Atypical miRNA ortholog loss

Here are several cases of miRNAs with atypical ortholog loss. Atypical loss means that either the loss of one or more orthologs is not common within a specific clade or there there is evidence for cross-mapping of small RNAseq reads from the species with the lost ortholog onto functional orthologs of related species. Many of these loss events are singleton cases and occur within simulans, yakuba, and persimilis primarily. There are however instances of clade specific losses which mostly occur in the obscura lineage. For many of the species below I was able to find the correct ortholog by searching the genome assembly trace reads.

droSim1

miRNA Name	Query Species	Result	Experiment Needed	Experiment Decision	Sequence
dme-mir-2282	Dm3	Found	N	NA	>dme-mir-2882-dm3 TGGGGCAGTGGAGCTGCCTGCATAACGCTGTGGCTTTCCGCTTCTATTTTTCGTTCACTGCCTTGTGC----TTCGTTGGAAAATCGGTGAGCTAAAAATAGAATTCGGTTGCCACGCTTCAAGGGAAATGAGGAAAATTA >zdo07f06.g1:327-468 TGGGGCAGTGGAGCTGCCTGCATAAGGCTGTGGCTTTCCGCTTCTATTTTTCGTTCACTGCCTTGTGCTTGCTTCGTTGGAAAATCGGTGAGCTAAAAATAGATTTCGGTTGCCACGCTTCAAGGGAAATGAGGAAAATTA These ortholog lie in a gap of the simulans assembly (chr3L:2949116-2949968). The sequence was not found in the w501 strain's shotgun reads which comprises the bulk of the droSim1 assembly. It was found in the 6 other strains.
dme-mir-2283	Dm3	Found	N	NA	>dme-mir-2283-dm3 GAAAACTCACTCCTGAGCTGCTCCGAAAATATCA-----------TGAATACGACAATTATGTGCACATCATTTAGTATACGTGATATTTTAGGAGCAGCTAAATTATATTTAAAGT >wcy63a10.b1:18-120 ---------------AGTTGTTCCGAAAAAATCAATTTTAATGACAGGATGGGCAGATAATTGGGACATCATGTTGTATACGTAATATTTTAGGAGCAGCTCAATTATATTTAAAGT This ortholog should lie in a region of the genome filled by a gap (chr3R:15989680-15990054). The ortholog was only found in the nc48 strain and in none of the other 6 strains.
dme-mir-2495	Dm3	Found	N	NA	>dme-mir-2495-dm3 CGTATGTATTATACATGTATGATCACATTGGCTTGTGGGCGTGGCACTTCAATTGGGCTGACTTTGCCTGGCTGGAAAAGCGGCACCTAATTGAAATGCCCGCCGATCAGTGCTCGATTACGATCGACACAAGCGACATTTTCGT----------------- >zds89d09.b1:77-211 CATACGTAC-ATACATGTATGATC-CATTGGCTTGTGGGCGCGGCACTTCAATTGGGCTGACTTTGCCTGGCTGGAAAAGCGGCACCTAATTGAAATGCCCGCCAAT--------------------------CGACATTTTCGTTTAATTTCGTTTCTAGC The ortholog was only found in 3 of the 7 strains and not in w501. This dme miRNA lies in an intron of the gene qtc. The genome browser indicate that the simulans alignment in this area is ambiguous as evident from chains in the browser screenshot below. As it appears, the ortholog for qtc would lie between a rearrangement event with chrU, chr3R, and chr2L_random: Flybase reports an ortholog of dsim\GD23320 for qtc, but this annotation was recently withdrawn. No ortholog is reported for sechelia but for erecta and yakuba.
dme-mir-274	Dm3	Found	N	NA	The correct ortholog was found in all the 7 strains. The correct simulans ortholog should lie in a gap region of the assembly (chr3L:11019633-11019769). >dme-mir-274-dm3 AGTTTTGGTGACGAATCCTGTGTTGCAGTTTCGTTTTGTGACCGACACTAACGGGTAATTGTTTGGCCGCCAGGATTACTCGTTTTTGCGATCACAAATTATGAAATTGCAGCAAAACTCAACGAAATTG >zdo16e12.g1:275-405 AGTTTTAGTGACAAATCCTGTGTTGCAGTTTCGTTTTGTGACCGACACTAACGGGTAATTGTTTGGCCACCAGGATTACTCGTTTTTGCGATCACAAATCATGAAATTGCAGCAAAATGCAACAAAAATG
dme-mir-280	Dm3	Found	N	NA	The correct ortholog was found in all 7 strains. This ortholog should lie in a gap region of the simulans reference assembly (chr2R:2844465-2844618). >dme-mir-280-dm3 CTGACGTGTGTATGCTGGCTTTTATGTATTTACGTTGCATATGAAATGATATTTATAGTAAACAGATTATTTTATATGCAGGTATATGCAAGTCGAGGTCCTCCACACTGCACTCGCCGCCTCGA >zee54c10.b1:599-724 CTGACGTGTGTGTGCTGGCTTTTATGTATTTACGTTGCATATGAAATGATATTTATAGTAAACAGATTATTTTATATGCAGGTATATGCAAGTCGAGGTCCTGCACACTGCACTCGCCGCCTCGA
dme-mir-281-2	Dm3	Found	N	NA	The correct ortholog was fully found in 3 of the 7 strains, and partly found in the remaining 4. This ortholog should lie upstream to the dme-mir-281-1 ortholog, but this region is actually filled by a gap (chr2R_random:2147066-2148065). The MAF actually reports the 281-2 and 281-1 orthologs as the same, but this is clearly not the case apparent from the discordant substitution patterns. >dme-mir-281-2-dm3 CTTTCAGATCCTCCGCGAATTGTGAAATGAAGAGAGCTATCCGTCGACAGTCAAGTTAAGACCGATTGTAATACTGTCATGGAATTGCTCTCTTTGTATAACATTCGAAAGGCGACGATTAT >zdp42d09.g1:294-416 CTTTCAGATCCTCCGCGAATTGTGAAATGAAGAGAGCTATCCGTCGACAGTCAAGTTAAGACCGATTGTAATACTGTCATGGAATTGCTCTCTTTGTATAACATTCGAAAGGCGACGATTAT
