob3I+S2F0YW9rYSwgRS48L2F1dGhvcj48YXV0aG9y
PktlaWdodGxleSwgUC4gRC48L2F1dGhvcj48YXV0aG9yPktoZXJhZHBvdXIsIFAuPC9hdXRob3I+
PGF1dGhvcj5LaXJrbmVzcywgRS4gRi48L2F1dGhvcj48YXV0aG9yPktvZXJpY2gsIEwuIEIuPC9h
dXRob3I+PGF1dGhvcj5LcmlzdGlhbnNlbiwgSy48L2F1dGhvcj48YXV0aG9yPkt1ZHJuYSwgRC48
L2F1dGhvcj48YXV0aG9yPkt1bGF0aGluYWwsIFIuIEouPC9hdXRob3I+PGF1dGhvcj5LdW1hciwg
Uy48L2F1dGhvcj48YXV0aG9yPkt3b2ssIFIuPC9hdXRob3I+PGF1dGhvcj5MYW5kZXIsIEUuPC9h
dXRob3I+PGF1dGhvcj5MYW5nbGV5LCBDLiBILjwvYXV0aG9yPjxhdXRob3I+TGFwb2ludCwgUi48
L2F1dGhvcj48YXV0aG9yPkxhenphcm8sIEIuIFAuPC9hdXRob3I+PGF1dGhvcj5MZWUsIFMuIEou
PC9hdXRob3I+PGF1dGhvcj5MZXZlc3F1ZSwgTC48L2F1dGhvcj48YXV0aG9yPkxpLCBSLjwvYXV0
aG9yPjxhdXRob3I+TGluLCBDLiBGLjwvYXV0aG9yPjxhdXRob3I+TGluLCBNLiBGLjwvYXV0aG9y
PjxhdXRob3I+TGluZGJsYWQtVG9oLCBLLjwvYXV0aG9yPjxhdXRob3I+TGxvcGFydCwgQS48L2F1
dGhvcj48YXV0aG9yPkxvbmcsIE0uPC9hdXRob3I+PGF1dGhvcj5Mb3csIEwuPC9hdXRob3I+PGF1
dGhvcj5Mb3pvdnNreSwgRS48L2F1dGhvcj48YXV0aG9yPkx1LCBKLjwvYXV0aG9yPjxhdXRob3I+
THVvLCBNLjwvYXV0aG9yPjxhdXRob3I+TWFjaGFkbywgQy4gQS48L2F1dGhvcj48YXV0aG9yPk1h
a2Fsb3dza2ksIFcuPC9hdXRob3I+PGF1dGhvcj5NYXJ6bywgTS48L2F1dGhvcj48YXV0aG9yPk1h
dHN1ZGEsIE0uPC9hdXRob3I+PGF1dGhvcj5NYXR6a2luLCBMLjwvYXV0aG9yPjxhdXRob3I+TWNB
bGxpc3RlciwgQi48L2F1dGhvcj48YXV0aG9yPk1jQnJpZGUsIEMuIFMuPC9hdXRob3I+PGF1dGhv
cj5NY0tlcm5hbiwgQi48L2F1dGhvcj48YXV0aG9yPk1jS2VybmFuLCBLLjwvYXV0aG9yPjxhdXRo
b3I+TWVuZGV6LUxhZ28sIE0uPC9hdXRob3I+PGF1dGhvcj5NaW54LCBQLjwvYXV0aG9yPjxhdXRo
b3I+TW9sbGVuaGF1ZXIsIE0uIFUuPC9hdXRob3I+PGF1dGhvcj5Nb250b290aCwgSy48L2F1dGhv
cj48YXV0aG9yPk1vdW50LCBTLiBNLjwvYXV0aG9yPjxhdXRob3I+TXUsIFguPC9hdXRob3I+PGF1
dGhvcj5NeWVycywgRS48L2F1dGhvcj48YXV0aG9yPk5lZ3JlLCBCLjwvYXV0aG9yPjxhdXRob3I+
TmV3ZmVsZCwgUy48L2F1dGhvcj48YXV0aG9yPk5pZWxzZW4sIFIuPC9hdXRob3I+PGF1dGhvcj5O
b29yLCBNLiBBLjwvYXV0aG9yPjxhdXRob3I+TyZhcG9zO0dyYWR5LCBQLjwvYXV0aG9yPjxhdXRo
b3I+UGFjaHRlciwgTC48L2F1dGhvcj48YXV0aG9yPlBhcGFjZWl0LCBNLjwvYXV0aG9yPjxhdXRo
b3I+UGFyaXNpLCBNLiBKLjwvYXV0aG9yPjxhdXRob3I+UGFyaXNpLCBNLjwvYXV0aG9yPjxhdXRo
b3I+UGFydHMsIEwuPC9hdXRob3I+PGF1dGhvcj5QZWRlcnNlbiwgSi4gUy48L2F1dGhvcj48YXV0
aG9yPlBlc29sZSwgRy48L2F1dGhvcj48YXV0aG9yPlBoaWxsaXBweSwgQS4gTS48L2F1dGhvcj48
YXV0aG9yPlBvbnRpbmcsIEMuIFAuPC9hdXRob3I+PGF1dGhvcj5Qb3AsIE0uPC9hdXRob3I+PGF1
dGhvcj5Qb3JjZWxsaSwgRC48L2F1dGhvcj48YXV0aG9yPlBvd2VsbCwgSi4gUi48L2F1dGhvcj48
YXV0aG9yPlByb2hhc2thLCBTLjwvYXV0aG9yPjxhdXRob3I+UHJ1aXR0LCBLLjwvYXV0aG9yPjxh
dXRob3I+UHVpZywgTS48L2F1dGhvcj48YXV0aG9yPlF1ZXNuZXZpbGxlLCBILjwvYXV0aG9yPjxh
dXRob3I+UmFtLCBLLiBSLjwvYXV0aG9yPjxhdXRob3I+UmFuZCwgRC48L2F1dGhvcj48YXV0aG9y
PlJhc211c3NlbiwgTS4gRC48L2F1dGhvcj48YXV0aG9yPlJlZWQsIEwuIEsuPC9hdXRob3I+PGF1
dGhvcj5SZWVuYW4sIFIuPC9hdXRob3I+PGF1dGhvcj5SZWlseSwgQS48L2F1dGhvcj48YXV0aG9y
PlJlbWluZ3RvbiwgSy4gQS48L2F1dGhvcj48YXV0aG9yPlJpZWdlciwgVC4gVC48L2F1dGhvcj48
YXV0aG9yPlJpdGNoaWUsIE0uIEcuPC9hdXRob3I+PGF1dGhvcj5Sb2JpbiwgQy48L2F1dGhvcj48
YXV0aG9yPlJvZ2VycywgWS4gSC48L2F1dGhvcj48YXV0aG9yPlJvaGRlLCBDLjwvYXV0aG9yPjxh
dXRob3I+Um96YXMsIEouPC9hdXRob3I+PGF1dGhvcj5SdWJlbmZpZWxkLCBNLiBKLjwvYXV0aG9y
PjxhdXRob3I+UnVpeiwgQS48L2F1dGhvcj48YXV0aG9yPlJ1c3NvLCBTLjwvYXV0aG9yPjxhdXRo
b3I+U2FsemJlcmcsIFMuIEwuPC9hdXRob3I+PGF1dGhvcj5TYW5jaGV6LUdyYWNpYSwgQS48L2F1
dGhvcj48YXV0aG9yPlNhcmFuZ2EsIEQuIEouPC9hdXRob3I+PGF1dGhvcj5TYXRvLCBILjwvYXV0
aG9yPjxhdXRob3I+U2NoYWVmZmVyLCBTLiBXLjwvYXV0aG9yPjxhdXRob3I+U2NoYXR6LCBNLiBD
LjwvYXV0aG9yPjxhdXRob3I+U2NobGVua2UsIFQuPC9hdXRob3I+PGF1dGhvcj5TY2h3YXJ0eiwg
Ui48L2F1dGhvcj48YXV0aG9yPlNlZ2FycmEsIEMuPC9hdXRob3I+PGF1dGhvcj5TaW5naCwgUi4g
Uy48L2F1dGhvcj48YXV0aG9yPlNpcm90LCBMLjwvYXV0aG9yPjxhdXRob3I+U2lyb3RhLCBNLjwv
YXV0aG9yPjxhdXRob3I+U2lzbmVyb3MsIE4uIEIuPC9hdXRob3I+PGF1dGhvcj5TbWl0aCwgQy4g
RC48L2F1dGhvcj48YXV0aG9yPlNtaXRoLCBULiBGLjwvYXV0aG9yPjxhdXRob3I+U3BpZXRoLCBK
LjwvYXV0aG9yPjxhdXRob3I+U3RhZ2UsIEQuIEUuPC9hdXRob3I+PGF1dGhvcj5TdGFyaywgQS48
L2F1dGhvcj48YXV0aG9yPlN0ZXBoYW4sIFcuPC9hdXRob3I+PGF1dGhvcj5TdHJhdXNiZXJnLCBS
LiBMLjwvYXV0aG9yPjxhdXRob3I+U3RyZW1wZWwsIFMuPC9hdXRob3I+PGF1dGhvcj5TdHVyZ2ls
bCwgRC48L2F1dGhvcj48YXV0aG9yPlN1dHRvbiwgRy48L2F1dGhvcj48YXV0aG9yPlN1dHRvbiwg
Ry4gRy48L2F1dGhvcj48YXV0aG9yPlRhbywgVy48L2F1dGhvcj48YXV0aG9yPlRlaWNobWFubiwg
Uy48L2F1dGhvcj48YXV0aG9yPlRvYmFyaSwgWS4gTi48L2F1dGhvcj48YXV0aG9yPlRvbWltdXJh
LCBZLjwvYXV0aG9yPjxhdXRob3I+VHNvbGFzLCBKLiBNLjwvYXV0aG9yPjxhdXRob3I+VmFsZW50
ZSwgVi4gTC48L2F1dGhvcj48YXV0aG9yPlZlbnRlciwgRS48L2F1dGhvcj48YXV0aG9yPlZlbnRl
ciwgSi4gQy48L2F1dGhvcj48YXV0aG9yPlZpY2FyaW8sIFMuPC9hdXRob3I+PGF1dGhvcj5WaWVp
cmEsIEYuIEcuPC9hdXRob3I+PGF1dGhvcj5WaWxlbGxhLCBBLiBKLjwvYXV0aG9yPjxhdXRob3I+
VmlsbGFzYW50ZSwgQS48L2F1dGhvcj48YXV0aG9yPldhbGVueiwgQi48L2F1dGhvcj48YXV0aG9y
PldhbmcsIEouPC9hdXRob3I+PGF1dGhvcj5XYXNzZXJtYW4sIE0uPC9hdXRob3I+PGF1dGhvcj5X
YXR0cywgVC48L2F1dGhvcj48YXV0aG9yPldpbHNvbiwgRC48L2F1dGhvcj48YXV0aG9yPldpbHNv
biwgUi4gSy48L2F1dGhvcj48YXV0aG9yPldpbmcsIFIuIEEuPC9hdXRob3I+PGF1dGhvcj5Xb2xm
bmVyLCBNLiBGLjwvYXV0aG9yPjxhdXRob3I+V29uZywgQS48L2F1dGhvcj48YXV0aG9yPldvbmcs
IEcuIEsuPC9hdXRob3I+PGF1dGhvcj5XdSwgQy4gSS48L2F1dGhvcj48YXV0aG9yPld1LCBHLjwv
YXV0aG9yPjxhdXRob3I+WWFtYW1vdG8sIEQuPC9hdXRob3I+PGF1dGhvcj5ZYW5nLCBILiBQLjwv
YXV0aG9yPjxhdXRob3I+WWFuZywgUy4gUC48L2F1dGhvcj48YXV0aG9yPllvcmtlLCBKLiBBLjwv
YXV0aG9yPjxhdXRob3I+WW9zaGlkYSwgSy48L2F1dGhvcj48YXV0aG9yPlpkb2Jub3YsIEUuPC9h
dXRob3I+PGF1dGhvcj5aaGFuZywgUC48L2F1dGhvcj48YXV0aG9yPlpoYW5nLCBZLjwvYXV0aG9y
PjxhdXRob3I+WmltaW4sIEEuIFYuPC9hdXRob3I+PGF1dGhvcj5CYWxkd2luLCBKLjwvYXV0aG9y
PjxhdXRob3I+QWJkb3VlbGxlaWwsIEEuPC9hdXRob3I+PGF1dGhvcj5BYmR1bGthZGlyLCBKLjwv
YXV0aG9yPjxhdXRob3I+QWJlYmUsIEEuPC9hdXRob3I+PGF1dGhvcj5BYmVyYSwgQi48L2F1dGhv
cj48YXV0aG9yPkFicmV1LCBKLjwvYXV0aG9yPjxhdXRob3I+QWNlciwgUy4gQy48L2F1dGhvcj48
YXV0aG9yPkFmdHVjaywgTC48L2F1dGhvcj48YXV0aG9yPkFsZXhhbmRlciwgQS48L2F1dGhvcj48
YXV0aG9yPkFuLCBQLjwvYXV0aG9yPjxhdXRob3I+QW5kZXJzb24sIEUuPC9hdXRob3I+PGF1dGhv
cj5BbmRlcnNvbiwgUy48L2F1dGhvcj48YXV0aG9yPkFyYWNoaSwgSC48L2F1dGhvcj48YXV0aG9y
PkF6ZXIsIE0uPC9hdXRob3I+PGF1dGhvcj5CYWNoYW50c2FuZywgUC48L2F1dGhvcj48YXV0aG9y
PkJhcnJ5LCBBLjwvYXV0aG9yPjxhdXRob3I+QmF5dWwsIFQuPC9hdXRob3I+PGF1dGhvcj5CZXJs
aW4sIEEuPC9hdXRob3I+PGF1dGhvcj5CZXNzZXR0ZSwgRC48L2F1dGhvcj48YXV0aG9yPkJsb29t
LCBULjwvYXV0aG9yPjxhdXRob3I+Qmx5ZSwgSi48L2F1dGhvcj48YXV0aG9yPkJvZ3VzbGF2c2tp
eSwgTC48L2F1dGhvcj48YXV0aG9yPkJvbm5ldCwgQy48L2F1dGhvcj48YXV0aG9yPkJvdWtoZ2Fs
dGVyLCBCLjwvYXV0aG9yPjxhdXRob3I+Qm91cnpndWksIEkuPC9hdXRob3I+PGF1dGhvcj5Ccm93
biwgQS48L2F1dGhvcj48YXV0aG9yPkNhaGlsbCwgUC48L2F1dGhvcj48YXV0aG9yPkNoYW5uZXIs
IFMuPC9hdXRob3I+PGF1dGhvcj5DaGVzaGF0c2FuZywgWS48L2F1dGhvcj48YXV0aG9yPkNodWRh
LCBMLjwvYXV0aG9yPjxhdXRob3I+Q2l0cm9lbiwgTS48L2F1dGhvcj48YXV0aG9yPkNvbGx5bW9y
ZSwgQS48L2F1dGhvcj48YXV0aG9yPkNvb2tlLCBQLjwvYXV0aG9yPjxhdXRob3I+Q29zdGVsbG8s
IE0uPC9hdXRob3I+PGF1dGhvcj5EJmFwb3M7QWNvLCBLLjwvYXV0aG9yPjxhdXRob3I+RGF6YSwg
Ui48L2F1dGhvcj48YXV0aG9yPkRlIEhhYW4sIEcuPC9hdXRob3I+PGF1dGhvcj5EZUdyYXksIFMu
PC9hdXRob3I+PGF1dGhvcj5EZU1hc28sIEMuPC9hdXRob3I+PGF1dGhvcj5EaGFyZ2F5LCBOLjwv
YXV0aG9yPjxhdXRob3I+RG9vbGV5LCBLLjwvYXV0aG9yPjxhdXRob3I+RG9vbGV5LCBFLjwvYXV0
aG9yPjxhdXRob3I+RG9yaWNlbnQsIE0uPC9hdXRob3I+PGF1dGhvcj5Eb3JqZSwgUC48L2F1dGhv
cj48YXV0aG9yPkRvcmplZSwgSy48L2F1dGhvcj48YXV0aG9yPkR1cGVzLCBBLjwvYXV0aG9yPjxh
dXRob3I+RWxvbmcsIFIuPC9hdXRob3I+PGF1dGhvcj5GYWxrLCBKLjwvYXV0aG9yPjxhdXRob3I+
RmFyaW5hLCBBLjwvYXV0aG9yPjxhdXRob3I+RmFybywgUy48L2F1dGhvcj48YXV0aG9yPkZlcmd1
c29uLCBELjwvYXV0aG9yPjxhdXRob3I+RmlzaGVyLCBTLjwvYXV0aG9yPjxhdXRob3I+Rm9sZXks
IEMuIEQuPC9hdXRob3I+PGF1dGhvcj5GcmFua2UsIEEuPC9hdXRob3I+PGF1dGhvcj5GcmllZHJp
Y2gsIEQuPC9hdXRob3I+PGF1dGhvcj5HYWRib2lzLCBMLjwvYXV0aG9yPjxhdXRob3I+R2Vhcmlu
LCBHLjwvYXV0aG9yPjxhdXRob3I+R2VhcmluLCBDLiBSLjwvYXV0aG9yPjxhdXRob3I+R2lhbm5v
dWtvcywgRy48L2F1dGhvcj48YXV0aG9yPkdvb2RlLCBULjwvYXV0aG9yPjxhdXRob3I+R3JhaGFt
LCBKLjwvYXV0aG9yPjxhdXRob3I+R3JhbmRib2lzLCBFLjwvYXV0aG9yPjxhdXRob3I+R3Jld2Fs
LCBTLjwvYXV0aG9yPjxhdXRob3I+R3lhbHRzZW4sIEsuPC9hdXRob3I+PGF1dGhvcj5IYWZleiwg
Ti48L2F1dGhvcj48YXV0aG9yPkhhZ29zLCBCLjwvYXV0aG9yPjxhdXRob3I+SGFsbCwgSi48L2F1
dGhvcj48YXV0aG9yPkhlbnNvbiwgQy48L2F1dGhvcj48YXV0aG9yPkhvbGxpbmdlciwgQS48L2F1
dGhvcj48YXV0aG9yPkhvbmFuLCBULjwvYXV0aG9yPjxhdXRob3I+SHVhcmQsIE0uIEQuPC9hdXRo
b3I+PGF1dGhvcj5IdWdoZXMsIEwuPC9hdXRob3I+PGF1dGhvcj5IdXJodWxhLCBCLjwvYXV0aG9y
PjxhdXRob3I+SHVzYnksIE0uIEUuPC9hdXRob3I+PGF1dGhvcj5LYW1hdCwgQS48L2F1dGhvcj48
YXV0aG9yPkthbmdhLCBCLjwvYXV0aG9yPjxhdXRob3I+S2FzaGluLCBTLjwvYXV0aG9yPjxhdXRo
b3I+S2hhemFub3ZpY2gsIEQuPC9hdXRob3I+PGF1dGhvcj5LaXNuZXIsIFAuPC9hdXRob3I+PGF1
dGhvcj5MYW5jZSwgSy48L2F1dGhvcj48YXV0aG9yPkxhcmEsIE0uPC9hdXRob3I+PGF1dGhvcj5M
ZWUsIFcuPC9hdXRob3I+PGF1dGhvcj5MZW5ub24sIE4uPC9hdXRob3I+PGF1dGhvcj5MZXRlbmRy
ZSwgRi48L2F1dGhvcj48YXV0aG9yPkxlVmluZSwgUi48L2F1dGhvcj48YXV0aG9yPkxpcG92c2t5
LCBBLjwvYXV0aG9yPjxhdXRob3I+TGl1LCBYLjwvYXV0aG9yPjxhdXRob3I+TGl1LCBKLjwvYXV0
aG9yPjxhdXRob3I+TGl1LCBTLjwvYXV0aG9yPjxhdXRob3I+TG9reWl0c2FuZywgVC48L2F1dGhv
cj48YXV0aG9yPkxva3lpdHNhbmcsIFkuPC9hdXRob3I+PGF1dGhvcj5MdWJvbmphLCBSLjwvYXV0
aG9yPjxhdXRob3I+THVpLCBBLjwvYXV0aG9yPjxhdXRob3I+TWFjRG9uYWxkLCBQLjwvYXV0aG9y
PjxhdXRob3I+TWFnbmlzYWxpcywgVi48L2F1dGhvcj48YXV0aG9yPk1hcnUsIEsuPC9hdXRob3I+
