Elgin, Sarah C.R.

et al,

Mardis, Elaine R

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   W. Leung et al. 1 SI   Drosophila Muller F elements maintain a distinct set of genomic properties over 40 million years of evolution  Wilson Leunga, … Sarah C. R. Elgina,*  aDepartment of Biology, Washington University in St. Louis, St. Louis, MO 63130  *Corresponding author: Washington University in St. Louis, Campus Box 1037, One Brookings Drive, St. Louis, MO 63130-4899. Phone: (314) 935-5348. Email: selgin@wustl.edu   DOI: 10.1534/g3.114.015966    W. Leung et al. 2 SI  SUPPORTING FIGURES     W. Leung et al. 3 SI  Figure S1   Defining the analysis regions for D. melanogaster, D. erecta, D. mojavensis, and D. grimshawi based on changes in repeat density. The bar in the "Region" track corresponds to the region analyzed in this study. The grayscale in the "Repeat Density" track corresponds to the results of sliding window analysis (1 kb window and 0.5 kb step size) of total transposon density using a species-specific repeat library. Darker regions in this track correspond to regions with higher repeat density. One of the characteristics of heterochromatin is its high repeat density and the selections of the euchromatic reference regions at the base of the D element correspond to regions with mostly uniform low repeat density juxtaposed with regions with high repeat density. The Genome Browser screenshots of the base of the D elements show the region that spans from the start of the analysis region to the end of the assembled scaffold.   W. Leung et al. 4 SI  Figure S2   Sliding window analysis of the distribution of tandem repeats shows that the portion of the D. erecta D element closer to the telomere has a higher tandem repeat density than the portion of the D element closer to the centromere. Tandem repeats on the D element are identified by Tandem Repeats Finder (TRF) and the density of tandem repeats (TRF density) is calculated using a 1 kb sliding window with 500 bp step size. (Grayscale: darker regions have a higher density of tandem repeats (range from 0 to 10%).) The sliding window analysis shows that the distal half of the D. erecta D element (red box) has higher density of tandem repeats than the proximal half. The D. melanogaster D element has a lower density of tandem repeats than the D. erecta D element, and it does not show the same skew in the density of tandem repeats.      W. Leung et al. 5 SI   Figure S3   Composition of repeat fragments identified by RepeatMasker using species-specific repeat libraries. Approximately half of the transposon fragments identified by RepeatMasker on the D. melanogaster (45.9%), D. erecta (45.1%), and D. mojavensis (53.3%) F elements have sequence similarity to DINE-1 elements. Only 8.3% of the transposon fragments identified on the D. grimshawi F have similarity to DINE-1 elements. The D. erecta euchromatic reference regions are enriched in DINE-1 elements compared to the euchromatic regions in the other species.   0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Percentage Species: Region DINE DNA Transposon LINE LTR Unclassified Retroelements Unclassified Muller F Elements Muller D Elements    W. Leung et al. 6 SI   Figure S4   The F element shows higher repeat density than the D euchromatic reference region irrespective of the repeat library used with RepeatMasker. In congruence with the results obtained using the combined repeat library, repeat density estimates using species-specific ReAS repeat libraries show that the F elements have a higher transposon density than the D elements in all four species. Unlike other de novo repeat algorithms, ReAS identifies repeats by finding k-mers that occur at a high frequency within genomic reads. Hence the quality of the ReAS library is less likely to be affected by misassemblies.   