dme-mir-306	Dme		N	NA	The correct ortholog was found in its entirety in 6 of the 7 strains, and partially found in md106. >dme-mir-306-dm3 GTGAATAGTTTAAAAGTCCACTCGATGGCTCAGGTACTTAGTGACTCTCAATGCTTTTGACATTTTGGGGGTCACTCTGTGCCTGTGCTGCCAGTGGGACATAATCTACAAATAA >zdv96e12.b1:525-640 GTGAATAGTTTAAAAGTCCACTCGATGGCTCAGGTACTTAGTGACTCTCAATGCCTTTGACATTTTGGGGGTCACTCTGTGCCTGTGCTGCCAGTGGGACATAATCTACAAATAA This miRNA lie in the same intron of the grp gene as 79 and 9b, and one intron downstream of 9c. The genome browser show that this region's alignment with simulans is ambiguous: Flybase also does not report an ortholog for grp in simulans, however one exists for sechellia (GM17194).
dme-mir-79	Dm3	Found	N	NA	The correct ortholog was only found in 4 of the 7 species. It was found partially in 1 (c1674) and not found in 2 (sim4 & md106). >dme-mir-79-dm3 ATGAGTGCCTTAGAGTGAAGCTGACTTGCCATTGCTTTGGCGCTTTAGCTGTATGATAGATTTAAACTACTTCATAAAGCTAGATTACCAAAGCATTGGCTTCTGCAGGTCAATCGTCAGAAACAAT >wck39a12.g1:600-726 ATGAGTGCGTCAGAGTGTAGCTGACTTACCATTGCTTTGGCGCTTTAGCTGTATGATAGATTTAAACTACTTCATAAAGCTAGATTACCAAAGCATTGGCTTGTGCAGATCAATCGTCAGAAACAA-
dme-mir-971	Dm3	Found	N	NA	>dme-mir-971-dm3 TGCATGTGAGAGAATTCCGTGGCTGGCATCGCTCGCTGTAAATTGTAATCATCAAAGCGTTTTCTCAGAGCCGCTTGGTGTTACTTCTTACAGTGAGTGTGCCAGTCCGTACACAGAAAGAAAACC >zec46h04.g1:44-170 TGCATGTGAGAGAATTCCGTGGCTGGCATCGCTCGCTGTAAATTGTAATCATCAAAGCGTTTTCTCAGAGCCGCTTGGTGTTACTTCTTACAGTGAGTGTGCCAGTCCGTACACAGAAAGAAAACC This reads lie in a gap in the simulans assembly (chrX:9859862-9862861). The sequence was not found in the w501 strain which comprises the bulk of the droSim1 assembly. It was found in the 6 other strains.
dme-mir-9b	Dm3	Found	N	NA	The best ortholog was found in the nc48 strain. Other suboptimal hits were found in c1674, md106, md199, and sim6. No hits found in sim4 or w501 >dme-mir-9b-dm3 TGTTGCTCTTTTGTTTGCATATTATTTGCTCTTTGGTGATTTTAGCTGTATGGTGTTTATGTATATTCCATAGAGCTTTATTACCAAAAACCAAATGGTTTCTGCATTATGTTTGAGTTGA >vom21g07.g1:730-849 TGTTGCTCTTTTGTTTGCATATTATTTGCTCTTTGGTGATTTTAGCTGTATGGTGTATAAGT--ATTCCATAGAGCTTTATTACCAAAAACCAAATGGTTTCTGCATTATGCTTGAGTTGA
dme-mir-9c	Dm3	Found	N	NA	>dme-mir-9c-dm3 CTTGCACTATTTATCATTTTTGCTGTTTCTTTGGTATTCTAGCTGTAGATTGTTTCACGCACATTGTATATCATCTAAAGCTTTTATACCAAAGCTCCAGCTTAAATTGCTTAACATGATAT >zdv96e12.b1:15-131 CTTGCACTATTTATCATTTTTGCTGTTTCTTTGGTATTCTAGCTGTAGATTGTTTCACGCACATTGTATATCATCTAAAGCTTTTATACCAAAGCTCCAGCTTAAATTGCTTAACA------ All strains contained the ortholog.
dme-mir-4954	Dm3	Found	N	NA	This miRNA lies in an intron of the dm3 gene actn. Actn has no reported ortholog in simulans. The Alignment in this area in simulans is also ambiguous; there are several chains in chrU, chr3L and chrX that comprise the surrounding syntenic region. The ortholog was only found in w501 strain, partially in nc48, and none of the other 5 strains >dme-mir-4954-dm3 ATATAAGTGTTCGATTTGGCGCTTGGAATCGATACCCGAGCCATGATAGATTGAAGTCAACCCAATCGATCGCGGTTCGAGTGCTCGAGTCTTGTGCGCCGGCATGCTTGAGATGTCCACTA >vgb65a08_5.b1:263-385 ATATAAGTGTTCGATTTGGCGCTTGGAATCGATACCCGAGCCCTGATAGATTGAAGTCAACCCAATCGATCGCGGTTCGGGTGCTCGAGTCCTGTGCGCCGGCATGCTTGAGATGTCCACTA
dme-mir-4958	Dm3	Found	N	NA	The ortholog was only found in md106 strain and in none of the other 6. >dme-mir-4958-dm3 GTCCGAACCGTACGATCTCTGGCAGCTGCAGTTCCGTTTCCGGAGCGGGATCTGGAACGGGCTCTGGTTCTAGATCAGGATCCAATCATGCACCCGGTCCAGGTACCGCTCCAGGTCCCGTTCCCGGAAACGGTGCCACCGCCAATGCAGCTGCAGCGGCATTCA >wgm54c09.g1:392-557 GTCCGAACCGCACGATCTCTGGCAGCTGCAGTTCCGTTTCCGCAGCGGGATCTGGGACGGGCTCTGCTTCTAGATCAGGATCCAATCATGCACCCGGTCCAGGTGCCGCTCCAGGTCCCGTACCCGGAAGCGGAGCCACCGCCAATGCAGCTGCAGCGGCATTCA
dme-mir-4961	Dm3	Found	N	NA	This miRNA is on the 3' UTR of the CG3056 gene. The ortholog for this gene is dsim\GD16553. This gene is very short (1KB), and had no similarity with 4961/dme. On the browser, the syntenic region of this miRNA would lie in a gap (chrX:979887-981047). However, searching the reads, the ortholog is found: >dme-mir-4961-dm3 AGATATTACCCAGAAGCGATATCCAATAGTAGCCAACTCTCTCGCTCTCTATGTGTATGTATGTATCTTGCTATCCATATATGTATATCCATATCAGAGAGCCAGGATTGGTTGAGACCATACGATATAACCCGAAACCACACTGGCCAGGAATAGCAAATCCA >wjj76b12.b1:303-467 AGATATTACCCAGAAGCGATATCCAATAGTAGCCAACTCTCTCGCTCTCTATGTGAATGTATGTATCTTGCTATCCATATATGTATATCCATATCAGAGAGCCAGGATTGGTTGAGACCATACGATATAACCCGAAACCATACTGGCCAGGAATAGCAAATCCA The best hits were found in all the strains except for w501 and c1674. In w510 only a partial hit was found.
dme-mir-4971	Dm3	Found	N	NA	This miRNA actually lies in the CDS region of msl-2 gene. No ortholog in simulans is reported but an ortholog in sechelia exists. >dme-mir-4971-dm3 GGAAGGGCCAACTTCTCGGCCCTCGACACGGTGGATGAGCTTGTCAGTGGCGGATCCAGGAGCAATTCTGCCGCTGGCGACAGATCATCGGCCACTGACAATGCCCATTCACTGTTCGAGGAGATCATGTCGGGCTCGGATG >wop21h10.b1:281-423 GGAAGGGCCAATTTCTCGGCTCTCGACACGGTTGATGAGCTTGTCAGTGGCGGATCCAGGAGCAATTCTGCCGCTGGCGACAGATCATCGGCCACTGACAATGCCCAGTCACTGTTCGAGGAGATTATGTCGGGCTCGGATG

