All predicted exons (71,788) from the 'official gene set' of the recently sequenced genome of the red flour beetle, Tribolium castaneum, were used to calculate independent designs of long dsRNAs for the Tribolium genome (exons longer 560 nt were additionally splitted in two sequences, resulting in overall 80,671 input sequences). Tribolium has become an important model organism for developmental and evolutionary studies and is suitable for RNAi by injection of long dsRNAs. Information on predicted genes and sequences was obtained from BeetleBase that integrates sequence annotations for Tribolium.
NEXT-RNAi HTML ouputs are available here
Overall 71,293 designs were obtained, covering 99.4% of the genome. 92.9% of all genes are covered by at least one design that does not show homology of 19 nt or longer to any other gene. 83.2% of all genes are additionally covered by at least one second, independent design.
beetlebase_exons_split.zip (6.6MB) containing exon target sequences as input file (-i input) (parsed from Tribolium GFF files).
>TC016216-RA-E3 GGCACGCACATCTCAGTCCTGCCAACGGAAATGATTTTGCACATTTTGCGGTGGGTGGTGAGTGCGGAG CTGGATTTGAGGTCGCTGGAGATGTTTTCGATGGTGTGTCGGGGCTTTTATTTGTGCGCACGTGACCCG GAGGTGTGGCGGTTGGCGTGCCTGAG >TC008956-RA-E3_1 GCTGCACTTCCGACCGAAGACGCCGCCCCCCGACGAAGAGTCCCCGTGCAGTTTGCTCCCGTTCAGCGA GAGTTATTTCGTCCCCGGTGCCAACATGTCGTTCTCAATCGAGGTCCAGTCGTTGGAAACGCCAAGTAC ACTCCTGGACACTTCCGCTTTCTTAACCGATCCATTCACGTTTGATAATTTGGACAGTATAGAAGACGT ACCGGTAACCGACACGCGGCAATCCAGTGATAAAATCGTGCAAACGGGCATTAACTTGAACGTTAATGT CGGTCTCAATACGCTTACTCCTAGTGTTAACG >TC008956-RA-E3_2 ATAATAGTAGTAGTGTTTTTTCAAATAGTAACGCGAACCTCACTGATAGTATCAAGCTCGAGGATGCGA CGTCATCGTCGACGTCGGACGAAAACGACGAAAGGACTATCGGATGTTCGAGGTTTTACGTGTCGGATT TGGGCGTCACGAAGAAAAATCAAACGTCCAAAACGCAAACCGTTACGAGTACCTTGACTAGTCCTAAGT TTAATAGTGTCAACGCCGTGTCTGAGGGATGGAGTAGGAGTACAGCCACCGAAGGGCCCTTGACTAACA CCAACGGACTGCTCTCGCAGTTTGGCCTGGATG
beetlebase_targetgroups.tab (732KB) defining which BeetleBase transcripts belong to the same gene (headers Target and TargetGroup) (TARGETGROUPS option)
Target TargetGroup TxnName GeneName TC004355-RA TC004355 TC004355-RA GLEAN_04355 TC004356-RA TC004356 TC004356-RA GLEAN_04356 TC004357-RA TC004357 TC004357-RA GLEAN_04357 TC004358-RA TC004358 TC004358-RA GLEAN_04358 TC004359-RA TC004359 TC004359-RA GLEAN_04359 TC004360-RA TC004360 TC004360-RA GLEAN_04360
Bowtie database/index containing annotated BeetleBase transcripts for specificity calculations (-d input):
The Bowtie database/index (GENOMEBOWTIE option) for mapping of reagents to the Tribolium genome:
Bowtie indexes for genomes are also available through the Bowtie webpage.
Transcriptome FASTA file to evaluate the homology of the designs using Blast (HOMOLOGY option):
perl nextrnai.pl -i beetlebase_exons_split.fa -s 2500 -r d -d beetlebase_transcript -e NO -o options.txt -n Tcas_3.0
Descriptions for start parameters used are available here.
DESIGNWINDOW=80,250 DESIGNNUM=50 OUTPUTNUM=1 SIRNALENGTH=19 EFFICIENCY=SIR,0 REDESIGN=ON BOWTIE=/usr/bin/ TARGETGROUPS=beetlebase_targetgroups.tab PRIMER3=/usr/bin/ GENOMEBOWTIE=beetlebase_genome GFF=GFF3 GBROWSEBASE=http://www.dkfz.de/signaling/cgi-bin/gbrowse_img/beetlebase/ GBROWSETRACK=GENE+TXN AFF=YES LOWCOMPEVAL=/usr/bin/ CANEVAL=6 HOMOLOGY=/usr/bin/,beetlebase_transcript.fa,1e-10 RANKD=SPEC
Descriptions for all options used are available here.