The sequence of the Drosophila melanogaster genome is available for several years and gene models are well annotated through computational and experimental strategies. NEXT-RNAi was used to design multiple independent long dsRNAs covering all Drosophila genes based on the latest genome release (FlyBase release 5.24). Each design was intended to target all splice variants of a given target gene. Overall approx. 74,900 common regions for 14,898 annotated coding and non-coding genes were selected as target sites, resulting in an average of about 5 regions per gene. The Drosophila transcriptome was used as database to evaluate siRNA specificities.
NEXT-RNAi results
NEXT-RNAi HTML ouputs are available here
Overall ~70,149 designs were obtained, covering 99.4% of the genome. 83 gene models could not be targeted due to low complexity regions that are not suitable for primer design. 90.7% of all genes are covered by a design that does not show homology of 19 nt or longer to any other gene. 88.7% of all genes are covered by at least one second, independent design.
Input files and settings used
Input FASTA file
COMMON_REGIONS_r5.24_ALL_EXTENDED_split_long.zip (8.2 MB) containing target sequences as input file (-i input).
>FBgn0029994_cr4 AGTGTTTACTTTGCTCAATATTCAGTCCGAATTCGTTAACAGCCGCTAACGGTTTGCTCCGGCTGCTCC GATCCCTTCCTATATACGTATATATATATATAGATATATATATACATTAACGATCCTCGCAGGATTTAG CCAAATACACGAAACATGTCGAAAGTGACGCAAAG >FBgn0037191_cr1_1 CAAGACTTTTAATGTAGTTTTAATTTTTGAACTAATCGACCGTGTACATTTAGTAGTAACATTTTTTTC ATATTGGCTAAGGATGAACAAAAGCCAAAAGTCCCTGGCGGACGACATGGCCTCGACGCCTTTCGGCAA GGAGGTGCTGATTGGCAACTGGGCGGAGCGCCGCTACGCAGTCGAGGAGCAGAGCAATGCCATCCTGCC AGGACTCCGTGTGAGTGGTTGCGAGCTCCATAGGTCGCTCTGCCACGACACCTACACTACTGCACCCTT CGTTGGCGCCGAGGCAGTACCCTTGTTTGTGGATCATCGCAAATTGGCTTACAAGAACTTTATAAGGAA TAGGCGATCTAGCTTGAACCTGGTGGACGATGAGCTACTCAAGAGGAACTTCACTACCA
Targetgroup file (tab-delimited)
FBtr-FBgn-CG-Txn-Name-r5.24.tab (1.4MB) defining which FlyBase transcripts belong to the same gene (headers Target and TargetGroup) (TARGETGROUPS option)
Target TargetGroup #1 #2 #3 #4 FBtr0085315 FBgn0039596 CG10000-RA CG10000-RA CG10000 CG10000 FBtr0085316 FBgn0039595 AR-2-RA CG10001-RA CG10001 AR-2 FBtr0085321 FBgn0000659 fkh-RA CG10002-RA CG10002 fkh FBtr0300259 FBgn0000659 fkh-RB CG10002-RB CG10002 fkh FBtr0082507 FBgn0037972 CG10005-RA CG10005-RA CG10005 CG10005
Intended target file
COMMON_REGIONS_r5.24_ALL_EXTENDED_split.intended (2.1MB) defining intended gene targets of queried identifiers (helps NEXT-RNAi to show intended target of a long dsRNA as first target in case it has multiple perfect targets) (INTENDED option). Here the intended gene targets are equal to the defined TargetGroup.
Query Intended FBgn0029994_cr1 FBgn0029994 FBgn0029994_cr2 FBgn0029994 FBgn0029994_cr3 FBgn0029994 FBgn0029994_cr4 FBgn0029994 FBgn0037191_cr1_1 FBgn0037191 FBgn0037191_cr1_2 FBgn0037191 FBgn0036810_cr1_1 FBgn0036810
Bowtie database/index for off-target evaluation
Bowtie database/index containing annotated FlyBase (release 5.24) transcripts for specificity calculations (-d input):
dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta.tar.gz (55MB)
Bowtie database/index for mapping of reagents
The Bowtie database/index (GENOMEBOWTIE option) for mapping of reagents to the Drosophila genome:
dmel-all-chromosome-r5.24.tar.gz (154MB)
Bowtie indexes for genomes are also available through the Bowtie webpage.
Feature file with UTR locations
Tab-delimited feature file containing FlyBase mappings of UTRs to chromosomes that is used to calculate the 'UTR-content' (FEATURE option) of designed reagents: dmel-all-UTRs-r5.24.zip (65MB)
UTRID FeatureName FeatureLoc FeatureStart FeatureEnd UTR_1 UTR 2RHet 2901830 2901830 UTR_2 UTR 2RHet 1335191 1335191 UTR_3 UTR 2RHet 198854 198854
FASTA file for homology evaluation
Transcriptome FASTA file to evaluate the homology of the designs using Blast (HOMOLOGY option):
dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta.zip (14MB)
FASTA file containing miRNAs (miRBase)
FASTA file containing miRNAs downloaded from miRBase used to avoid conserved miRNA seeds from long dsRNA designs (MIRSEED option):
miRBase_r14_Dmel.fa (16KB)
Design criteria
Start of program
perl nextrnai.pl -i COMMON_REGIONS_r5.24_ALL_EXTENDED_split_long.fa -s 2500 -r d -d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta -e NO -o options.txt -n Dmel_r524
Descriptions for start parameters used are available here.
Options file
DESIGNWINDOW=80,250 DESIGNNUM=50 OUTPUTNUM=1 SIRNALENGTH=19 EFFICIENCY=SIR,0 REDESIGN=ON INTRON=90 BOWTIE=/usr/bin/ TARGETGROUPS=FBtr-FBgn-CG-Txn-Name-r5.24.tab INTENDED=COMMON_REGIONS_r5.24_ALL_EXTENDED_split.intended PRIMER3=/usr/bin/ GENOMEBOWTIE=dmel-all-chromosome-r5.24 GFF=GFF3 GBROWSEBASE=http://www.dkfz.de/signaling/cgi-bin/gbrowse_img/flybase/ GBROWSETRACK=GENE+TXN AFF=YES LOWCOMPEVAL=/usr/bin/ CANEVAL=6 HOMOLOGY=/usr/bin/,dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta,1e-10 FEATURE=dmel-all-UTRs-r5.24.tab MIRSEED=7,miRBase_r14_Dmel.fa RANKD=SPEC
Descriptions for all options used are available here.