droYak2

miRNA Name	Query Species	Result	Fragment to clone	Experiment Needed	Experiment Decision	Sequence
dme-mir-12	dm3	Found.		N	NA	>dme-mir-12-dm3 AAGGAGCAGCGTCTGTACGGTTGAGTATTACATCAGGTACTGGTGTGCCTTAAATCCAACAACCAGTACTTATGTCATACTACGCCGTGCACGGATCGCACTAA >gnl\|ti\|371645083:614-718 AAGGAGCAGCGTCTGTACGGTTGAGTATTACATCAGGTACTGGTGTGCCTTAAATCCAACAACCAGTACTTATGTCATACTACGCCGTGCACGGATCGCACTAA
dme-mir-972	dm3, droEre2	Not found	chrX:11892021-11893235 (1214nt)	Y	Requenced - Found the 973 and 974, but not 972	chrX:11892021-11893235 has a 1214nt gap. The multiple alignment suggest that all three yakuba miRNA ortholog should lie within this gap, however, my search of the reads did not turn up any hits. I should note that the assembly shotgun reads extend about 17-694nt into this gap, so it is mostly covered. Given that the miRNA downstream of this gap (975-977) has yakuba orthologs, it's very suspicious have this 972-974 miRNA sub-cluster loss.
dme-mir-973	droSec1, droEre2	Not found
dme-mir-974	droSec1, droEre2	Not found
dme-mir-983-1	droSec1, droEre2	Not found		N	Genuine loss event.	983-1/dme and 983-2/dme should lie in intron 5 of gene CG3626/dme. The ortholog for this gene is GRE16342/dyak. Searched this gene by BLAST for the mature and star sequence for both sechelia and erecta orthologs but only found hits of length 7 and not 21-23nt. Only singleton reads (6 in total) from the yakuba small RNA head and female-body libraries reads mapped to the sechellia or erecta ortholog. This looks like a genuine loss event.
dme-mir-983-2	droSec1, droEre2	Not found		N	Genuine loss event.
dme-mir-997	droSec1, droEre2	Not found	Approximate genomic coordinates unknown. Clone with primers designed with dme-mir-997/dsec ortholog.	Y	Nothing done	A search of the droYak2 genome assembly with the droSec1 ortholog found 15 hits, but these hits only captured the star sequence partially and did not span the mature sequence. droEre2 ortholog search within the droYak2 genome assembly did not return any hits. >dme-mir-997-droSec1 CAATAAATATGTAGTTTTAGATACTCGCCAGTCAGGATGCTCTGTCAATGAATTTAGTATGCCCAAACTCGAAGGAGTTTCACCTCCATAGGAGCGGCAGACCTGGAGAAGTTTTCAGAGCCAACAAAATTCATATGATGATGCATTTTCAGTCTCTGAAAATTTCTTCAGCAGAAGTTGATTTTAGCGAAGTGAAGCTCATTCGATTTTGATCATACTAACATTTGTGGATGCTTGGATCGTCAGTTTGGTGGAATATT >gnl\|ti\|386447040:428-513 CG-------------------------------------------------------------------------------------------------------------------------ACACAATTTATTTAATGATGCATTTTCAGTCCCTGAAATCTTCTTCAGTAAAAGTGGATTTTTGTAAAGTTGAGCTCATTCGA------------------------------------------------------ The erecta ortholog has many substitutions that don't agree closely with the simulans nor sechellia ortholog, however this ortholog folds into a long hairpin and has reads within the mature and star regions indicative of RNase III cleavage. Do the yakuba reads map to the sechellia ortholog? Yes. 14 yakuba small RNA reads mapped to the sechellia ortholog, however all were 19nt long. Since the orthologs reported in the MAF and by LASTZ searches do not appear confident and the hit reported from trace reads search return a gappy, non-confident sequence, we are unsure what the correct region to PCR would be. This ortholog appears to be lost. V052_mapped_droSec1.sam:V052_199748_1 16 V052_mapped_droSec1.sam:V052_455157_1 16 V058_mapped_droSec1.sam:V058_51901_5 16 V058_mapped_droSec1.sam:V058_161246_2 16 V058_mapped_droSec1.sam:V058_169511_2 16 V058_mapped_droSec1.sam:V058_701167_1 16 V058_mapped_droSec1.sam:V058_891376_1 16 V058_mapped_droSec1.sam:V058_1231934_1 16 V058_mapped_droSec1.sam:V058_1365009_1 16 V058_mapped_droSec1.sam:V058_1866832_1 16 M026_mapped_droSec1.sam:M026_181245_1 16 M026_mapped_droSec1.sam:M026_330923_1 16 M043_mapped_droSec1.sam:M043_137968_23 16 M043_mapped_droSec1.sam:M043_707351_3 0 M043_mapped_droSec1.sam:M043_2823216_1 0 M043_mapped_droSec1.sam:M043_3233721_1 16 M043_mapped_droSec1.sam:M043_5112769_1 0 V120_mapped_droSec1.sam:V120_98271_7 0 V120_mapped_droSec1.sam:V120_961113_1 16 V120_mapped_droSec1.sam:V120_1656829_1 0 dme-mir-997_droSec1 65 25 19M * 0 0 AACCTCGAAGCAGTTTCACXT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:2A7G8 dme-mir-997_droSec1 43 25 19M 0 0 TGTCAATGAATGCAGTATGXT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:11T0T6 dme-mir-997_droSec1 65 25 19M 0 0 AAACTCTAAGCAGTTTCACXT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:6G3G8 dme-mir-997_droSec1 65 25 19M 0 0 AACCTCGAAGCAGTTTCACXT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:2A7G8 dme-mir-997_droSec1 66 25 18M 0 0 AACTCTAAGCAGTTTCAC XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:5G3G8 dme-mir-997_droSec1 129 25 18M 0 0 ATTCAGATGGTGATGCAT XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:5T3A8 dme-mir-997_droSec1 83 37 5M1I12M 0 0 CATCCGATAGGAGCGGCA XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:1C15 dme-mir-997_droSec1 66 25 18M 0 0 ACCTCGAAGCAGTTTCAC XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:1A7G8 dme-mir-997_droSec1 66 25 18M 0 0 AGCTCGAAGCAGTTTCAC XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:1A7G8 dme-mir-997_droSec1 65 25 19M 0 0 AAACTCAAAGCAGTTTCACXT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:6G3G8 dme-mir-997_droSec1 197 25 18M 0 0 GCTCAATCGACTTTGATC XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:5T4T7 dme-mir-997_droSec1 129 25 18M 0 0 ATTCAGATGGTGATGCAT XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:5T3A8 dme-mir-997_droSec1 129 25 18M 0 0 ATTCAGATGGTGATGCAT XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:5T3A8 dme-mir-997_droSec1 135 25 19M 0 0 ATGATGATGATTTTTCAGTXT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:9C0A8 dme-mir-997_droSec1 238 25 18M 0 0 GATCGTCAGTGTGCTGGA XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:10T2G4 dme-mir-997_droSec1 13 25 21M 0 0 AGCTTTAGATACTCGCGAGTC * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:2T13C4 dme-mir-997_droSec1 223 37 9M1I8M * 0 0 ATCTGTGGATTGCTTGGA XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:2T14 dme-mir-997_droSec1 135 25 19M 0 0 ATGATGATGATTTTTCAGT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:9C0A8 dme-mir-997_droSec1 141 25 19M * 0 0 ATGCATTTTCAGTCTTTGC * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:15C2A0 dme-mir-997_droSec1 116 37 12M1I5M * 0 0 CAGAGCCAACAACAATTA * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:16C0