0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% Repeat Density Species: Region % Unclassified % Unclassified Retroelements % LTR % LINE % DNA Transposons % DINE Muller F Elements Muller D Elements    W. Leung et al. 7 SI  Figure S5   Scaffolds assembled from unmapped RNA-Seq reads show some of the helitrons are being actively transcribed in the D. mojavensis genome. CD-Search (Marchler-Bauer et al. 2011) of a scaffold assembled from unmapped RNA-Seq reads (SRR166835_unmapped_15807) against the NCBI Conserved Domains database shows a full-length match to the Helitron_like_N domain (pfam 14214). This domain contains a helicase and is commonly found at the N-terminus of helitrons.   1 125 250 375 500 625 750 824 Helitron_like_N SRR166835_unmapped_15807 Helitron_like_N Conserved Domain Length: 184;  Bit Score: 255.76;  E-value: 4.09e-84 SRR166835_unmapped_15807 Helitron_like_N SRR166835_unmapped_15807 Helitron_like_N SRR166835_unmapped_15807 CD-Search Alignment:    W. Leung et al. 8 SI           W. Leung et al. 9 SI    Figure S6   Violin plots, Nc versus CAI scatterplots, and melting temperature profiles for all of the analysis regions. These plots are the same as the figures in the manuscript but they also include the extended and telomeric regions from the D. erecta D element (D. ere: D (ext.) and D. ere: D (tel.), respectively). These plots show that F element genes have different characteristics from genes found in the D element euchromatic reference regions. The violin plots also show that the genes in the three regions (base, extended, and telomeric) of the D. erecta D element have similar characteristics. These gene characteristics are distinct from those observed on the D. erecta F element.  Median 9 bp Sequence Melting Temperature (℃) Translation Start Relative Position 5 10 15 20 -2kb 0 3kb +2kb Coding Span 2kb 2kb 3kb M *D. mel: F D. ere: F D. moj: F D. gri: F D. mel: D (base) D. ere: D (base) D. ere: D (ext.) D. ere: D (tel.) D. moj: D (base) D. gri: D (base)    W. Leung et al. 10 SI  Figure S7   Heat map analysis of codon bias on the F elements shows that genes on the D. mojavensis F element exhibit a different pattern of codon bias (measured using CAI and Nc) than D. melanogaster, D. erecta, and D. grimshawi. The D. mojavensis orthologs of Thd1, Ephrin, sv, Actbeta, and Rfabg show higher CAI (blue) than those genes in other Drosophila species (red), indicating more optimal codon usage. RpS3A and ATPsyn-beta exhibit the highest CAI and lowest Nc, indicating that they are under the strongest selective pressure among all the F element genes in the four Drosophila species. The order of the genes in the heat map is determined by Ward hierarchical clustering.      W. Leung et al. 11 SI  Figure S8   Possible gene inversion scenarios for the D. melanogaster, D. mojavensis, and D. grimshawi F elements as determined by GRIMM. Each box in the diagram corresponds to a gene that is shared between the source and target genomes while each row represents an inversion step. The color boxes in each row correspond to the size of the inverted region at that step. (Top) GRIMM estimates that it requires a minimum of 31 inversions to transform the gene order and orientation seen in the D. melanogaster F element to that seen in the D. mojavensis F element (72 genes in common). (Middle) A minimum of 33 inversions is required to transform the D. melanogaster F element gene order and orientation to that seen in the D. grimshawi F element (73 genes in common). (Bottom) A minimum of seven inversions is required to transform the D. mojavensis F element gene order and orientation to that seen in the D. grimshawi F element (78 genes in common).    