Eight large-scale RNAi libraries designed for the Drosophila genome (Ambion, HD2, HFA/DRSC v1.0, DRSC v2.0, OpenBiosystems v1/v2, MRC, NIGFLY and VDRC) were re-annotated using the FlyBase annotations of release 5.24.
NEXT-RNAi results
HTML reports
NEXT-RNAi HTML ouputs for the library evaluations are available here:
Pairwise comparisons of libraries
We used NEXT-RNAi results to identify the number of genes that could be targeted with independent designs through pairwise combinations of all Drosophila RNAi libraries. A summary report for all pairwise combinations is contained in libComparison_report.txt (44KB).
A detailed analysis for each combination of two libraries mapping reagents available for a certain target gene in one library to the reagents for this gene available in the other library (including information whether these reagents are 'completely', 'partially' or 'not' independent ) is available for download (libComparison.tar.gz (15MB).
Input files and settings used
Input FASTA files
Ambion, DRSC v2.0 and HD2 libraries were evaluated using long dsRNA sequences as input; HFA/DRSC v1.0, OpenBiosystems v1/v2, MRC and VDRC libraries were evaluated using both, primer and long dsRNA sequences as input and the NIGFLY library was evaluated starting from primers.
- Ambion long dsRNAs: ambion_amplicon.fa (3.3MB)
- HD2 long dsRNAs: HD2Designs_Batch123.fasta (7.2MB)
- HFA/DRSC v1.0 primer and long dsRNAs: HFA_primer.fasta (1.4MB), HFA_dsRNA.fasta (8.6MB)
- DRSC v2.0 long dsRNAs: v2_seqs_MOD.fa (11MB)
- OpenBiosystems v1/v2 primer and long dsRNAs: ob_v01_v02_primer.fasta (960KB), ob_v01_v02_dsRNA.fasta (7.2MB)
- MRC primer and long dsRNAs: mrc_primer.fa (1.1MB), mrc_amplicon.fa (7.4MB)
- NIGFLY primer: nigfly_primer.fa (740KB)
- VDRC primer and long dsRNAs: vdrc_primer.fa (1.1MB), vdrc_amplicon.fa
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)
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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 |
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)
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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)
Evaluation criteria
Start of program
Ambion
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perl nextrnai.pl -i ambion_amplicon.fa -s 14000 -r d -d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta -e DSRNA -o options.txt -n Dmelr524_AMB |
HD2
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perl nextrnai.pl -i HD2Designs_Batch123.fasta -s 14000 -r d -d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta -e DSRNA -o options.txt -n Dmelr524_HD2 |
HFA
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perl nextrnai.pl -i HFA_dsRNA.fasta+HFA_primer.fasta -s 14000 -r d -d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta -e DSRNA+OLIGO -o options.txt -n Dmelr524_HFA |
DRSC v2.0
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perl nextrnai.pl -i v2_seqs_MOD.fa -s 14000 -r d -d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta -e DSRNA -o options.txt -n Dmelr524_DRSCv2 |
OpenBiosystems v1/v2
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perl nextrnai.pl -i ob_v01_v02_dsRNA.fasta+ob_v01_v02_primer.fasta -s 14000 -r d -d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta -e DSRNA+OLIGO -o options.txt -n Dmelr524_OBS_v01_v02 |
MRC
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perl nextrnai.pl -i mrc_amplicon.fa+mrc_primer.fa -s 14000 -r d -d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta -e DSRNA+OLIGO -o options.txt -n Dmelr524_MRC |
NIGFLY
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perl nextrnai.pl -i nigfly_primer.fa -s 15000 -r d -d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta -e OLIGO -o options.txt -n Dmelr524_NIGFLY |
VDRC
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perl nextrnai.pl -i vdrc_amplicon.fa+vdrc_primer.fa -s 14000 -r d -d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta -e DSRNA+OLIGO -o options.txt -n Dmelr524_VDRC |
Descriptions for start parameters used are available here.
Options file
The same options file was used for all evaluation runs.
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DESIGNWINDOW=1,10000 SIRNALENGTH=19 EFFICIENCY=SIR,63 BOWTIE=/usr/bin/ TARGETGROUPS=FBtr-FBgn-CG-Txn-Name-r5.24.tab PRIMER3=/usr/bin/ GENOMEBOWTIE=dmel-all-chromosome-r5.24 BLAT=/usr/bin/ BLATPROGRAM=gfClient BLATHOST=b110-cellarray3 BLATPORT=3501 GENOMEFASTA=dmel-all-chromosome-r5.24.fasta 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 |
Descriptions for all options used are available here.