dp4

miRNA Name	Query Species	Result	Fragment to clone	Experiment Needed	Experiment Decision	Sequence/Notes
dme-mir-313	droAna3	Not found	chr3:8678956-8680852 (1897nt) This region should emcompass dme-mir-992/dpse - dps-mir-2507b	Y	Cluster will be resequenced	The alignment suggest that 313/dpse should lie adjacent to 312/dpse, similar to how 313/dper lies adjacent to 312/dper. However, searching the dp4 assembly with the annanasae ortholog did not turn up any hits. In pseudobscura, the 310-313 cluster has many adjacent miRBase annotated miRNAs. With so many new annotations, this locus may contain many duplicate miRNAs and thus poorly assembled. Therefore, resequencing is needed to clarify if 313/dpse is genuinely missing.
dme-mir-955	droAna3	N	chrXR_group8:3250437-3253723 - (3286nt)	N	Confidently lost in the obscura subgroup	955/dmel is an intergenic miRNA. The flanking genes in melanogaster have orthologs in pseudobscura as well. Similarly, the 955/pse ortholog is also intergenic and flanked by these orthologous genes. A search of the pseudoobscura sRNAseq libraries mapped to the ananassae and willistoni ortholog turned up few reads. Are are the reads mapping to the willitoni ortholog: V043_mapped_droWil1.sam:V043_73838_4 16 dme-mir-955_droWil1 62 37 6M1I11M * 0 0 TTTGTTGTTCTCCAATGG * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:11T5 V043_mapped_droWil1.sam:V043_149175_2 0 dme-mir-955_droWil1 35 25 22M * 0 0 CATCGTGCACAGGTTTGAGTGT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:8T0T12 V043_mapped_droWil1.sam:V043_169017_2 4 dme-mir-955_droWil1 116 25 20M * 0 0 AACAGCCTAAAAAGTACAAT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:12T2G4 V043_mapped_droWil1.sam:V043_1068829_1 0 dme-mir-955_droWil1 16 25 18M * 0 0 CATTAAGGATGGCTGGCT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:1G6T9 V043_mapped_droWil1.sam:V043_1265617_1 0 dme-mir-955_droWil1 35 25 23M * 0 0 CATCGTGCAGAGGTTTGAGTGTC * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:8T0T13 M040_mapped_droWil1.sam:M040_2435810_2 0 dme-mir-955_droWil1 61 25 18M * 0 0 TTTTGTTTTCGCTAATGG * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:0G9T7 M040_mapped_droWil1.sam:M040_3794921_1 0 dme-mir-955_droWil1 16 25 18M * 0 0 CATTAAGGATGGCTGGCT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:1G6T9 M040_mapped_droWil1.sam:M040_3943027_1 16 dme-mir-955_droWil1 49 37 8M1D11M * 0 0 TTGAGTGTTTCGTTCGTTT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:8^C6T4 M040_mapped_droWil1.sam:M040_4187516_1 0 dme-mir-955_droWil1 51 25 18M * 0 0 GAGTGACTTCCTTTGTTT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:5T4G7 M040_mapped_droWil1.sam:M040_4279699_1 4 dme-mir-955_droWil1 117 25 19M * 0 0 ACAGCCTAAAAAGTACAAT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:11T2G4 M040_mapped_droWil1.sam:M040_4468108_1 0 dme-mir-955_droWil1 62 37 5M1I13M * 0 0 TTTGTGTTTCTCAAATGGC * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:11T6 M040_mapped_droWil1.sam:M040_5795261_1 0 dme-mir-955_droWil1 43 37 12M1I5M * 0 0 TTAGGATTGAGTCGTCTT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:5T11 M040_mapped_droWil1.sam:M040_9979667_1 0 dme-mir-955_droWil1 24 37 9M2I9M * 0 0 TTGGCTGGCCGTCCATCGTG * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:0 XO:i:1 XG:i:2 MD:Z:18 This miRNA looks confidently lost in obscura lineage.
dme-mir-963	droAna3, droWil1	Not found		N	Confidently lost	See notes in persimilis. I checked how many pseudoobscura reads mapped to the willistoni and ananassae orthologs, but found very few did and they did not map perfectly. Here are the reads mapping to the willistoni ortholog: V112_mapped_droWil1.sam:V112_335143_2 16 dme-mir-963_droWil1 123 25 20M * 0 0 TTTTCGTTAAGCCCTACACA * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:5T8A5 V112_mapped_droWil1.sam:V112_349332_2 0 dme-mir-963_droWil1 95 25 21M * 0 0 TAGCTTTGTTTCGTATAGGAC * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:2C10A7 V112_mapped_droWil1.sam:V112_941426_1 16 dme-mir-963_droWil1 123 25 21M * 0 0 TTTTCGTTAAGCCCAACACAA * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:5T14T0 V112_mapped_droWil1.sam:V112_1298991_1 16 dme-mir-963_droWil1 122 25 21M * 0 0 TTTTTCGTTAAGCCCAACACA * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:0G5T14 V043_mapped_droWil1.sam:V043_198574_1 0 dme-mir-963_droWil1 27 37 10M1I7M * 0 0 TAATTCTAGTGCTAATAC * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:14A2 V051_mapped_droWil1.sam:V051_563156_1 16 dme-mir-963_droWil1 73 25 19M * 0 0 TGGAGCCTGAAACATCCGT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:2A13T2 M040_mapped_droWil1.sam:M040_4092641_1 16 dme-mir-963_droWil1 22 37 9M1I10M * 0 0 AATTCTAATCTCTCGTCTAA * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:12A6 M040_mapped_droWil1.sam:M040_4593573_1 0 dme-mir-963_droWil1 68 37 10M1I7M * 0 0 ATATTTGGAGACCTGAAA * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:7A9 M040_mapped_droWil1.sam:M040_6556843_1 0 dme-mir-963_droWil1 42 37 21M * 0 0 ACAAGGTACATATCAGGTTGT * XT:A:U NM:i:0 X0:i:1 X1:i:0 XM:i:0 XO:i:0 XG:i:0 MD:Z:21 M040_mapped_droWil1.sam:M040_6571014_1 20 dme-mir-963_droWil1 143 37 12M1D7M * 0 0 TTTGTTTTTACGCCAACAC * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:10A1^C7 M040_mapped_droWil1.sam:M040_7361873_1 20 dme-mir-963_droWil1 147 25 18M * 0 0 TTTTTAAGGCGAACACAT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:8C1C7 M040_mapped_droWil1.sam:M040_9413604_1 0 dme-mir-963_droWil1 84 25 22M * 0 0 ACATCTGTATATACCTTTGTTC * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:10C10T0 This miRNA appears confidently lost in the obscura lineage.
dme-mir-973 - 978	--	Not found	Given that both flanking coding genes are on different chromsomes, we may be able to define forward and reverse primers on different chrsomosomes, however it is still unsure how large the intermediate region will be.	Y	Nothing done	These miRNAs lie in the intergenic region between CG32532/dmel and Grip84/dmel. CG32532/dmel has the ortholog GA23014/dpse (chrXL_group1a:5357217-5385309) and Grip84/dmel has the ortholog GA17771/dpse (chrXL_group1e:12505277-12510040 +). These two orthlogous genes lie on different chromsomes. In fact, GA17771/dpse lies 15Kb from the end of chrXL_group1e. It is highly suspicioius that these miRNA orthologs are missing given the choppy assembly in this region.