PGF1dGhvcj5NYXR0aGV3cywgQy48L2F1dGhvcj48YXV0aG9yPk1jQ3Vza2VyLCBXLjwvYXV0aG9y
PjxhdXRob3I+TWNEb25vdWdoLCBTLjwvYXV0aG9yPjxhdXRob3I+TWVodGEsIFQuPC9hdXRob3I+
PGF1dGhvcj5NZWxkcmltLCBKLjwvYXV0aG9yPjxhdXRob3I+TWVuZXVzLCBMLjwvYXV0aG9yPjxh
dXRob3I+TWloYWksIE8uPC9hdXRob3I+PGF1dGhvcj5NaWhhbGV2LCBBLjwvYXV0aG9yPjxhdXRo
b3I+TWlob3ZhLCBULjwvYXV0aG9yPjxhdXRob3I+TWl0dGVsbWFuLCBSLjwvYXV0aG9yPjxhdXRo
b3I+TWxlbmdhLCBWLjwvYXV0aG9yPjxhdXRob3I+TW9udG1heWV1ciwgQS48L2F1dGhvcj48YXV0
aG9yPk11bHJhaW4sIEwuPC9hdXRob3I+PGF1dGhvcj5OYXZpZGksIEEuPC9hdXRob3I+PGF1dGhv
cj5OYXlsb3IsIEouPC9hdXRob3I+PGF1dGhvcj5OZWdhc2gsIFQuPC9hdXRob3I+PGF1dGhvcj5O
Z3V5ZW4sIFQuPC9hdXRob3I+PGF1dGhvcj5OZ3V5ZW4sIE4uPC9hdXRob3I+PGF1dGhvcj5OaWNv
bCwgUi48L2F1dGhvcj48YXV0aG9yPk5vcmJ1LCBDLjwvYXV0aG9yPjxhdXRob3I+Tm9yYnUsIE4u
PC9hdXRob3I+PGF1dGhvcj5Ob3ZvZCwgTi48L2F1dGhvcj48YXV0aG9yPk8mYXBvcztOZWlsbCwg
Qi48L2F1dGhvcj48YXV0aG9yPk9zbWFuLCBTLjwvYXV0aG9yPjxhdXRob3I+TWFya2lld2ljeiwg
RS48L2F1dGhvcj48YXV0aG9yPk95b25vLCBPLiBMLjwvYXV0aG9yPjxhdXRob3I+UGF0dGksIEMu
PC9hdXRob3I+PGF1dGhvcj5QaHVua2hhbmcsIFAuPC9hdXRob3I+PGF1dGhvcj5QaWVycmUsIEYu
PC9hdXRob3I+PGF1dGhvcj5Qcmllc3QsIE0uPC9hdXRob3I+PGF1dGhvcj5SYWdodXJhbWFuLCBT
LjwvYXV0aG9yPjxhdXRob3I+UmVnZSwgRi48L2F1dGhvcj48YXV0aG9yPlJleWVzLCBSLjwvYXV0
aG9yPjxhdXRob3I+UmlzZSwgQy48L2F1dGhvcj48YXV0aG9yPlJvZ292LCBQLjwvYXV0aG9yPjxh
dXRob3I+Um9zcywgSy48L2F1dGhvcj48YXV0aG9yPlJ5YW4sIEUuPC9hdXRob3I+PGF1dGhvcj5T
ZXR0aXBhbGxpLCBTLjwvYXV0aG9yPjxhdXRob3I+U2hlYSwgVC48L2F1dGhvcj48YXV0aG9yPlNo
ZXJwYSwgTi48L2F1dGhvcj48YXV0aG9yPlNoaSwgTC48L2F1dGhvcj48YXV0aG9yPlNoaWgsIEQu
PC9hdXRob3I+PGF1dGhvcj5TcGFycm93LCBULjwvYXV0aG9yPjxhdXRob3I+U3BhdWxkaW5nLCBK
LjwvYXV0aG9yPjxhdXRob3I+U3RhbGtlciwgSi48L2F1dGhvcj48YXV0aG9yPlN0YW5nZS1UaG9t
YW5uLCBOLjwvYXV0aG9yPjxhdXRob3I+U3RhdnJvcG91bG9zLCBTLjwvYXV0aG9yPjxhdXRob3I+
U3RvbmUsIEMuPC9hdXRob3I+PGF1dGhvcj5TdHJhZGVyLCBDLjwvYXV0aG9yPjxhdXRob3I+VGVz
ZmF5ZSwgUy48L2F1dGhvcj48YXV0aG9yPlRob21zb24sIFQuPC9hdXRob3I+PGF1dGhvcj5UaG91
bHV0c2FuZywgWS48L2F1dGhvcj48YXV0aG9yPlRob3VsdXRzYW5nLCBELjwvYXV0aG9yPjxhdXRo
b3I+VG9waGFtLCBLLjwvYXV0aG9yPjxhdXRob3I+VG9wcGluZywgSS48L2F1dGhvcj48YXV0aG9y
PlRzYW1sYSwgVC48L2F1dGhvcj48YXV0aG9yPlZhc3NpbGlldiwgSC48L2F1dGhvcj48YXV0aG9y
PlZvLCBBLjwvYXV0aG9yPjxhdXRob3I+V2FuZ2NodWssIFQuPC9hdXRob3I+PGF1dGhvcj5XYW5n
ZGksIFQuPC9hdXRob3I+PGF1dGhvcj5XZWlhbmQsIE0uPC9hdXRob3I+PGF1dGhvcj5XaWxraW5z
b24sIEouPC9hdXRob3I+PGF1dGhvcj5XaWxzb24sIEEuPC9hdXRob3I+PGF1dGhvcj5ZYWRhdiwg
Uy48L2F1dGhvcj48YXV0aG9yPllvdW5nLCBHLjwvYXV0aG9yPjxhdXRob3I+WXUsIFEuPC9hdXRo
b3I+PGF1dGhvcj5aZW1iZWssIEwuPC9hdXRob3I+PGF1dGhvcj5aaG9uZywgRC48L2F1dGhvcj48
YXV0aG9yPlppbW1lciwgQS48L2F1dGhvcj48YXV0aG9yPlp3aXJrbywgWi48L2F1dGhvcj48YXV0
aG9yPkphZmZlLCBELiBCLjwvYXV0aG9yPjxhdXRob3I+QWx2YXJleiwgUC48L2F1dGhvcj48YXV0
aG9yPkJyb2NrbWFuLCBXLjwvYXV0aG9yPjxhdXRob3I+QnV0bGVyLCBKLjwvYXV0aG9yPjxhdXRo
b3I+Q2hpbiwgQy48L2F1dGhvcj48YXV0aG9yPkduZXJyZSwgUy48L2F1dGhvcj48YXV0aG9yPkdy
YWJoZXJyLCBNLjwvYXV0aG9yPjxhdXRob3I+S2xlYmVyLCBNLjwvYXV0aG9yPjxhdXRob3I+TWF1
Y2VsaSwgRS48L2F1dGhvcj48YXV0aG9yPk1hY0NhbGx1bSwgSS48L2F1dGhvcj48L2F1dGhvcnM+
PC9jb250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5EZXBhcnRtZW50IG9mIE1vbGVjdWxhciBCaW9s
b2d5IGFuZCBHZW5ldGljcywgQ29ybmVsbCBVbml2ZXJzaXR5LCBJdGhhY2EsIE5ldyBZb3JrIDE0
ODUzLCBVU0EuIGFjMzQ3QGNvcm5lbGwuZWR1PC9hdXRoLWFkZHJlc3M+PHRpdGxlcz48dGl0bGU+
RXZvbHV0aW9uIG9mIGdlbmVzIGFuZCBnZW5vbWVzIG9uIHRoZSBEcm9zb3BoaWxhIHBoeWxvZ2Vu
eTwvdGl0bGU+PHNlY29uZGFyeS10aXRsZT5OYXR1cmU8L3NlY29uZGFyeS10aXRsZT48YWx0LXRp
dGxlPk5hdHVyZTwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2RpY2FsPjxmdWxsLXRpdGxlPk5h
dHVyZTwvZnVsbC10aXRsZT48YWJici0xPk5hdHVyZTwvYWJici0xPjwvcGVyaW9kaWNhbD48YWx0
LXBlcmlvZGljYWw+PGZ1bGwtdGl0bGU+TmF0dXJlPC9mdWxsLXRpdGxlPjxhYmJyLTE+TmF0dXJl
PC9hYmJyLTE+PC9hbHQtcGVyaW9kaWNhbD48cGFnZXM+MjAzLTE4PC9wYWdlcz48dm9sdW1lPjQ1
MDwvdm9sdW1lPjxudW1iZXI+NzE2NzwvbnVtYmVyPjxlZGl0aW9uPjIwMDcvMTEvMTM8L2VkaXRp
b24+PGtleXdvcmRzPjxrZXl3b3JkPkFuaW1hbHM8L2tleXdvcmQ+PGtleXdvcmQ+Q29kb24vZ2Vu
ZXRpY3M8L2tleXdvcmQ+PGtleXdvcmQ+RE5BIFRyYW5zcG9zYWJsZSBFbGVtZW50cy9nZW5ldGlj
czwva2V5d29yZD48a2V5d29yZD5Ecm9zb3BoaWxhLypjbGFzc2lmaWNhdGlvbi8qZ2VuZXRpY3Mv
aW1tdW5vbG9neS9tZXRhYm9saXNtPC9rZXl3b3JkPjxrZXl3b3JkPkRyb3NvcGhpbGEgUHJvdGVp
bnMvZ2VuZXRpY3M8L2tleXdvcmQ+PGtleXdvcmQ+KkV2b2x1dGlvbiwgTW9sZWN1bGFyPC9rZXl3
b3JkPjxrZXl3b3JkPkdlbmUgT3JkZXIvZ2VuZXRpY3M8L2tleXdvcmQ+PGtleXdvcmQ+R2VuZXMs
IEluc2VjdC8qZ2VuZXRpY3M8L2tleXdvcmQ+PGtleXdvcmQ+R2Vub21lLCBJbnNlY3QvKmdlbmV0
aWNzPC9rZXl3b3JkPjxrZXl3b3JkPkdlbm9tZSwgTWl0b2Nob25kcmlhbC9nZW5ldGljczwva2V5
d29yZD48a2V5d29yZD4qR2Vub21pY3M8L2tleXdvcmQ+PGtleXdvcmQ+SW1tdW5pdHkvZ2VuZXRp
Y3M8L2tleXdvcmQ+PGtleXdvcmQ+TXVsdGlnZW5lIEZhbWlseS9nZW5ldGljczwva2V5d29yZD48
a2V5d29yZD4qUGh5bG9nZW55PC9rZXl3b3JkPjxrZXl3b3JkPlJOQSwgVW50cmFuc2xhdGVkL2dl
bmV0aWNzPC9rZXl3b3JkPjxrZXl3b3JkPlJlcHJvZHVjdGlvbi9nZW5ldGljczwva2V5d29yZD48
a2V5d29yZD5TZXF1ZW5jZSBBbGlnbm1lbnQ8L2tleXdvcmQ+PGtleXdvcmQ+U2VxdWVuY2UgQW5h
bHlzaXMsIEROQTwva2V5d29yZD48a2V5d29yZD5TeW50ZW55L2dlbmV0aWNzPC9rZXl3b3JkPjwv
a2V5d29yZHM+PGRhdGVzPjx5ZWFyPjIwMDc8L3llYXI+PHB1Yi1kYXRlcz48ZGF0ZT5Ob3YgODwv
ZGF0ZT48L3B1Yi1kYXRlcz48L2RhdGVzPjxpc2JuPjE0NzYtNDY4NyAoRWxlY3Ryb25pYykmI3hE
OzAwMjgtMDgzNiAoTGlua2luZyk8L2lzYm4+PGFjY2Vzc2lvbi1udW0+MTc5OTQwODc8L2FjY2Vz
c2lvbi1udW0+PHdvcmstdHlwZT5Db21wYXJhdGl2ZSBTdHVkeSYjeEQ7UmVzZWFyY2ggU3VwcG9y
dCwgTi5JLkguLCBFeHRyYW11cmFsJiN4RDtSZXNlYXJjaCBTdXBwb3J0LCBOb24tVS5TLiBHb3Ym
YXBvczt0PC93b3JrLXR5cGU+PHVybHM+PHJlbGF0ZWQtdXJscz48dXJsPmh0dHA6Ly93d3cubmNi
aS5ubG0ubmloLmdvdi9wdWJtZWQvMTc5OTQwODc8L3VybD48L3JlbGF0ZWQtdXJscz48L3VybHM+
PGVsZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjEwLjEwMzgvbmF0dXJlMDYzNDE8L2VsZWN0cm9uaWMt
cmVzb3VyY2UtbnVtPjxsYW5ndWFnZT5lbmc8L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48L0Vu
ZE5vdGU+
ADDIN EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5DbGFyazwvQXV0aG9yPjxZZWFyPjIwMDc8L1llYXI+PFJl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ADDIN EN.CITE.DATA (Clark et al. 2007) plus the eight additional species sequenced for this project. We first aligned 250 loci that were included in the orthologous gene set used in a recent drosophilid phylogeny ADDIN EN.CITE <EndNote><Cite><Author>Obbard</Author><Year>2012</Year><RecNum>8</RecNum><DisplayText>(Obbard et al. 2012)</DisplayText><record><rec-number>8</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">8</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Obbard, D. J.</author><author>Maclennan, J.</author><author>Kim, K. W.</author><author>Rambaut, A.</author><author>O'Grady, P. M.</author><author>Jiggins, F. M.</author></authors></contributors><auth-address>Institute of Evolutionary Biology, and Centre for Infection Immunity and Evolution, University of Edinburgh, Edinburgh, United Kingdom.</auth-address><titles><title>Estimating divergence dates and substitution rates in the Drosophila phylogeny</title><secondary-title>Mol Biol Evol</secondary-title><alt-title>Molecular biology and evolution</alt-title></titles><periodical><full-title>Mol Biol Evol</full-title><abbr-1>Molecular biology and evolution</abbr-1></periodical><alt-periodical><full-title>Mol Biol Evol</full-title><abbr-1>Molecular biology and evolution</abbr-1></alt-periodical><pages>3459-73</pages><volume>29</volume><number>11</number><edition>2012/06/12</edition><dates><year>2012</year><pub-dates><date>Nov</date></pub-dates></dates><isbn>1537-1719 (Electronic)
0737-4038 (Linking)</isbn><accession-num>22683811</accession-num><urls><related-urls><url>;(Obbard et al. 2012). Each of these loci was unavailable for one or more of the 20 species. We ranked all loci by the number of available taxa and retained 41 loci that were present for at least 75% of the species. Each locus was then aligned separately with the MUSCLE aligner ADDIN EN.CITE <EndNote><Cite><Author>Edgar</Author><Year>2004</Year><RecNum>2</RecNum><DisplayText>(Edgar 2004)</DisplayText><record><rec-number>2</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">2</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Edgar, R. C.</author></authors></contributors><auth-address>bob@</auth-address><titles><title>MUSCLE: multiple sequence alignment with high accuracy and high throughput</title><secondary-title>Nucleic Acids Res</secondary-title><alt-title>Nucleic acids research</alt-title></titles><periodical><full-title>Nucleic Acids Res</full-title><abbr-1>Nucleic acids research</abbr-1></periodical><alt-periodical><full-title>Nucleic Acids Res</full-title><abbr-1>Nucleic acids research</abbr-1></alt-periodical><pages>1792-7</pages><volume>32</volume><number>5</number><edition>2004/03/23</edition><keywords><keyword>Algorithms</keyword><keyword>Amino Acid Motifs</keyword><keyword>Amino Acid Sequence</keyword><keyword>Internet</keyword><keyword>Molecular Sequence Data</keyword><keyword>Reproducibility of Results</keyword><keyword>Sequence Alignment/*methods</keyword><keyword>Sequence Analysis, Protein/*methods</keyword><keyword>*Software</keyword><keyword>Time Factors</keyword></keywords><dates><year>2004</year></dates><isbn>1362-4962 (Electronic)
