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 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).
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)
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.
Bowtie database/index for off-target evaluation
Bowtie database/index containing annotated FlyBase (release 5.24) transcripts for specificity calculations (-d input):
Bowtie database/index for mapping of reagents
The Bowtie database/index (GENOMEBOWTIE option) for mapping of reagents to the Drosophila genome:
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)
FASTA file for homology evaluation
Transcriptome FASTA file to evaluate the homology of the designs using Blast (HOMOLOGY option):
FASTA file containing miRNAs (miRBase)
Start of program
Descriptions for start parameters used are available here.
Descriptions for all options used are available here.