droPer1

miRNA Name	Query Species	Result	Fragment to clone	Experiment Needed	Experiment Decision	Sequence/Notes
dme-mir-2500	dp4	Found		N	NA	Dm3 miRNA lies within CG3764/dme. This gene has an ortholog reported in dp4, namely dpse\GA17671, but none reported for droPer1 according to Flybase. According to the nets and chains track for droPer1, the ortholog should lie in a stretch of ambiguity characters (N's) at position super_29:1057504-1057925. Searching the droPer1 trace reads with the dp4 ortholog correctly locates the ortholog. >dme-mir-2500-dp4 AAGAACTTTTTGCAGGTAAGCCATCACCAGCATTGAAAGCCATTGAAATGCGTCTAAATAGCAGTCTCTCTCCTCCCACAGATCATGTTCCAGGCC >gnl\|ti\|780630171:515-611 AAGAACTTTTTGCAGGTAAGCCATCACCAGCATTGAAAGCCATTGAAATGCGTCTAAATAGCAGTCTCTCTCCTCCCACAGATCATGTTCCAGGCC
dme-mir-285	dp4	Found		N	NA	This miRNA is intergenic and there appear to be no nearby genes. The correct persimilis ortholog is found by searching the persimilis trace reads for the pseudobscura sequence via LASTZ. >dme-mir-285-dp4 TCTTACAAT-TACAGTTCGAATCGAAGAAC-TGAGATCGATTGGTGCATAGATATCAAGAGGACTCGCTAATTTTCAACTCTAGCACCATTCGAAATCAGTGCTTTTGATGAGAACCATTCAACAGA >gnl\|ti\|738578230:636-763 TCTTACAATTTACAGTGGGAATCGAAGAACCTGAGATCGATTGGTGCATAGATATCAAGAGGACTCGATAATTTTCAACTCTAGCACCATTCGAAATCAGTGCTTTTGATGAGAACCATTCAACAGA
dme-mir-304	Dp4, droWil1	Not found	super_26:1090127-1093182 (3056nt)	Y	1st round of resequencing indicates that the ortholog may lie immediately before the gap, but the quality of the returned sequence was poor.	A search of the persimilis trace data with the dp4 or droWil1 orthologs did not return any hits. This miRNA lies in an intron of Gmap/dmel. It is unusual that flybase did not report an ortholog for this gene in persimilis but did for pseudobscura (GA22886/dpse). 304 lies on the same intron as 283/dme and 12/dme and the droPer1 ortholog for these were found; this is the first peice of evidence to suggest that the ortholog exists. The flanking synthenic regions of dp4's ortholog offer some clues as to where the droPer1 ortholog may lie. droPer1 ortholog may lie in a gap region located at super_26:1091883-1092236. This gap is 353nt long. The two neighboring assembly read only go 77nt and 28nt into the gap from either side, but it seem like the miRNA lies more toward the middle of this gap and thus uncovered by reads. Does the persimillis reads map to the pseudobscura lineage? Yes. Reads from all persimilis libraries align to the dp4 ortholog. For example V042_1404_168, V111_2403_579, and V050_394_390. These reads are all 23nt in length and contain the DNA sequence "TAATCTCAATTTGTAAATGTGAG". These reads are unmapped to the droPer1 genome assembly genome assembly. The DNA sequence of these reads also do not mapp to the trace reads for droPer1.
dme-mir-311	Dp4	Found		N	Cluster will be resequenced anyways	>dme-mir-311-dp4 CATCGAGCTGCTTTGATTTGTAGGCCGTGGTTCTTGCAAATACGGATTCATAACGTATTGCACTAGCCCCGGTCCAAAAAACAATAGCAACGCCGGCAACAGCAAA >gnl\|ti\|732560583:591-697 CATCGAGCTGCTTTGATTTGTAGGCCGTGGTTCTTGCAAATACGAATTCATAACGTATTGCACTAGCCCCGGTCCAAAAAACAATAGCAACGCCGGCAACAGCAAA When this droPer1 sequence is blatted against the droPer1 assembly, reads pileup nicely on both mature and star regions (super_4:2,566,608-2,566,713). The MAF does not report this region as the ortholog to the dm3 sequence. Instead, the MAF reports that this sequence partly belongs to 312/dper. 312/dper looks genuinely missing and is confident in pseudobscura, willistoni, and grimshawi.
dme-mir-312	Dp4	Found		N	Cluster will be resequenced anyways	312/dper looks genuinely missing but is confident in pseudobscura, willistoni, and grimshawi. Searching with the 312/dpse actually finds the correct persimilis sequence which also turns out to be 313/dpse. >dme-mir-312-dp4 GGGCAATACTGTGTTGTATTTCACCAGTATTGCACACCCACTGGCCTGAAAGTGCCTACTGCTGGGTTCAAA >gnl\|ti\|732455623:23-94_313_dpse GGGCAATACTGTGTTGTATTTGAACAGTATTGCACACCCACTGGCCTGAAAGTGCCTACTGCTGGGTTCAA-
dme-mir-313	droAna3	Not found	super_4:2565122-2567706 (2584nt)	Y	Cluster will be resequenced	Similar to 313/dpse, this ortholog is also missing. Searching the droPer1 assembly with the annanasae ortholog also did not turn up any hits. Given that species that outgroup the obscura lineage contain orthologs for this miRNA, resequencing is need to validate if the 313/dper ortholog is genuinely lost. It also would not hurt to resequence this enter cluster within both obscura species.
dme-mir-955	droAna3, droWil1	Not found	super_65:50349-56133 (5784nt)	N	Confidently lost in obscura subgroup.	955/dmel lies in an intergenic region. The flanking coding genes to this miRNA in melanogaster has no reported ortholog in persimilis. However, it is unusual to have orthologs reported in pseudobscura and not in persimilis. Looking at the persimilis assembly suggest that the homologous flanking genes may lie in the gap regions below.. Searching the trace data with droAna3 or droWil1 orthologs turned up very limited number of reads. Here are the reads mapping to the willistoni ortholog: V111_mapped_droWil1.sam:V111_801966_1 0 dme-mir-955_droWil1 35 25 23M * 0 0 CATCGTGCAGAGGTTTGAGTGTC * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:8T0T13 V111_mapped_droWil1.sam:V111_974686_1 0 dme-mir-955_droWil1 35 25 19M * 0 0 CATCGTGCAGAGGTTTGAG * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:8T0T9 V042_mapped_droWil1.sam:V042_610002_1 4 dme-mir-955_droWil1 