0305-1048 (Linking)</isbn><accession-num>15034147</accession-num><work-type>Comparative Study
Evaluation Studies</work-type><urls><related-urls><url>;(Edgar 2004) and gap regions and regions of low alignment score were removed using the “automated” heuristic implemented in trimAL ADDIN EN.CITE <EndNote><Cite><Author>Capella-Gutierrez</Author><Year>2009</Year><RecNum>3</RecNum><DisplayText>(Capella-Gutierrez et al. 2009)</DisplayText><record><rec-number>3</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">3</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Capella-Gutierrez, S.</author><author>Silla-Martinez, J. M.</author><author>Gabaldon, T.</author></authors></contributors><auth-address>Comparative Genomics Group, Bioinformatics and Genomics Programme, Centre for Genomic Regulation, 88 08003 Barcelona, Spain.</auth-address><titles><title>trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses</title><secondary-title>Bioinformatics</secondary-title></titles><periodical><full-title>Bioinformatics</full-title></periodical><pages>1972-3</pages><volume>25</volume><number>15</number><edition>2009/06/10</edition><keywords><keyword>Algorithms</keyword><keyword>Computational Biology/*methods</keyword><keyword>*Phylogeny</keyword><keyword>Sequence Alignment/*methods</keyword><keyword>Sequence Analysis, Protein</keyword><keyword>*Software</keyword><keyword>User-Computer Interface</keyword></keywords><dates><year>2009</year><pub-dates><date>Aug 1</date></pub-dates></dates><isbn>1367-4811 (Electronic)
1367-4803 (Linking)</isbn><accession-num>19505945</accession-num><work-type>Research Support, Non-U.S. Gov't</work-type><urls><related-urls><url>;(Capella-Gutierrez et al. 2009). Individual sequence alignments for each locus were concatenated using in-house perl scripts. Phylogenetic analysis of the total dataset (20 taxa, 41 loci, 63,254 nucleotide positions, ~18% missing data) was conducted using MrBayes 3.2 ADDIN EN.CITE <EndNote><Cite><Author>Ronquist</Author><Year>2012</Year><RecNum>4</RecNum><DisplayText>(Ronquist et al. 2012)</DisplayText><record><rec-number>4</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">4</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Ronquist, F.</author><author>Teslenko, M.</author><author>van der Mark, P.</author><author>Ayres, D. L.</author><author>Darling, A.</author><author>Hohna, S.</author><author>Larget, B.</author><author>Liu, L.</author><author>Suchard, M. A.</author><author>Huelsenbeck, J. P.</author></authors></contributors><auth-address>Department of Biodiversity Informatics, Swedish Museum of Natural History, SE-10405 Stockholm, Sweden. fredrik.ronquist@nrm.se</auth-address><titles><title>MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space</title><secondary-title>Syst Biol</secondary-title><alt-title>Systematic biology</alt-title></titles><periodical><full-title>Syst Biol</full-title><abbr-1>Systematic biology</abbr-1></periodical><alt-periodical><full-title>Syst Biol</full-title><abbr-1>Systematic biology</abbr-1></alt-periodical><pages>539-42</pages><volume>61</volume><number>3</number><edition>2012/02/24</edition><dates><year>2012</year><pub-dates><date>May</date></pub-dates></dates><isbn>1076-836X (Electronic)
1063-5157 (Linking)</isbn><accession-num>22357727</accession-num><work-type>Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.</work-type><urls><related-urls><url>;(Ronquist et al. 2012) under the GTR+I+gamma model (estimated values of model parameters were: r(A<->C): 0.113; r(A<->G): 0.255; r(A<->T): 0.146; r(C<->G): 7.85E-2; r(C<->T): 0.326; r(G<->T): 8.134E-2; pi(A): 0.267; pi(C): 0.241; pi(G): 0.24; pi(T): 0.252; alpha: 1.246; pinvar : 0.294). Two Markov Chain Monte Carlo (MCMC) chains converged to stationarity (standard deviation of split frequencies = 0.000000) within 5x103 moves. Our phylogeny for the 20 Drosophila species is derived from the posterior distribution of topologies and branch lengths from 1x107 MCMC steps. In all subsequent analyses, we used the patristic distance between each pair or species (that is, the combined branch length separating these species on the phylogeny, expressed in substitutions per site (ss) estimated under the empirical nucleotide substitution model detailed above) as the measure of evolutionary divergence between these species. We used a conversion benchmark of 0.016 substitutions per million years ADDIN EN.CITE <EndNote><Cite><Author>Sharp</Author><Year>1989</Year><RecNum>175</RecNum><DisplayText>(Sharp and Li 1989)</DisplayText><record><rec-number>175</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">175</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Sharp, P. M.</author><author>Li, W. H.</author></authors></contributors><auth-address>Department of Genetics, Trinity College, Dublin, Ireland.</auth-address><titles><title>On the rate of DNA sequence evolution in Drosophila</title><secondary-title>J Mol Evol</secondary-title><alt-title>Journal of molecular evolution</alt-title></titles><periodical><full-title>J Mol Evol</full-title><abbr-1>Journal of molecular evolution</abbr-1></periodical><alt-periodical><full-title>J Mol Evol</full-title><abbr-1>Journal of molecular evolution</abbr-1></alt-periodical><pages>398-402</pages><volume>28</volume><number>5</number><edition>1989/05/01</edition><keywords><keyword>Animals</keyword><keyword>*Base Sequence</keyword><keyword>Codon</keyword><keyword>DNA/genetics</keyword><keyword>DNA, Mitochondrial/genetics</keyword><keyword>Drosophila melanogaster/*genetics</keyword><keyword>Genes</keyword><keyword>Mammals/genetics</keyword><keyword>Mutation</keyword><keyword>*Phylogeny</keyword><keyword>Species Specificity</keyword><keyword>Time Factors</keyword></keywords><dates><year>1989</year><pub-dates><date>May</date></pub-dates></dates><isbn>0022-2844 (Print)
0022-2844 (Linking)</isbn><accession-num>2501501</accession-num><work-type>Comparative Study
Research Support, U.S. Gov't, P.H.S.</work-type><urls><related-urls><url>;(Sharp and Li 1989) to estimate ss from divergence times given in million years. To estimate half-life, we used the N0 and λ parameters in the exponential regression line y=N0e-λx for aligned and expressed elements where: y was the percent aligned and expressed for each element type in each non-melanogaster species; x was phylogenetic distance from D. melanogaster; N0 was the percent of aligned and expressed elements in D. melanogaster; and λ was the decay constant of percent aligned and expressed. Half-life was where percent aligned and expressed fell to N0/2.Whole-genome alignments between D. melanogaster and other species were performed using lastz ADDIN EN.CITE <EndNote><Cite><Author>Harris</Author><Year>2007</Year><RecNum>37</RecNum><DisplayText>(Harris 2007)</DisplayText><record><rec-number>37</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">37</key></foreign-keys><ref-type name="Thesis">32</ref-type><contributors><authors><author>Harris, R.</author></authors></contributors><titles><title>Improved pairwise alignment of genomic DNA</title></titles><volume>Ph.D.</volume><dates><year>2007</year></dates><publisher>The Pennsylvania State University</publisher><urls></urls></record></Cite></EndNote>(Harris 2007) according to UCSC Genome Browser manual pages PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5NZXllcjwvQXV0aG9yPjxZZWFyPjIwMTI8L1llYXI+PFJl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 EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5NZXllcjwvQXV0aG9yPjxZZWFyPjIwMTI8L1llYXI+PFJl
Y051bT43MDwvUmVjTnVtPjxEaXNwbGF5VGV4dD4oTWV5ZXIgZXQgYWwuIDIwMTIpPC9EaXNwbGF5
VGV4dD48cmVjb3JkPjxyZWMtbnVtYmVyPjcwPC9yZWMtbnVtYmVyPjxmb3JlaWduLWtleXM+PGtl
eSBhcHA9IkVOIiBkYi1pZD0id3BwYTByMnYwc2RkejZlNXBwNXYyMDBqZXIwdHJhZjJ4OXBlIj43
MDwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYtdHlwZSBuYW1lPSJKb3VybmFsIEFydGljbGUiPjE3
PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxhdXRob3JzPjxhdXRob3I+TWV5ZXIsIEwuIFIuPC9h
dXRob3I+PGF1dGhvcj5ad2VpZywgQS4gUy48L2F1dGhvcj48YXV0aG9yPkhpbnJpY2hzLCBBLiBT
LjwvYXV0aG9yPjxhdXRob3I+S2Fyb2xjaGlrLCBELjwvYXV0aG9yPjxhdXRob3I+S3VobiwgUi4g
TS48L2F1dGhvcj48YXV0aG9yPldvbmcsIE0uPC9hdXRob3I+PGF1dGhvcj5TbG9hbiwgQy4gQS48
L2F1dGhvcj48YXV0aG9yPlJvc2VuYmxvb20sIEsuIFIuPC9hdXRob3I+PGF1dGhvcj5Sb2UsIEcu
PC9hdXRob3I+PGF1dGhvcj5SaGVhZCwgQi48L2F1dGhvcj48YXV0aG9yPlJhbmV5LCBCLiBKLjwv
YXV0aG9yPjxhdXRob3I+UG9obCwgQS48L2F1dGhvcj48YXV0aG9yPk1hbGxhZGksIFYuIFMuPC9h
dXRob3I+PGF1dGhvcj5MaSwgQy4gSC48L2F1dGhvcj48YXV0aG9yPkxlZSwgQi4gVC48L2F1dGhv
cj48YXV0aG9yPkxlYXJuZWQsIEsuPC9hdXRob3I+PGF1dGhvcj5LaXJrdXAsIFYuPC9hdXRob3I+
PGF1dGhvcj5Ic3UsIEYuPC9hdXRob3I+PGF1dGhvcj5IZWl0bmVyLCBTLjwvYXV0aG9yPjxhdXRo
b3I+SGFydGUsIFIuIEEuPC9hdXRob3I+PGF1dGhvcj5IYWV1c3NsZXIsIE0uPC9hdXRob3I+PGF1