W. Leung et al. 12 SI   Figure S9   Net alignment of the whole genome assemblies from five Drosophila species against the D. mojavensis F element shows a single region (i.e. hotspot) where five of the six D. melanogaster wanderer genes are found. A similar hotspot on the D. grimshawi F element contains three of the four wanderer genes relative to D. melanogaster and one wanderer gene (yellow-h) relative to D. mojavensis. We have changed the color of the Net alignments to reflect the assignment of genomic scaffolds to Muller elements determined by previous studies (Schaeffer et al. 2008; Koerich et al. 2008).    W. Leung et al. 13 SI SUPPORTING TABLES  Table S1   Regions used in the comparative analyses Label Database (Assembly) Region in assembly First gene in region Last gene in region D. mel: F dm3 (Release 5) chr4:24053-1274855 JYalpha Caps D. mel: D (base) dm3 (Release 5) chr3L:21645371–22948385 CG43980 CG32461 D. ere: F DereWGS (CAF1) scaffold_4512:1–1283782 Caps CG40625 D. ere: D (base) DereWGS (CAF1) scaffold_4784:21301945–22602847 CG43980 CG32461 D. ere: D (ext.) DereWGS (CAF1) scaffold_4784:19933404–21301944 CG7017 Atox1 D. ere: D (tel.) DereWGS (CAF1) scaffold_4784:1461828–2709938 stet Fife D. moj: F DmojImproved (GEP) improved_6498:1348734–3038944 CG31999 Cyp1_alpha D. moj: D (base) DmojImproved (GEP) improved_6680:21061438–22011287 CG14826 CG11370 D. gri: F DgriImproved (GEP) improved_F:28556–1228705 CG32850 sv D. gri: D (base) DgriImproved (CAF1) scaffold_15110:22264226–23522110 W fal        W. Leung et al. 14 SI Table S2   Sequence improvement statistics A. Number of gaps closed by sequence improvement  Region # Gaps (Improved) Total gap size (Improved) # Gaps (CAF1) Total gap size (CAF1) Difference # gaps Difference gap size Dgri: F 13 13,670 56 42,648 -43 -28,978 Dmoj: D (base) 0 0 7 1,765 -7 -1,765 Dmoj: F 1 1,166 23 14,891 -22 -13,725 Total 14 14,836 86 59,304 -72 -44,468  B. Number of changes between the improved and CAF1 assemblies  Region # Changes Total size of changes # Single base changes % Single base changes Dgri: F 151 56,000* 56 37.1% Dmoj: D (base) 42 2,791 18 42.9% Dmoj: F 116 15,500 53 45.7% Total 309 74,291 127 41.1%  * The first 40 kb of the D. grimshawi F element scaffold in the CAF1 assembly contains a major misassembly. The discrepancies in this region account for 25/151 changes and cover a total of 24,303 bases.       W. Leung et al. 15 SI  Table S3   Comparison of GLEAN-R gene predictions and the most comprehensive isoform (i.e. isoform with the largest coding region) annotated by the GEP A. GLEAN-R gene models that match the comprehensive isoform annotated by the GEP  Region # Match # Mismatch # GEP annotations # GLEAN-R annotations % Match  Dere: F 53 28 78 81 65.4% Dmoj: F 26 55 81 81 32.1% Dgri: F 36 56 79 92 39.1% Dere: D (base) 84 37 110 121 69.4% Dere: D (ext.) 141 81 207 222 63.5% Dere: D (tel.)  108 62 162 170 63.5% Dmoj: D (base)  57 42 84 99 57.6% Dgri: D (base)  47 34 77 81 58.0% Total 552 395 878 947 58.3%   B. GLEAN-R coding exons that match the coding exons in the comprehensive isoform annotated by the GEP  Region # Match # Mismatch # GEP Annotations # GLEAN-R annotations % Match Dere: F 510 56 597 566 90.1% Dmoj: F 451 108 624 559 80.7% Dgri: F 513 122 629 635 80.8% Dere: D (base) 