118 25 18M * 0 0 CAGCCTAAAAAGTACAAT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:10T2G4 M021_mapped_droWil1.sam:M021_65469_10 16 dme-mir-955_droWil1 62 37 6M1I11M * 0 0 TTTGTTGTTCTCCAATGG * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:11T5 M021_mapped_droWil1.sam:M021_104734_6 0 dme-mir-955_droWil1 16 25 18M * 0 0 CATTAAGGATGGCTGGCT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:1G6T9 M021_mapped_droWil1.sam:M021_1027206_1 0 dme-mir-955_droWil1 10 37 12M1D7M * 0 0 CTTTTGCGTTAAGTTGACT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:12^G4G2 M042_mapped_droWil1.sam:M042_1599213_1 16 dme-mir-955_droWil1 99 37 5M1D15M * 0 0 GAGACGCAAATCGAGAAACA * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:5^G2C12 M042_mapped_droWil1.sam:M042_1665682_1 0 dme-mir-955_droWil1 49 37 3M1I14M * 0 0 TTGAAGTGTCCTCGTTTG * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:9T7 This miRNA looks confidently lost in the obscura subgroup.
dme-mir-956	Dp4	Found		N	NA	>dme-mir-956-dp4 ATGTGCACTTCTACTGATCGTTATCGTGTTTGGAATGGTCTCGTTAGCTAACGGATGAGCAACTGCTTGCGCGCATTGGCCAAATGCATTTCGCACCGGGATGGATGTGGATGTGGCTGGATGTGGTGTCCCCAAGCACAGCCAGGTGTAGTTTCGAGACCACTCTAATCCATTGCACCGCCCACCCATTTATGCAAA >gnl\|ti\|732454354:721-914 ATGTGCACTTCTACTGATCGTTATCGTGTTTGGAATGGTCTCGTTAGCTAACGGATGAGCAACTGCTTGCGCGCATTGG-CAAATGCATTTCGCACCG-GATGGATGTGGATGTGGCTGGATGTG-TGTTCCCAAGCACAGCCA-GTGTAGTTTCGAGACCACTCTA-TCCATTGCACCGCCCACCCATTTATGCAAA
dme-mir-963	droAna3, droWil1	Not found		N	Confidently lost in obscura lineage.	This miRNA lie within the gene CG31646/dme. Flybase does not report an ortholog for this gene in the obscura lineage. The adjacent 964 has somewhat questionable orthologs, but it does have good read pileup on the mature and star strands. Searching with the ananassae and willistoni orthologs in persimilis assembly reads returned very few reads which did not map perfectly. Here are the reads mapping to the willistoni ortholog: V111_mapped_droWil1.sam:V111_95073_7 0 dme-mir-963_droWil1 42 25 24M * 0 0 ACAAGGTACATATCAGAGTGTTTC * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:16G0T6 V111_mapped_droWil1.sam:V111_171720_3 0 dme-mir-963_droWil1 41 25 24M * 0 0 AACAAGGTACATATCAGAGTGTTT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:17G0T5 V111_mapped_droWil1.sam:V111_799750_1 0 dme-mir-963_droWil1 42 25 23M * 0 0 ACAAGGTACATATCAGAGTGTTT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:16G0T5 V111_mapped_droWil1.sam:V111_1107330_1 16 dme-mir-963_droWil1 23 25 20M * 0 0 ATCCTAATTCTCGTCTAAAA * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:2T8A8 V111_mapped_droWil1.sam:V111_1107436_1 0 dme-mir-963_droWil1 42 37 24M * 0 0 ACAAGGTAAATATCAGGTTGTTTC * XT:A:U NM:i:1 X0:i:1 X1:i:0 XM:i:1 XO:i:0 XG:i:0 MD:Z:8C15 V042_mapped_droWil1.sam:V042_243826_1 20 dme-mir-963_droWil1 147 23 7M1I10M * 0 0 TTTTTAACGCCCAACACA * XT:A:U NM:i:1 X0:i:1 X1:i:1 XM:i:0 XO:i:1 XG:i:1 MD:Z:17 XA:Z:dme-mir-963_droWil1,-126,7M1I10M,2; V057_mapped_droWil1.sam:V057_1010865_1 16 dme-mir-963_droWil1 75 25 18M * 0 0 AAACCTCAAACATCTGTA * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:2G3G11 V057_mapped_droWil1.sam:V057_1016836_1 16 dme-mir-963_droWil1 75 25 18M * 0 0 AAGCCTGAACCATATGTA * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:9A3C4 M021_mapped_droWil1.sam:M021_406362_2 0 dme-mir-963_droWil1 60 37 4M1I13M * 0 0 TGTTGTCGTTTATTTGAA * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:8A8 M021_mapped_droWil1.sam:M021_715756_1 0 dme-mir-963_droWil1 92 37 4M1I15M * 0 0 ATCTGATCTTTGTTTCGAAT * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:5C13 M042_mapped_droWil1.sam:M042_1047908_1 0 dme-mir-963_droWil1 79 37 12M1I7M * 0 0 CTGGAACATCTGCTATCTAC * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:1 XO:i:1 XG:i:1 MD:Z:3A15 M042_mapped_droWil1.sam:M042_3478115_1 0 dme-mir-963_droWil1 117 25 19M * 0 0 AAAGAAATTTCTTTAAGCC * XT:A:U NM:i:2 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 MD:Z:5G0T12 This miRNA appears confidently lost in the obscura lineage.
dme-mir-973 - 978	droAna3, droWil1	Not found	super_14:1988476-2012995 (24519nt) Will have to PCR out this region in several fragments.	Y	Nothing done	These miRNAs lie in the intergenic region between CG32532/dmel and Grip84/dmel. CG32532/dmel has the ortholog GL14778/dper (super_14:2010114-2033134) and Grip84/dmel has the ortholog GL14777/dper (scaffold_14:1985718-1990982). Unlike the pseudobscura orthologs which lie on separate chromosomes, both of these orthologous coding genes lie on the same scaffold. However, there is no reads within this ~24kb region indicative of RNase III cleavage: It is still uncertain whether these miRNAs are confidently lost in the obscura lineage. Some dp4 and droPer1 adult male-body reads do map to the droAna3 and droWil1 orthologs for all of these miRNAs suggesting that the correct obscura orthologs remain to be found.
dme-mir-4984	Dp4	Found		N	NA	>dme-mir-4984-dp4 TTAAAATAGTTGCAGCCCACAA-------GTCACATGCCGCCGCCGGCGCATTGGACATTGGAGGTGTGAAAACCAATATTCGCTTCAGGTCCAATCACGTTTCAAGATCGAGGTGAATTCTTTGACGTATTCGCTTGGCAAATGACACGTTTTGGACCTGCAATTTCCAGCATTTACTTTCATT >gnl\|ti\|733036400:69-254 TTAAAATAGTTGCAGCCCACAAGTCACAAGTCACATGCCGCCGCCGGCGCATTGGACATTGGAGGTGTGAAAACCAATATTCGCTTCAGGTCCAATCACGTTTCAAGATCGAGGTGAATTCTTTGACGTATTCGCTTGGCAAATGACACGTTTTGGACCTGCAATTTCCAGCATTTACTTTCATT Read gnl\|ti\|733036400 overlap with the adjacent gap at position super_1:4362270-4362410