dGhvcj5HdXJ1dmFkb28sIEwuPC9hdXRob3I+PGF1dGhvcj5Hb2xkbWFuLCBNLjwvYXV0aG9yPjxh
dXRob3I+R2lhcmRpbmUsIEIuIE0uPC9hdXRob3I+PGF1dGhvcj5GdWppdGEsIFAuIEEuPC9hdXRo
b3I+PGF1dGhvcj5EcmVzemVyLCBULiBSLjwvYXV0aG9yPjxhdXRob3I+RGlla2hhbnMsIE0uPC9h
dXRob3I+PGF1dGhvcj5DbGluZSwgTS4gUy48L2F1dGhvcj48YXV0aG9yPkNsYXdzb24sIEguPC9h
dXRob3I+PGF1dGhvcj5CYXJiZXIsIEcuIFAuPC9hdXRob3I+PGF1dGhvcj5IYXVzc2xlciwgRC48
L2F1dGhvcj48YXV0aG9yPktlbnQsIFcuIEouPC9hdXRob3I+PC9hdXRob3JzPjwvY29udHJpYnV0
b3JzPjxhdXRoLWFkZHJlc3M+Q2VudGVyIGZvciBCaW9tb2xlY3VsYXIgU2NpZW5jZSBhbmQgRW5n
aW5lZXJpbmcsIFNjaG9vbCBvZiBFbmdpbmVlcmluZywgVW5pdmVyc2l0eSBvZiBDYWxpZm9ybmlh
IFNhbnRhIENydXogKFVDU0MpLCBTYW50YSBDcnV6LCBDQSA5NTA2NCwgVVNBLCBDZW50cmUgZm9y
IEdlbm9taWMgUmVndWxhdGlvbiAoQ1JHKSwgQy8gRHIuIEFpZ3VhZGVyLCA4OCwgMDgwMDMgQmFy
Y2Vsb25hLCBTcGFpbiwgQ2VudGVyIGZvciBDb21wYXJhdGl2ZSBHZW5vbWljcyBhbmQgQmlvaW5m
b3JtYXRpY3MsIEh1Y2sgSW5zdGl0dXRlcyBvZiB0aGUgTGlmZSBTY2llbmNlcywgUGVubnN5bHZh
bmlhIFN0YXRlIFVuaXZlcnNpdHksIFVuaXZlcnNpdHkgUGFyaywgUEEgMTY4MDIgYW5kIEhvd2Fy
ZCBIdWdoZXMgTWVkaWNhbCBJbnN0aXR1dGUsIFVDU0MsIFNhbnRhIENydXosIENBIDk1MDY0LCBV
U0EuPC9hdXRoLWFkZHJlc3M+PHRpdGxlcz48dGl0bGU+VGhlIFVDU0MgR2Vub21lIEJyb3dzZXIg
ZGF0YWJhc2U6IGV4dGVuc2lvbnMgYW5kIHVwZGF0ZXMgMjAxMzwvdGl0bGU+PHNlY29uZGFyeS10
aXRsZT5OdWNsZWljIEFjaWRzIFJlczwvc2Vjb25kYXJ5LXRpdGxlPjxhbHQtdGl0bGU+TnVjbGVp
YyBhY2lkcyByZXNlYXJjaDwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2RpY2FsPjxmdWxsLXRp
dGxlPk51Y2xlaWMgQWNpZHMgUmVzPC9mdWxsLXRpdGxlPjxhYmJyLTE+TnVjbGVpYyBhY2lkcyBy
ZXNlYXJjaDwvYWJici0xPjwvcGVyaW9kaWNhbD48YWx0LXBlcmlvZGljYWw+PGZ1bGwtdGl0bGU+
TnVjbGVpYyBBY2lkcyBSZXM8L2Z1bGwtdGl0bGU+PGFiYnItMT5OdWNsZWljIGFjaWRzIHJlc2Vh
cmNoPC9hYmJyLTE+PC9hbHQtcGVyaW9kaWNhbD48ZWRpdGlvbj4yMDEyLzExLzE3PC9lZGl0aW9u
PjxkYXRlcz48eWVhcj4yMDEyPC95ZWFyPjxwdWItZGF0ZXM+PGRhdGU+Tm92IDE1PC9kYXRlPjwv
cHViLWRhdGVzPjwvZGF0ZXM+PGlzYm4+MTM2Mi00OTYyIChFbGVjdHJvbmljKSYjeEQ7MDMwNS0x
MDQ4IChMaW5raW5nKTwvaXNibj48YWNjZXNzaW9uLW51bT4yMzE1NTA2MzwvYWNjZXNzaW9uLW51
bT48dXJscz48cmVsYXRlZC11cmxzPjx1cmw+aHR0cDovL3d3dy5uY2JpLm5sbS5uaWguZ292L3B1
Ym1lZC8yMzE1NTA2MzwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxlY3Ryb25pYy1yZXNv
dXJjZS1udW0+MTAuMTA5My9uYXIvZ2tzMTA0ODwvZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+PGxh
bmd1YWdlPkVuZzwvbGFuZ3VhZ2U+PC9yZWNvcmQ+PC9DaXRlPjwvRW5kTm90ZT4A
ADDIN EN.CITE.DATA (Meyer et al. 2012). Briefly, genomic sequences from each non-melanogaster species were split into 5 MB segments with faSplit (parameters: size -oneFile 5000000 -extra=10000), and pairwise alignment was performed against D. melanogaster with lastz (parameters: --masking=50 --hspthresh=2200 --ydrop=3400 --gappedthresh=4000 --inner=2000). These alignments were converted to Pattern Space Layout (PSL) format and lifted to chromosomes with lavToPsl and liftUp. PSL alignments were chained with axtChain (parameters: -linearGap=medium -psl), combined with chainMergeSort and chainSplit, and converted to alignment nets with the chainNet. Based on alignment nets, liftOver chain files that convert annotations from D. melanogaster to other species were created with the netChainSubset executable.We also used the UCSC genome browser liftover software PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5LZW50PC9BdXRob3I+PFllYXI+MjAwMjwvWWVhcj48UmVj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ADDIN EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5LZW50PC9BdXRob3I+PFllYXI+MjAwMjwvWWVhcj48UmVj
TnVtPjM2PC9SZWNOdW0+PERpc3BsYXlUZXh0PihLZW50IGV0IGFsLiAyMDAyKTwvRGlzcGxheVRl
eHQ+PHJlY29yZD48cmVjLW51bWJlcj4zNjwvcmVjLW51bWJlcj48Zm9yZWlnbi1rZXlzPjxrZXkg
YXBwPSJFTiIgZGItaWQ9IndwcGEwcjJ2MHNkZHo2ZTVwcDV2MjAwamVyMHRyYWYyeDlwZSI+MzY8
L2tleT48L2ZvcmVpZ24ta2V5cz48cmVmLXR5cGUgbmFtZT0iSm91cm5hbCBBcnRpY2xlIj4xNzwv
cmVmLXR5cGU+PGNvbnRyaWJ1dG9ycz48YXV0aG9ycz48YXV0aG9yPktlbnQsIFcuIEouPC9hdXRo
b3I+PGF1dGhvcj5TdWduZXQsIEMuIFcuPC9hdXRob3I+PGF1dGhvcj5GdXJleSwgVC4gUy48L2F1
dGhvcj48YXV0aG9yPlJvc2tpbiwgSy4gTS48L2F1dGhvcj48YXV0aG9yPlByaW5nbGUsIFQuIEgu
PC9hdXRob3I+PGF1dGhvcj5aYWhsZXIsIEEuIE0uPC9hdXRob3I+PGF1dGhvcj5IYXVzc2xlciwg
RC48L2F1dGhvcj48L2F1dGhvcnM+PC9jb250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5EZXBhcnRt
ZW50IG9mIE1vbGVjdWxhciwgQ2VsbHVsYXIsIGFuZCBEZXZlbG9wbWVudGFsIEJpb2xvZ3ksIFVu
aXZlcnNpdHkgb2YgQ2FsaWZvcm5pYSwgU2FudGEgQ3J1eiwgQ0EgOTUwNjQsIFVTQS4ga2VudEBi
aW9sb2d5LnVjc2MuZWR1PC9hdXRoLWFkZHJlc3M+PHRpdGxlcz48dGl0bGU+VGhlIGh1bWFuIGdl
bm9tZSBicm93c2VyIGF0IFVDU0M8L3RpdGxlPjxzZWNvbmRhcnktdGl0bGU+R2Vub21lIFJlczwv
c2Vjb25kYXJ5LXRpdGxlPjxhbHQtdGl0bGU+R2Vub21lIHJlc2VhcmNoPC9hbHQtdGl0bGU+PC90
aXRsZXM+PHBlcmlvZGljYWw+PGZ1bGwtdGl0bGU+R2Vub21lIFJlczwvZnVsbC10aXRsZT48YWJi
ci0xPkdlbm9tZSByZXNlYXJjaDwvYWJici0xPjwvcGVyaW9kaWNhbD48YWx0LXBlcmlvZGljYWw+
PGZ1bGwtdGl0bGU+R2Vub21lIFJlczwvZnVsbC10aXRsZT48YWJici0xPkdlbm9tZSByZXNlYXJj
aDwvYWJici0xPjwvYWx0LXBlcmlvZGljYWw+PHBhZ2VzPjk5Ni0xMDA2PC9wYWdlcz48dm9sdW1l
PjEyPC92b2x1bWU+PG51bWJlcj42PC9udW1iZXI+PGVkaXRpb24+MjAwMi8wNi8wNTwvZWRpdGlv
bj48a2V5d29yZHM+PGtleXdvcmQ+Q2FsaWZvcm5pYTwva2V5d29yZD48a2V5d29yZD4qRGF0YWJh
c2UgTWFuYWdlbWVudCBTeXN0ZW1zPC9rZXl3b3JkPjxrZXl3b3JkPkRhdGFiYXNlcywgR2VuZXRp
Yzwva2V5d29yZD48a2V5d29yZD5HZW5lIEV4cHJlc3Npb248L2tleXdvcmQ+PGtleXdvcmQ+R2Vu
ZXM8L2tleXdvcmQ+PGtleXdvcmQ+Kkdlbm9tZSwgSHVtYW48L2tleXdvcmQ+PGtleXdvcmQ+SHVt
YW5zPC9rZXl3b3JkPjxrZXl3b3JkPlJOQSwgTWVzc2VuZ2VyPC9rZXl3b3JkPjxrZXl3b3JkPlNl
cXVlbmNlIEhvbW9sb2d5LCBOdWNsZWljIEFjaWQ8L2tleXdvcmQ+PGtleXdvcmQ+U29mdHdhcmU8
L2tleXdvcmQ+PGtleXdvcmQ+VW5pdmVyc2l0aWVzL3RyZW5kczwva2V5d29yZD48L2tleXdvcmRz
PjxkYXRlcz48eWVhcj4yMDAyPC95ZWFyPjxwdWItZGF0ZXM+PGRhdGU+SnVuPC9kYXRlPjwvcHVi
LWRhdGVzPjwvZGF0ZXM+PGlzYm4+MTA4OC05MDUxIChQcmludCkmI3hEOzEwODgtOTA1MSAoTGlu
a2luZyk8L2lzYm4+PGFjY2Vzc2lvbi1udW0+MTIwNDUxNTM8L2FjY2Vzc2lvbi1udW0+PHdvcmst
dHlwZT5SZXNlYXJjaCBTdXBwb3J0LCBOb24tVS5TLiBHb3YmYXBvczt0JiN4RDtSZXNlYXJjaCBT
dXBwb3J0LCBVLlMuIEdvdiZhcG9zO3QsIE5vbi1QLkguUy4mI3hEO1Jlc2VhcmNoIFN1cHBvcnQs
IFUuUy4gR292JmFwb3M7dCwgUC5ILlMuPC93b3JrLXR5cGU+PHVybHM+PHJlbGF0ZWQtdXJscz48
dXJsPmh0dHA6Ly93d3cubmNiaS5ubG0ubmloLmdvdi9wdWJtZWQvMTIwNDUxNTM8L3VybD48L3Jl
bGF0ZWQtdXJscz48L3VybHM+PGN1c3RvbTI+MTg2NjA0PC9jdXN0b20yPjxlbGVjdHJvbmljLXJl
c291cmNlLW51bT4xMC4xMTAxL2dyLjIyOTEwMi4gQXJ0aWNsZSBwdWJsaXNoZWQgb25saW5lIGJl
Zm9yZSBwcmludCBpbiBNYXkgMjAwMjwvZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+PGxhbmd1YWdl
PmVuZzwvbGFuZ3VhZ2U+PC9yZWNvcmQ+PC9DaXRlPjwvRW5kTm90ZT5=
ADDIN EN.CITE.DATA (Kent et al. 2002), with alignment output in the form of liftover chain files, to project D. melanogaster annotation coordinates from modENCODE D. melanogaster transcriptome annotation version 2 (MDv2; ) to the reference genome of each other species. FlyBase r5.45 annotation (ftp.) was used as the baseline to identify elements that represent novel annotations. Aligned regions that spanned less than half, or more than double, the range of MDv2 annotated exons in D. melanogaster, including CDS, UTR and ncRNA exons, were removed to exclude low confidence conversions. We also aligned introns and intergenic regions of D. melanogaster with the same criteria as exons. Introns that have overlap with any exons were excluded. Intergenic regions are the complement to all genes, exons and introns. Intergenic regions shorter than RNA-Seq reads (75 nt) were excluded. We lifted-over a 20bp region around the splice junction, and required presence and correct positioning of the donor and acceptor motifs in the test species as present in D. melanogaster. For A-I editing site alignments, we lifted over the single base position that is edited in D. melanogaster, and required the base of the aligned region in the query genome to be “A” or “T”. ExonsAll RNA-seq protocols are found in the GEO entries. We analyzed all exons defined in the MDv2 annotation. For exons including the translation initiator or transcription termination site, the UTR segments were defined as “UTR exons” and the coding segments were grouped with “CDS exons.” Gene models with no coding regions were defined as “ncRNA exons”. Introns and intergenic regions show no overlap with transcript exons.All expression and comparative analysis was performed from the same set of alignments. To obtain alignments, RNA-Seq reads were trimmed to 75nt using shell scripts with the cut command. Trimmed reads were uniquely-mapped (-g 1 -r 150 --solexa1.3-quals) to respective genomes using TopHat 2 (v2.0.3) PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5UcmFwbmVsbDwvQXV0aG9yPjxZZWFyPjIwMDk8L1llYXI+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ADDIN EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5UcmFwbmVsbDwvQXV0aG9yPjxZZWFyPjIwMDk8L1llYXI+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ADDIN EN.CITE.DATA (Trapnell et al. 2009; Trapnell et al. 2012). We estimated expression of aligned elements in each sample (See Table S4 for identifiers) by quantifying reads that map within the relevant regions, and intersecting coordinates with RNA-Seq read alignments using the coverageBed (v2.7.1) command in BedTools ADDIN EN.CITE <EndNote><Cite><Author>Quinlan</Author><Year>2010</Year><RecNum>12</RecNum><DisplayText>(Quinlan and Hall 2010)</DisplayText><record><rec-number>12</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">12</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Quinlan, A. R.</author><author>Hall, I. M.</author></authors></contributors><auth-address>Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. aaronquinlan@</auth-address><titles><title>BEDTools: a flexible suite of utilities for comparing genomic features</title><secondary-title>Bioinformatics</secondary-title></titles><periodical><full-title>Bioinformatics</full-title></periodical><pages>841-2</pages><volume>26</volume><number>6</number><edition>2010/01/30</edition><keywords><keyword>Genome</keyword><keyword>Genomics/*methods</keyword><keyword>Internet</keyword><keyword>*Software</keyword></keywords><dates><year>2010</year><pub-dates><date>Mar 15</date></pub-dates></dates><isbn>1367-4811 (Electronic)