288 49 324 337 85.5% Dere: D (ext.)  737 99 827 836 88.2% Dere: D (tel.) 613 84 711 697 87.9% Dmoj: D (base) 305 73 353 378 80.7% Dgri: D (base) 231 48 277 279 82.8% Total 3648 639 4342 4287 85.1%        W. Leung et al. 16 SITable S4   Results of the Tallymer to determine the fraction of unique k-mers in each analysis region A. F elements  K-mer Dmel: F Dere: F Dmoj: F Dgri: F 8 0.015 0.015 0.006 0.018 9 0.203 0.196 0.148 0.221 10 0.525 0.515 0.472 0.540 11 0.751 0.742 0.717 0.751 12 0.870 0.862 0.843 0.858 13 0.927 0.921 0.900 0.911 14 0.953 0.949 0.925 0.938 15 0.965 0.961 0.937 0.953 16 0.970 0.967 0.943 0.962 17 0.973 0.970 0.947 0.967 18 0.975 0.972 0.949 0.971 19 0.976 0.974 0.952 0.973 20 0.977 0.975 0.954 0.975  B. Euchromatic reference regions from the D elements  K-mer Dmel: D (base) Dere: D (base) Dere: D (ext.) Dere: D (tel.) Dmoj: D (base) Dgri: D (base) 8 0.002 0.002 0.002 0.003 0.022 0.016 9 0.121 0.124 0.117 0.136 0.237 0.199 10 0.501 0.501 0.488 0.511 0.578 0.517 11 0.786 0.787 0.779 0.795 0.804 0.754 12 0.916 0.918 0.915 0.924 0.911 0.882 13 0.965 0.968 0.968 0.971 0.957 0.943 14 0.982 0.985 0.987 0.986 0.976 0.969 15 0.988 0.991 0.993 0.992 0.984 0.981 16 0.990 0.993 0.995 0.993 0.987 0.986 17 0.991 0.994 0.996 0.994 0.989 0.989 18 0.992 0.995 0.997 0.994 0.990 0.991 19 0.992 0.995 0.997 0.995 0.991 0.992 20 0.992 0.996 0.997 0.995 0.992 0.992          W. Leung et al. 17 SI Table S5   Statistics on the DINE-1 fragments identified by the species-specific library that do not overlap with known repeats in the RepBase library. Region DINE-1 elements that do not overlap with RepBase repeats (count) DINE-1 elements that do not overlap with RepBase repeats (bp) DINE-1 elements identified by the species-specific library (bp) Percentage of DINE-1 elements that do not overlap with RepBase repeats (bp) Dmel: F 278 12,479 161,138 7.7% Dere: F 209 8,225 144,399 5.7% Dmoj: F 602 40,587 449,309 9.0% Dgri: F 120 8,524 19,533 43.6% Dmel: D (base) 62 2,999 31,903 9.4% Dere: D (base) 64 2,265 41,760 5.4% Dere: D (ext.) 33 1,589 21,657 7.3% Dere: D (tel.) 25 15,470 34,476 44.9% Dmoj: D (base) 161 9,782 33,262 29.4% Dgri: D (base) 123 8,256 19,751 41.8%        W. Leung et al. 18 SITable S6   Gene density for the different analysis regions Region # Genes Region size (bp) # Genes / Mb Dmel: F 79 1250803 63 Dere: F 77 1283782 60 Dmoj: F 81 1690211 48 Dgri: F 79 1200150 66 Dmel: D (base) 121 1303015 93 Dere: D (base) 110 1300903 85 Dere: D (ext.) 207 1368541 151 Dere: D (tel.) 161 1248111 129 Dmoj: D (base) 84 949850 88 Dgri: D (base) 76 1257885 60        W. Leung et al. 19 SI SUPPORTING FILES  Available for download at http://www.g3journal.org/lookup/suppl/doi:10.1534/g3.114.015966/-/DC1 File S1 Supplemental text and methods  File S2 Summary of gene structure changes compared to D. melanogaster  File S3 WindowMasker, tandem repeats, and simple repeats statistics  File S4 Transposon density estimates using different transposon libraries  File S5 Statistics on RepBase transposons that overlap with DINE-1 fragments  File S6 Kruskal-Wallis rank sum test results for different gene characteristics  File S7 Codon usage heat map  File S8  List of participating courses  File S9  Improved sequences and annotations in GenBank ASN.1 format  

Drosophila Muller F elements maintain a distinct set of genomic properties over 40 million years of evolution

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Drosophila Muller F elements maintain a distinct set of genomic properties over 40 million years of evolution

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