droWil1

miRNA Name

Query Species

Result

Experiment Needed

Experiment decision

Sequence/Notes

dme-mir-4977

droPer1

Not found

This miRNA is not expressed outside the melanogaster-subgroup even though all of the orthologs have a hairpin fold. Not sure what the use is to find the true hairpin-forming willistoni ortholog then.

Searching the willistoni trace reads with the persimilis ortholog as query with LASTZ did not turn up any hits.

This miRNA lies in the 5' UTR of the gene Eaf. The reported flybase ortholog for this gene is dwil/GK22015. Searching the entire dwil/GK22015 gene body with the persimilis sequence via LASTZ did not turn up any hits either. Apart from this, the droWil1 ortholog reported in the mutiple alignment seems accurate given the high net which it belongs to and the high synteny of the flanking sequence. It appears that the willistoni ortholog simply diverged too fast into a lesser-structured sequence compared to its neighboring species on the phylogeny.

Do the willistoni reads map to to the 4977/dper or 4977/dvir orthologs?

There very few reads that map to either the persimllis or virillis orthologs. The ones that do map are not perfect.

CONCLUSION: This ortholog looks genuinely loss in droWil. There seems to be two insertions of simple-repeats into the droWil1 ortholog that makes it not fold like the nicer folds that droPer1 or droVir3 have. Keep in mind that these other neiboring species may have nice hairpin structures, however there are no sRNAseq reads to support that these orthologs are functional as miRNAs.

droVir3

miRNA Name	Query Species	Result	Fragment for cloning	Experiment Needed	Experiment decision	Sequence/Notes
dme-mir-312	droGri2	Found	scaffold_12875:13668426-13669695 (1269nt)	Y	Cluster will be sequenced	Searching with the grimshawi ortholog against the virilis genome trace reads found the sequence that is supposed to be 313/dvir. >dme-mir-312-droGri2 AAGCAAATCTTCAGTTTTTCGGCTGTGGTTAGTGTCAATTCTTTTTAT-----TTGACAAGCATTGCACTCTTCACGGCCGGATAAAT-GAGGGTCT >gnl\|ti\|391667200:98-186_313_droVir3 A------TCGCTAGTTTTTCGGCTGTGGTCAGTGTCAATTCTTTTTTTTTATTTTTACAAGTATTGCACTTTTCACGGCCGGAAAAATTGAGA-T-- This observation is actally also true for persimilis where the 313/dper ortholog was found when searching for the 312/dper ortholog with 312/dp4 query.
dme-mir-959	droWil1	Not Found	scaffold_12963:3614884-3616293 (1409nt)	Y	Confidently lost	Searching for the correct droVir3 ortholog with the droWil1 ortholog did not turn up any hits. It appears that 959, 960, and 961 are all lost in D. virilis. For 959 at least, the reported mojavensis and grimshawi orthologs are confident although neither of these orthologs has any small RNA reads 4 virilis reads mapped onto the 959/dwil ortholog. It would still be good to validate that this miRNA is lost in the virilis group.
dme-mir-960	droMoj3, droWil1	Not found		Y	Confidently lost	Searching for the droWil1 or droMoj3 ortholog in droVir3 trace reads did not turn up any hits. Do the virillis reads map to to the 960/dmoj ortholog? There were only 5 droVir3 sRNAseq reads accross 3 libraries (Embryo, Male body and [Head?]) that aligned to the droWil1 ortholog. This suggest that this ortholog may be genuinely loss, but it would still be good to PCR out this entire cluster region in droVir3 and resequence it.
dme-mir-961	droWil1	Not Found		Y	Confidently lost.	Searching for the correct droVir3 ortholog with the droWil1 ortholog did not turn up any hits. It appears that 959, 960, and 961 are all lost in D. virilis. For 961, the reported mojavensis and grimshawi orthologs are confident although neither of these orthologs has any small RNA reads. 11 virilis reads mapped onto the 961/dwil ortholog. It would still be good to validate that this miRNA is lost in the virilis group.
dme-mir-968	droWil1	Not found		Yes	Sequence the 1002 and 968 virilis orthologs	This miRNA is located antisense to an intron, however, I could not locate the gene or intron that emcompassed both this miRNA nor 1002, an adjacent miRNA. Searching for the correct droVir3 ortholog with the droWil1 ortholog was unsuccessful. It should be noted that 968/dmoj is also missing yet d68/dgri looks confident and has one 15 reads. Many virilis reads mapped to the grimshawi ortholog. For example the read V047_15_28649 corresponds to the mature sequence "TAAGTAGTAACCATTAAGAGGTTG".
dme-mir-1002	droWil1,droGri2	Not found		Yes	Sequence the 1002 and 968 virilis orthologs	Many virilis reads mapped to the willistoni ortholog. For example, the reads V047_109_2049 corresponds to the mature sequence "TTAAGTAGTTAATACAAGGGCGA". It appears that the star sequence may be V041_168952_1 which corresponds to "GCATTGTGTGAGCTACTTC"
dme-mir-978	droWil1, droGri2	Found		N	NA	>gnl\|ti\|376844870:534-649 A------------------------CAGGCACAGCCGTACTCTACGCTTTTGGGAACGAGCTCTTTGACGCACTCGGTTCCATTGCCGTTGAGTAGAGCTGTCCTGATGCATATCTCAACGT------ATTGATTCAAACAAGAA >dme-mir-978_droGri2 AAGTACGGCAGTTCGATAACCAACACAGGTCCAGCCGTATTCAGCGCTTTTGGAAGTAAGGAACATGATTCTCCTAGTTTCAATGCCGCTGAGTAAAGCTGTCCCAATGCAAATCGCAACGTAACTGAATCGAATCAATCAATAA The correct ortholog was found by searching the virilis trace reads with the grimshawi ortholog. A simlar search with the willistoni ortholog did not uncover any hits. Note that the mojavensis ortholog was not found.