1367-4803 (Linking)</isbn><accession-num>20110278</accession-num><work-type>Comparative Study
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't</work-type><urls><related-urls><url>;(Quinlan and Hall 2010). Raw coverage results were used for validation. We used read coverage as a criterion of validation of exons. Specifically, any exon was validated in a species if 95% of all the bases of its aligned region on coverageBed output using custom shell scripts had at least 1x coverage in at least one non D. melanogaster sample (Tables S6-S8). We applied the same criterion to introns and intergenic regions (Table S9-S10). Intergenic regions that had overlap with CAGE sites and K27Ac3 modifications were extracted by bedtools intersect. H3K27Ac annotation is based on E0-4_H3K27Ac-Set2_merged_dcc.gff from (accessible through DCC ID: modENCODE 970) PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5OZWdyZTwvQXV0aG9yPjxZZWFyPjIwMTE8L1llYXI+PFJl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ADDIN EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5OZWdyZTwvQXV0aG9yPjxZZWFyPjIwMTE8L1llYXI+PFJl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ADDIN EN.CITE.DATA (Negre et al. 2011). For abundance comparisons between samples and between species, normalized expression measures in the form of reads per kilobase per million mapped reads (RPKM) were generated by quantifying reads mapped within the exon, dividing by exon length, and dividing by the total number of mapped reads in millions. The non-redundant set of first coding exons in D. melanogaster annotation were sometimes used to minimize any bias in expression measurement due to incomplete gene models as indicated. Transcript profiles for all samples are provided in Table S5. Clustering of expression values was performed with the heatmap.2 command in the gplots package ADDIN EN.CITE <EndNote><Cite><Author>Bolker</Author><Year>2012</Year><RecNum>128</RecNum><DisplayText>(Bolker et al. 2012)</DisplayText><record><rec-number>128</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">128</key></foreign-keys><ref-type name="Artwork">2</ref-type><contributors><authors><author>Bolker, Ben</author><author>Warnes, Gregory R.</author><author>Bonebakker, Lodewijk</author><author>Gentleman, Robert</author><author>Liaw, Wolfgang Huber Andy</author><author>Lumley, Thomas</author><author>Maechler, Martin</author><author>Magnusson, Arni</author><author>Moeller, Steffen</author><author>Schwartz, Marc</author><author>Venables, Bill</author></authors></contributors><auth-address>: Various R programming tools for plotting data</title></titles><dates><year>2012</year></dates><urls></urls></record></Cite></EndNote>(Bolker et al. 2012), which performs hierarchical clustering via the hclust command. For samples with multiple replicates, the first replicate is presented, although using either replicate produced identical clustering.To determine an RPKM threshold for detection above background, we quantified expression in intergenic space to estimate a cutoff RPKM (CR). We took p as the probability that a nonfunctional element is aligned to, and expressed in, a certain sample from a non-melanogaster species. Thus, the probability, which is also false positive rate, that a nonfunctional element is aligned to and expressed in any of the 75 samples is PN=1-(1-p)75. To avoid 95% false positives (PN<0.05), no more than 0.068% (p <0.068%) of nonfunctional elements may have expression over CR. We split intergenic regions into 200 nt bins with 100 nt overlap to normalize for exon length (median = 201 nt). We defined “nonfunctional” intergenic regions as those that were aligned to, but not expressed (> 95% element coverage) in any non-melanogaster sample (N = 190,232 intergenic bins). These intergenic bins may have 75N aligned regions in non-melanogaster samples at most, while 9,228,499 (64.682%) aligned regions were not found. If nonfunctional elements are expressed randomly, their aligned regions should have a similar distribution of RPKM in non-melanogaster samples as in D. melanogaster samples. Therefore, we estimated CR based on the expression of nonfunctional regions in D. melanogaster samples. We investigated distribution of RPKM of intergenic bins in 6 D. melanogaster samples, and set CR = 1.47, as only 0.193% (0.068%/(1-64.682%)) of them have RPKM > 1.47. Gene-level expressionTo analyze gene expression within D. melanogaster, we used the calculated abundance estimates from complete MDv2 gene models with Cufflinks PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5UcmFwbmVsbDwvQXV0aG9yPjxZZWFyPjIwMTI8L1llYXI+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==
ADDIN EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5UcmFwbmVsbDwvQXV0aG9yPjxZZWFyPjIwMTI8L1llYXI+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==
ADDIN EN.CITE.DATA (Trapnell et al. 2012). We used the Cuffdiff command (version 2.0.2), supplying the MDv2 annotation and alignment files for D. melanogaster adults, with females as sample 1 and males as sample 2. We used upper quartile normalization and required Cuffdiff to report values for high abundance features (parameters -N --max-bundle-frags 2000000). TSSTotal RNA for D. melanogaster CAGE-Seq from dissected testis and ovary of D. melanogaster was prepared as previously described PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Ib3NraW5zPC9BdXRob3I+PFllYXI+MjAxMTwvWWVhcj48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==
ADDIN EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Ib3NraW5zPC9BdXRob3I+PFllYXI+MjAxMTwvWWVhcj48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==
ADDIN EN.CITE.DATA (Hoskins et al. 2011). RNA samples for D. pseudoobscura for CAGE experiments were the same as those used in RNA-Seq. CAGE libraries were prepared for sequencing on the Illumina platform as described ADDIN EN.CITE <EndNote><Cite><Author>Takahashi</Author><Year>2012</Year><RecNum>62</RecNum><DisplayText>(Takahashi et al. 2012)</DisplayText><record><rec-number>62</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">62</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Takahashi, H.</author><author>Lassmann, T.</author><author>Murata, M.</author><author>Carninci, P.</author></authors></contributors><auth-address>RIKEN Omics Science Center, RIKEN Yokohama Institute, Yokohama, Japan.</auth-address><titles><title>5' end-centered expression profiling using cap-analysis gene expression and next-generation sequencing</title><secondary-title>Nat Protoc</secondary-title><alt-title>Nature protocols</alt-title></titles><periodical><full-title>Nat Protoc</full-title><abbr-1>Nature protocols</abbr-1></periodical><alt-periodical><full-title>Nat Protoc</full-title><abbr-1>Nature protocols</abbr-1></alt-periodical><pages>542-61</pages><volume>7</volume><number>3</number><edition>2012/03/01</edition><keywords><keyword>Gene Expression Profiling/*methods</keyword><keyword>*Gene Library</keyword><keyword>Genomics/*methods</keyword><keyword>RNA Caps/genetics/*metabolism</keyword><keyword>Sequence Analysis, DNA/*methods</keyword></keywords><dates><year>2012</year><pub-dates><date>Mar</date></pub-dates></dates><isbn>1750-2799 (Electronic)
1750-2799 (Linking)</isbn><accession-num>22362160</accession-num><work-type>Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't</work-type><urls><related-urls><url>;(Takahashi et al. 2012) and sequenced (Illumina GAIIx) to generate 36 nt reads. Barcode sequence was trimmed and the 27 nt CAGE reads were aligned to the D. pseudoobscura genome using StatMap () and represented as a 1 bp CAGE site. We retained CAGE sites with >1 tag per million (TPM). To compare orthologous TSSs, we used the translation start sites of 1:1 orthologs of D. melanogaster and D. pseudoobscura from OrthoDB version 6 ADDIN EN.CITE <EndNote><Cite><Author>Waterhouse</Author><Year>2013</Year><RecNum>129</RecNum><DisplayText>(Waterhouse et al. 2013)</DisplayText><record><rec-number>129</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">129</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Waterhouse, R. M.</author><author>Tegenfeldt, F.</author><author>Li, J.</author><author>Zdobnov, E. M.</author><author>Kriventseva, E. V.</author></authors></contributors><auth-address>Department of Genetic Medicine and Development, University of Geneva Medical School, Swiss Institute of Bioinformatics, rue Michel-Servet 1, 1211 Geneva, Switzerland and Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.</auth-address><titles><title>OrthoDB: a hierarchical catalog of animal, fungal and bacterial orthologs</title><secondary-title>Nucleic Acids Res</secondary-title><alt-title>Nucleic acids research</alt-title></titles><periodical><full-title>Nucleic Acids Res</full-title><abbr-1>Nucleic acids research</abbr-1></periodical><alt-periodical><full-title>Nucleic Acids Res</full-title><abbr-1>Nucleic acids research</abbr-1></alt-periodical><pages>D358-65</pages><volume>41</volume><number>D1</number><edition>2012/11/28</edition><dates><year>2013</year><pub-dates><date>Jan 1</date></pub-dates></dates><isbn>1362-4962 (Electronic)
0305-1048 (Linking)</isbn><accession-num>23180791</accession-num><urls></urls><electronic-resource-num>10.1093/nar/gks1116</electronic-resource-num><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>(Waterhouse et al. 2013) (parsed from ). We associated CAGE sites with the nearest downstream gene AUG codon within 5kb because >90% of previously annotated D. melanogaster TSSs were within 5kb of the nearest AUG. For motif analysis we selected the CAGE site with the greatest tag frequency for each gene (File S1-File S8). We used Random Forests (RF) PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5CcmVpbWFuPC9BdXRob3I+PFllYXI+MjAwMTwvWWVhcj48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ADDIN EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5CcmVpbWFuPC9BdXRob3I+PFllYXI+MjAwMTwvWWVhcj48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ADDIN EN.CITE.DATA (Breiman 2001; Malley et al. 2011; Malley et al. 2012), Additional details on RF can be found at (Leo Breiman and Adele Cutler). seqLogo v.1.2 ADDIN EN.CITE <EndNote><Cite><Author>Bembom</Author><Year>2012</Year><RecNum>127</RecNum><DisplayText>(Bembom 2012)</DisplayText><record><rec-number>127</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">127</key></foreign-keys><ref-type name="Computer Program">9</ref-type><contributors><authors><author>Bembom, Oliver</author></authors></contributors><titles><title>seqLogo: Sequence logos for DNA sequence alignments</title></titles><dates><year>2012</year></dates><urls></urls></record></Cite></EndNote>(Bembom 2012) and K-means clustering to examine regions flanking CAGE sites for motifs. We used the selected CAGE sites upstream of orthologs as true sites for probability machines and CAGE sites in the last exons of genes (regardless of TPM) were taken as nulls. RF was applied to each sample type separately. All machines were trained on bootstrap draws and data sent to the machines was pre-balanced (100 resamples to train 100 RF machines, each with 1,000 trees). The "features" used as input to RF were tetramers, situated at each position of the 500nt segment (256 different tetramers per position). RF used tetramers and position separately to make splits at the nodes in its tree building. The position importance ranking from RF was stable across all samples and centered on the adjusted CAGE peak +1 position. Across all the trees in any forest, those terminal nodes where a declaration was made for each training instance of 80% or more for "true" was considered predictive. Analysis was performed in R with the randomForest package ADDIN EN.CITE <EndNote><Cite><Author>Liaw</Author><Year>2002</Year><RecNum>133</RecNum><DisplayText>(Liaw and Wiener 2002)</DisplayText><record><rec-number>133</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">133</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Liaw, Andy</author><author>Wiener, Matthew</author></authors></contributors><auth-address> and Regression by randomForest</title><secondary-title>R News</secondary-title></titles><periodical><full-title>R News</full-title></periodical><pages>18-22</pages><volume>2</volume><number>3</number><dates><year>2002</year></dates><urls></urls></record></Cite></EndNote>(Liaw and Wiener 2002). RNA elementsQuantification of splice junction coverage, and other splicing analysis described below, was performed with the Splicing Analysis Toolkit (Spanki) v.0.4.0 ( and ). Briefly, this program analyzes splicing at the junction level, by calculating read coverage over splice junctions and over exon-intron boundaries. Pairwise splicing events are defined from annotation using the AStalavista tool PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TYW1tZXRoPC9BdXRob3I+PFllYXI+MjAwODwvWWVhcj48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 EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TYW1tZXRoPC9BdXRob3I+PFllYXI+MjAwODwvWWVhcj48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 EN.CITE.DATA (Sammeth et al. 2008), parsed into their component splice junctions, and quantified using junction coverage. Coverage over junctions and estimates for intron retention, based on reads that span the exon-intron boundary, was performed with the spankijunc utility, using RNA-Seq alignment files as input. Qualitative analysis of splice junctions, including identification of donor/acceptor motifs, was performed with the annotate_junctions utility with transcript model annotation as input. Alternative splicing was quantified from junction coverage using the Percent Spliced In (PSI) metric, defined as the junction coverage of the inclusion form divided by the sum of the junction coverage of the inclusion and exclusion forms, normalizing each value by the number of sites. Calculations of PSI in non-melanogaster species were made using orthologous junctions identfied by alignment. For individual junctions, our minimum criterion for validation is one junction-spanning read in any non-melanogaster RNA-Seq sample. Validation results for splice junctions in each species are given in Table S13.For the validation of aligned editing sites, we extracted the base calling at the aligned editing sites with the mpileup command in samtools (v.0.1.18) ADDIN EN.CITE <EndNote><Cite><Author>Li</Author><Year>2009</Year><RecNum>132</RecNum><DisplayText>(Li et al. 2009)</DisplayText><record><rec-number>132</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">132</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Li, H.</author><author>Handsaker, B.</author><author>Wysoker, A.</author><author>Fennell, T.</author><author>Ruan, J.</author><author>Homer, N.</author><author>Marth, G.</author><author>Abecasis, G.</author><author>Durbin, R.</author></authors></contributors><auth-address>Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, CB10 1SA, UK, Broad Institute of MIT and Harvard, Cambridge, MA 02141, USA.</auth-address><titles><title>The Sequence Alignment/Map format and SAMtools</title><secondary-title>Bioinformatics</secondary-title></titles><periodical><full-title>Bioinformatics</full-title></periodical><pages>2078-9</pages><volume>25</volume><number>16</number><edition>2009/06/10</edition><keywords><keyword>Algorithms</keyword><keyword>Base Sequence</keyword><keyword>Computational Biology/*methods</keyword><keyword>Genome</keyword><keyword>Genomics</keyword><keyword>Molecular Sequence Data</keyword><keyword>Sequence Alignment/*methods</keyword><keyword>Sequence Analysis, DNA/*methods</keyword><keyword>*Software</keyword></keywords><dates><year>2009</year><pub-dates><date>Aug 15</date></pub-dates></dates><isbn>1367-4811 (Electronic)