droMoj3

miRNA Name	Query Species	Result	Fragment for Cloning	Experiment Needed	Experiment decision	Sequence/Notes
dme-mir-312	droGri2	Found	scaffold_6496:9090108-9091422 (1314nt)	Y	Cluster will be resequenced.	Searching with the grimshawi ortholog against the mojavensis genome trace reads found the sequence that is supposed to be 313/moj. >dme-mir-312-droGri2 AAGCAAATCTTCAGTTTTTCGGCTGTGGTTAGTGTCAATTCTTTTTATTTGACAAGCATTGCACTCTTCACGGCCGGATAAATGAGGGTCT >gnl\|ti\|490408270:305-377 -------------GTTTTCCGGTTGTGAATTGTGTCAATTCTTTTTATTTTACAAGTATTGCACTTTTCACGGCCGGAAAAAGGA------ This observation is actally also true for persimilis where the 313/dper ortholog was found when searching for the 312/dper ortholog with 312/dp4 query.
dme-mir-263b	droVir3, droGri2	Not found	Genomic coordinate is unknown. We may have to design primers using the virilis or grimshawi orthologs.	Y	Will be resequenced.	Searching with the droVir3 or droGri2 ortholog in the droMoj3 trace reads did not turn up any hits. No good chains exist for this miRNA in the droMoj3 assembly. This miRNA lies in an intron of the gene dme/CG32150. Flybase does not report an ortholog for this gene in mojavensis, however does report orthologs in virilus (dvir\GJ11223) and grimshawi (dgri\GH14363). I tried looking for flanking genes to dme/CG32150 and found two with flybase-reported orthologs in D. mojavensis- dmoj/GI13967 and dmoj/GI11303. Next I looked for gaps in the intermediate region of these two orthologs and found 5. The genomic coordinates for the top 3 are shown in the image below. We should seqeuence any of these regions. Do the mojavensis reads map to to the 263b/dvir ortholog? Yes. Many reads from the mojavensis library maps to the virilus ortholog. The most abundant uniquely mapped reads are V041_122_1442, V110_640_2972, and V049_149_2107. The reads all have the same 23nt DNA sequence "CTTGGCACTGGGAGAATTCACAG". Note that these reads did not map to the droMoj3 genome assembly. Even more surprising, the DNA sequence of these reads could not be found via LASTZ in the droMoj3 trace reads either.
dme-mir-968	droWil1	Not found	Actual genomic location is unknown. We may have to design primers using the willistoni ortholog.	Y	Nothing done	The MAF reports the 968/dmoj ortholog in the correct position. That is, upstream of the 1002 ortholog just as in every other species. However this ortholog has no reads: The mojavensis read V041_35_10366 mapped to the willistoni ortholog with 2 mismatches. This read is "TAAGTAGTAACCATTAAAAGGTCG" and corresponds to the mature miRNA for the other species. This is a good indication that the correct ortholog remains to be found. Searching for the correct droVir3 ortholog with the droWil1 ortholog was unsuccessful. It should be noted that 968/dvir is also missing yet 968/dgri looks confident and has 2 perfectly-mapped reads.
dme-mir-1002	droWil1, droGri2	Not found		Y	Nothing done	The mojavensis reads V041_58_4018 mapped to the willistoni ortholog and corresponds to the mature sequence "TTAAGTAGTTGATACAAAGGCGA" with 2 mismatches.
dme-mir-978	droWil1, droGri2	Not found	scaffold_6328:2896355-2898115 (1760nt)	Y	Nothing done	The correct mojavensis ortholog could not be found by searching the trace data with the willistoni or grimshawi orthologs. However, the virilis ortholog was found. Given that the grimshawi and virilis orthologs are confident, it is unlikely that the mojavensis ortholog is genuinely lost. Resequencing is needed for this miRNA. There are also many unannotated miRNAs in the vicinity of 978/dmoj. Could one of these be 978/dmoj?

droGri2

miRNA Name

Query Species

Result

Fragment to clone

Experiment Needed

Experiment decision

Sequence/Notes

dme-mir-974 -977

droMoj3

Not found

scaffold_15081:2040325-2051833

(11508nt)

Nothing done

The grimshawi ortholog is present in 973 and 978, however it is missing from many of the miRNAs that lie between these two miRNAs. Explicitly, these miRNAs include 974, 975, 976 and 977:

The 974/dmoj and 975/dmoj orthologs were used as queries in a search against the grimshawi assembly. No orthologs were found with either of these queries.

Several singleton reads from the grimshawi small RNA library mapped to the 974-977 orthologs. These may be suprious mappings, however it would still be good to validate via experiments if these miRNAs are genuinely missing from grimshawi.