1367-4803 (Linking)</isbn><accession-num>19505943</accession-num><work-type>Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't</work-type><urls><related-urls><url>;(Li et al. 2009), and compared them with the reference bases. Reads where base calling of the site is “G” and reference is “A”, or base calling of the site is “C” and reference is “T” were taken as evidence of editing, base calling that is the same as reference bases were taken as reference match, and other reads were excluded. We required at least two mapped reads, accounting for at least 5% of mapped reads at the aligned site, in at least one sample to show evidence of editing (ignoring direction in non-strand-specific reads), and at least 10 mapped reads with reference match. Complete validation results for editing sites are provided in Table S16. Informatics and statisticsAll statistical computation was performed in the R software environment ADDIN EN.CITE <EndNote><Cite><Author>R Core Team</Author><Year>2012</Year><RecNum>123</RecNum><DisplayText>(R Core Team 2012)</DisplayText><record><rec-number>123</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">123</key></foreign-keys><ref-type name="Artwork">2</ref-type><contributors><authors><author>R Core Team, </author></authors></contributors><auth-address>: A Language and Environment for Statistical Computing</title></titles><dates><year>2012</year></dates><pub-location>Vienna, Austria</pub-location><urls></urls></record></Cite></EndNote>(R Core Team 2012). Correlation (Pearson and Spearman) was calculated using the cor command in the stats package ADDIN EN.CITE <EndNote><Cite><Author>R Core Team</Author><Year>2012</Year><RecNum>123</RecNum><DisplayText>(R Core Team 2012)</DisplayText><record><rec-number>123</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">123</key></foreign-keys><ref-type name="Artwork">2</ref-type><contributors><authors><author>R Core Team, </author></authors></contributors><auth-address>: A Language and Environment for Statistical Computing</title></titles><dates><year>2012</year></dates><pub-location>Vienna, Austria</pub-location><urls></urls></record></Cite></EndNote>(R Core Team 2012). Visualizations of read coverage were generated by loading BAM files into the Integrative Genomics Viewer (IGV) v.2.1 ADDIN EN.CITE <EndNote><Cite><Author>Thorvaldsdottir</Author><Year>2012</Year><RecNum>124</RecNum><DisplayText>(Thorvaldsdottir et al. 2012)</DisplayText><record><rec-number>124</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">124</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Thorvaldsdottir, H.</author><author>Robinson, J. T.</author><author>Mesirov, J. P.</author></authors></contributors><titles><title>Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration</title><secondary-title>Brief Bioinform</secondary-title><alt-title>Briefings in bioinformatics</alt-title></titles><periodical><full-title>Brief Bioinform</full-title><abbr-1>Briefings in bioinformatics</abbr-1></periodical><alt-periodical><full-title>Brief Bioinform</full-title><abbr-1>Briefings in bioinformatics</abbr-1></alt-periodical><edition>2012/04/21</edition><dates><year>2012</year><pub-dates><date>Apr 19</date></pub-dates></dates><isbn>1477-4054 (Electronic)
1467-5463 (Linking)</isbn><accession-num>22517427</accession-num><urls><related-urls><url>;(Thorvaldsdottir et al. 2012). Sequence alignments were visualized using Jalview v.2.7 ADDIN EN.CITE <EndNote><Cite><Author>Waterhouse</Author><Year>2009</Year><RecNum>125</RecNum><DisplayText>(Waterhouse et al. 2009)</DisplayText><record><rec-number>125</rec-number><foreign-keys><key app="EN" db-id="wppa0r2v0sddz6e5pp5v200jer0traf2x9pe">125</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Waterhouse, A. M.</author><author>Procter, J. B.</author><author>Martin, D. M.</author><author>Clamp, M.</author><author>Barton, G. J.</author></authors></contributors><auth-address>School of Life Sciences Research, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.</auth-address><titles><title>Jalview Version 2--a multiple sequence alignment editor and analysis workbench</title><secondary-title>Bioinformatics</secondary-title></titles><periodical><full-title>Bioinformatics</full-title></periodical><pages>1189-91</pages><volume>25</volume><number>9</number><edition>2009/01/20</edition><keywords><keyword>Computational Biology/*methods</keyword><keyword>Databases, Protein</keyword><keyword>Proteins/*chemistry</keyword><keyword>Sequence Alignment/*methods</keyword><keyword>Sequence Analysis, Protein</keyword><keyword>*Software</keyword></keywords><dates><year>2009</year><pub-dates><date>May 1</date></pub-dates></dates><isbn>1367-4811 (Electronic)
1367-4803 (Linking)</isbn><accession-num>19151095</accession-num><work-type>Research Support, Non-U.S. Gov't</work-type><urls><related-urls><url>;(Waterhouse et al. 2009).Table S1: Sample and assembly information for new Drosophila genomesSpeciesDbiaDbipDeleDeugDficDkikDrhoDtakGeneral InfoStock Number 14023-0361.10 14024-0381.19 14027-0461.03 14026-0451.10 14025-0441.05 14028-0561.14 14029-0021.01 14022-0311.13 NCBI BioProject6230762313623156770962317623196766562321Inbreeding Generations111811111010013Strain 14023-0361.00 14024-0381.03 HK KB1 14025-0441.00 14028-0561.00 BaVi067 14022-0311.05Collection locationAri Ksatr, CambodiaChia-I, TaiwanHong Kong, ChinaKuala Belalong, BruneiTaiwanCaroline Island, ColombiaVietnamYun Shui, TaiwanCollection Year19671967unknown20021961unknownunknown1968Collector or DonorM. DelfinadounknownJohn TrueArtyom KoppunknownMarvin WassermanHisaki TakamoriLynn ThrockmortonSequence Generation and CoverageFragment18.3M(24.2X)18.7M (24.4X)18.0M (23.3X)17.1M (24.9X)16.3M (23.9X)14.2M (21.5X)11.4M (20.2X)18.9M (22.5X)3kb9.7M (8.6X)8.6M (9.7X)8.6M (10.7X)9.0M (12.8X)8.3M (10.1X)10.5M (10.0X)4.8M (8.3X)10.5M (11.7X)8kb3.0M (2.4X)2.0M (2.2X)3.1M (2.7X)2.6M (3.0X)1.9M (2.1X)3.3M (2.7X)1.2M (1.9X)2.3M (2.3X)Total31.0M (35.2X)29.3M (36.3X)29.7M (36.7X)28.7M (40.7X)26.5M (36.1X)28.0M (34.2X)17.4M (30.4X)31.7M (31.7X)Assembly StatisticsContig N50436 kb149 kb214 kb224 kb276 kb209 kb19 kb125 kbScaffold N503,128 kb663 kb1,714 kb977 kb1,049 kb911 kb45 kb390 kbAssembled bases180 Mb166 Mb171 Mb156 Mb151 Mb163 Mb195 Mb181 MbTable S2: Matrix of patristic distances.DanaDbiaDbipDeleDereDeugDficDgriDkikDmelDmojDperDpseDrhoDsecDsimDtakDvirDwilDyakDana0.000.570.130.540.630.610.601.020.560.621.120.690.690.540.620.620.571.011.020.62Dbia0.570.000.580.280.310.290.320.960.440.301.050.630.630.270.300.300.190.950.960.30Dbip0.130.580.000.550.630.610.601.020.570.631.120.700.700.540.630.620.581.021.020.63Dele0.540.280.550.000.330.310.300.930.410.331.020.600.600.170.330.320.280.920.930.33Dere0.630.310.630.330.000.320.371.010.490.121.110.690.680.330.120.120.311.011.010.10Deug0.610.290.610.310.320.000.350.990.470.321.090.670.660.310.320.310.290.990.990.32Dfic0.600.320.600.300.370.350.000.980.460.361.080.660.660.300.360.360.310.980.980.37Dgri1.020.961.020.931.010.990.980.000.941.010.510.860.860.921.001.000.960.410.881.01Dkik0.560.440.570.410.490.470.460.940.000.491.040.620.620.400.480.480.440.940.940.49Dmel0.620.300.630.330.120.320.361.010.490.001.100.680.680.320.050.050.301.001.010.12Dmoj1.121.051.121.021.111.091.080.511.041.100.000.960.961.021.101.101.050.370.981.11Dper0.690.630.700.600.690.670.660.860.620.680.960.000.010.600.680.680.630.860.860.68Dpse0.690.630.700.600.680.660.660.860.620.680.960.010.000.600.680.680.630.860.860.68Drho0.540.270.540.170.330.310.300.920.400.321.020.600.600.000.320.320.270.920.920.32Dsec0.620.300.630.330.120.320.361.000.480.051.100.680.680.320.000.020.301.001.000.12Dsim0.620.300.620.320.120.310.361.000.480.051.100.680.680.320.020.000.301.001.000.12Dtak0.570.190.580.280.310.290.310.960.440.301.050.630.630.270.300.300.000.950.960.30Dvir1.010.951.020.921.010.990.980.410.941.000.370.860.860.921.001.000.950.000.881.00Dwil1.020.961.020.931.010.990.980.880.941.010.980.860.860.921.001.000.960.880.001.01Dyak0.620.300.630.330.100.320.371.010.490.121.110.680.680.320.120.120.301.001.010.00Table S3: Summary of RNA-Seq sequencing depth Number of mapped RNA-Seq reads (millions)Reads mapping to aligned Dmel sequence (%)SpeciesAdult FemaleAdult MaleOvaryTestisFemale CarcassMale CarcassFemale HeadMale HeadMixed EmbryoTotal CDS exonsUTR exonsncRNA exonsintronsInter-genicDmel96.2103.4----11.49.1-220.0 64.32 21.39 25.86 3.34 1.08 Dsim151.7172.0167.9114.9173.760.8---807.1 46.57 18.56 0.91 1.63 0.38 Dyak91.896.5-------188.3 68.42 22.71 0.86 2.30 0.49 Deug60.151.6------201.6313.3 49.16 17.99 0.88 0.98 0.26 Dbia56.958.8------196.8312.5 40.34 15.29 0.37 0.84 0.12 Dtak49.452.3------197.1298.8 35.33 17.40 0.27 0.74 0.18 Dfic81.278.8------200.5360.4 45.18 17.05 0.28 0.76 0.13 Dele50.248.8------297.8396.7 53.82 19.44 0.48 1.35 0.25 Drho45.459.4------192.3297.2 50.17 18.96 0.27 0.82 0.13 Dkik34.247.2------168.6250.0 41.82 10.12 0.18 0.38 0.16 Dana92.176.8-------168.9 51.26 9.69 0.53 0.41 0.02 Dbip37.145.4------258.8341.3 58.78 13.71 0.19 0.60 0.05 Dpse133.3112.8357.5350.9250.3282.511.716.7-1515.9 50.03 9.27 0.78 0.40 0.02 Dmoj92.6101.9----21.130.5-246.1 56.69 7.11 0.09 0.33 0.01 Dvir130.380.0-------210.3 39.43 4.63 0.40 0.27 0.01 Table S4: Sample_identifiers and accessions.Key to headers for data file: Table_S4_sample_identifiers.xls. Species, strain, developmental stage, sex, tissue, and biological replicate are given for each sample. Information for the Gene Expression Ominibus (GEO) entries are given, including the sample ID and the sample accession. There are rows for each sample, with Dana_371.13_F_R1 as an example.Example: Sample_IDSpeciesStrainStageSexTissueReplicateGEO_sampleIDGEO_accessionDana_371.13_F_R1D. ananassae14024-0371.13AdultFemaleWhole1Whole_Dana_371.13_Female_Rep1GSM694275... all samplesTable S5: First CDS exon RPKM for each sample Key to headers for data file: Table_S5_first_CDS_RPKM.xls. Matrix of all RPKM values for first coding exons for orthologs in all samples, showing the MDv2 Id, chromosome location, nt start and stop positions, and strand. There are columns for each sample, with Dana_371.13_F_R1 shown as an example. There are rows for each MDv2 element.Example:Idchromstart (1-based)end(1-based)strandDana_371.13_F_R1.. all samplesmdcds_1chr2L1000404710004126+48.7389..… all exons…………….. Table S6: CDS exon validation and evolution (Table_S6_CDS_exon_validation.xls).Key to validation results for CDS exons in the MDv2 annotation. Unique identifier for event (Id), chromosome arm (chrom), element starting and ending position in the genome (1-based coordinates), strand, conservation index (CI) are shown. There are columns for each sample, with Dana_371.13_F_R1 shown as an example. RPKMs are given for elements that are aligned and expressed (#s). No RPKMs are given for elements not aligning with the Dmel element (NA) or showing <95% coverage (LC). There are rows for each MDv2 element.Example:Idchromstart (1-based)end(1-based)strandCIDana_371.13_F_R1.. all samplesmdcds_1chr2L1000404710004126+6.62248.7389.... all exons………………..Table S7: UTR exon validation and evolution (Table_S7_UTR_validation.xls).See Table S7 for key. Example:idchromstart (1-based)end(1-based)strandCIDana_371.13_F_R1.. all samplesmdutr_1chr2L1000142910001433-3.40528.8823.... all exons………………..Table S8: ncRNA exon validation and evolution (Table_S8_ncRNA_validation.xls).See Table S7 for key. Example:idchromstart (1-based)end(1-based)strandCIDana_371.13_F_R1.. all samplesmdncRNA_1chr2L1086219410862734-0NA.... all exons………………..Table S9: Intron validation and evolution (Table_S9_intron_validation.xls)Expression results for introns in MDv2 annotation that do not overlap exons. See Table S7 for key. Example:idchromstart (1-based)end(1-based)strandCIDana_371.13_F_R1.. all samplesmdintron_14chr2L1000641810006474+2.432LC..… all exons……………..Table S10: Intergenic validation and evolution (Table_S10_intergenic_validation.xls)Expression results for regions that do not overlap gene models. See Table S7 for key. Example:idchromstart (1-based)end(1-based)strandCIDana_371.13_F_R1.. all samplesmdintergenic_1chr2L17409.0NA..… all exons……………..Table S11: DNA element validation totals and rates. MDv2 annotationDNA elements% validated at distance (ss) from DmelParameterst1/2Type#>0.05>0.30>0.60N0λR2MyrsssCDS exon 62,210 98.296.688.595.60.30.87128.92.06ncRNA exon 1,816 61.747.430.946.81.20.8636.00.58Intron 43,443 51.736.319.232.61.80.8124.10.39UTR exon 64,955 86.373.136.576.21.90.9822.60.36Intergenic 10,995 15.310.82.69.82.90.8714.80.24Table S12: Promoter summary (Table_S12_promoter_summary.xls).Key to promoter analysis. Chromosome arm (chr), element starting and ending position in the genome (0-based coordinates), strand, the orthoDB ID for the protein-coding gene following the CAGE peak (orthoid), tags per million tags sequenced (TPM), the distance between the CAGE peak and first ATG of the CDS (Dist_AUG), promoter motif group type (Promoter_group), and the sample type (sample) are shown. There are rows for each CAGE peak. chrstartendstrandorthoidTPMDist_AUGPromoter groupsamplechr2L6704367044+EOG6STSR24.01058581aDmel.carcass...all CAGE peaksTable S13: Splice junction validation and evolution (Table_S13_splice_junction_validation.xls).Key to splicing analysis. The junction identifier is given according to genomic coordinates in D. melanogaster (Dmel). This identifier includes the chromosome and position of the first base of the intron on the each side, in 1-based coordinates (and inclusive). The lower coordinate number is always given first. The coordinates are separated by an underscore, with the chromosome at the beginning and the strand at the end, separated by colons. One junction is shown, but there are rows for each junction. Conservation index (CI) and validation results for each species are shown, where not aligned (0), aligned but not used (1), and aligned and used (2) are summarized. Four species are shown here, but there is a column for each species. The validation summary across all species uses the same codes.dmelCIdsimdyakdtakdbia… all speciessummarychr2L:10006418_10006474:+6.2582222…2… all junctions………………Table S14: RNA element validation totals and rates MDv2 annotationRNA elements% validated at distance (ss) from D. melanogasterParameterst1/2Type#>0.05>0.30>0.60N0λR2MyrsssGT-AG splice 63,524 87.780.968.779.40.40.95104.11.67GC-AG splice 788 70.358.041.958.40.70.9364.71.03A-I editing 972 70.257.741.641.11.00.50NDNDAT-AC splice 118 13.66.45.912.20.50.32NDNDTable S15: Splicing events (Table_S15_splicing_events.xls)Key to splicing analysis. Alternative-splicing results for whole adult female vs male. For each pairwise alternative-splicing event, the splicing difference is given in delta PSI (Percent Spliced In) for each species. Unique identifiers for each pairwise defined alternative event (envent_id), FlyBase gene name (gene_id), common abbreviation for gene (gene-name), basic type of alternative event (eventcode), an graph-based classification describing all possible types of alternative events from AStalavista output (structure), and the mutually exclusive splice junctions that compose the event (joinstring) are shown. There are columns for each female/male comparison (PSI value in females minus the PSI value in males, and ranges from -1 to 1). Note that each strain of Dsim is presented separately. event_idgene_idgene_nameeventcodestructurejoinstringASTA10442FBgn0028582lqfaltacceptor1-,2-chr3L:7528745_7528983:+;chr3L:7528745_7529103:+... all eventsContinued header. Dmel_deltaPSIDsim011_deltaPSIDsim198_deltaPSIDyak_deltaPSI… all species0.6740.8080.6560.796………………Table S16: RNA editing validation and evolution (Table_S16_editing_validation.xls).Key to editing analysis. Chromosome arm (chrom), nt position of edit (position), conservation index (CI) and sample ID are given. One example is shown, but here are columns for each sample and rows for each editing event, where not aligned (0), aligned but not edited (1), and aligned and edited (2) summarize the event.chromPosition (1-based)CIDana_371.13_F_R1... all sampleschr3L81134680.1730..… all editing events……..Table S17: Expression of conserved intergenic regions. Intergenic regions with K27Ac enrichmentIntergenic regions with CAGE peaksDistance (ss)ExpressedUnexpressedRatioP-valueExpressedUnexpressedRatioP-valueD > 053315480.364326720.2D>0.0549610570.53.05E-05**58717380.31.87E-07**D>0.33705310.7<2.2E-16**4347650.6<2.2E-16**D>0.659930.65.80E-04**521340.46.01E-03*Files S1 – S8:CAGE peak data in Browser Extensible Data (BED) format. Fields are specified below and an example shown. This is a standardized format that uses 0-based coordinates. For a description of the data standard for BED, see . A list of available files are shown below. ColumnDescription1chromosome2Peak start (0-based coordinates) 3Peak end (0-based coordinates)4The orthoDB ID for the protein-coding gene following the CAGE peak5TPM (tags per million) for the CAGE peak6strand7Distance between the CAGE peak and its downstream translation start site.Example:chr2L6704367044EOG6STSR24.01058+581See:File S1: CAGE peaks in D. melanogaster mixed-sex carcass (File_S1_CAGE_Dmel_FM_carcass.bed)File S2: CAGE peaks in D. melanogaster ovary (File_S2_CAGE_Dmel_ovary.bed)File S3: CAGE peaks in D. melanogaster testis, replicate 1 (File_S3_CAGE_Dmel_testis_rep1.bed)File S4: CAGE peaks in D. melanogaster testis, replicate 2 (File_S4_CAGE_Dmel_testis_rep2.bed)File S5: CAGE peaks in D. pseudoobscura female carcass (File_S5_CAGE_Dpse_F_carcass.bed)File S6: CAGE peaks in D. pseudoobscura male carcass (File_S6_CAGE_Dpse_M_carcass.bed)File S7: CAGE peaks in D. pseudoobscura ovary (File_S7_CAGE_Dpse_ovary.bed)File S8: CAGE peaks in D. pseudoobscura testis (File_S8_CAGE_Dpse_testis.bed) ................
................
In order to avoid copyright disputes, this page is only a partial summary.
To fulfill the demand for quickly locating and searching documents.
It is intelligent file search solution for home and business.
Related searches
- seton institute of reconstructive surgery
- michigan institute of real estate classes
- sports institute of tucson
- institute of physics iop
- institute of physics uk
- american institute of physics
- american institute of physics citation
- american institute of physics inc
- chicago institute of plastic surgery
- indian institute of public health
- nigerian institute of international affairs
- eric institute of education science