The mosquito Anopheles gambiae is widely studied to dissect mechanism of innate immunity in its response to Plasmodium falciparum. RNAi can be used in Anopheles tissue culture as well as in live mosquitoes for in vivo studies, leading to efficient depletion of target mRNAs. We used VectorBase annotations to generate input files for NEXT-RNAi. Specifically we designed long dsRNAs against 62,138 exons annotated for the mosquito genome (exons longer 560 nt were splitted in two sequences, resulting in overall 74,944 input sequences for NEXT-RNAi).
NEXT-RNAi results
NEXT-RNAi HTML ouputs are available here
Overall 68.855 designs were obtained, covering 95% of the genome. 89.2% of all genes are covered by at least one design that does not show homology of 19 nt or longer to any other gene. 90.1% of all genes are additionally covered by at least one second, independent design.
Input files and settings used
Input FASTA file
- agambiae_exons_split.zip (7.6MB) containing exon target sequences as input file (-i input).
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>AGAP007336:4_1 AATCAGACTCGACGGAGATGCTCGACTCGGAGACGGAACCGTGCCTCATGATGGACAACGTGCTCGAGG ACGTGACGATGCCGGACTCGCATTCCCACAACATGGTTTCGTCGTCGACCCGCATCTCCCAGATCGAGA TGCCATCGTCGCTCGAGAGTGGCGTTGGAGCGACCAGCAACGCGATGACGATGGCGCACGACGAATCGC TCGATAATCTTTCGCAGGCCATCGCTAACCGGCAGCAGATTGAGCTGCAGTCAAACATGATAGCTAGTG GTCGGTCGTCACGGTTCCACCATTCGGTCGGTAGCA >AGAP007336:4_2 ACAACGACCAGCAGCAGCACCAACAGCAACCGTCTCGCAACACACCGTCCGTGGTAACGTCGGCGGCGG CAGCTATGACGACCGCTCCGGACCACACGACCGCAAGCCACTCTTCGTCGCTGGACGACGAGGAGAACA GCTGCTGCGCGGAGGCGTCGATGGCCGAAATGCCGTCGATTTGCGAGTACTGCAACGCGCAGTCGCTGG TGGATCAGGTGATCGATGTGGACAATCTGATCACGAAGCTGCTGAAGGTGTTGCGGATCGTCCAGATGG ATAATGATCACTGCATACAGGAGCTGATCAATCATAA |
Targetgroup file (tab-delimited)
agambiae_targetgroups.tab (352KB) defining which VectoBase transcripts belong to the same gene (headers Target and TargetGroup) (TARGETGROUPS option
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Target TargetGroup AGAP000002-RA AGAP000002 AGAP000005-RA AGAP000005 AGAP000007-RA AGAP000007 AGAP000008-RA AGAP000008 AGAP000009-RC AGAP000009 AGAP000009-RB AGAP000009 AGAP000009-RA AGAP000009 AGAP000010-RA AGAP000010 |
Bowtie database/index for off-target evaluation
Bowtie database/index containing annotated VectorBase transcripts for specificity calculations (-d input):
agambiae_transcript.tar.gz (28MB)
Bowtie database/index for mapping of reagents
The Bowtie database/index (GENOMEBOWTIE option) for mapping of reagents to the Anopheles genome:
agambiae_genome.tar.gz (250MB)
Bowtie indexes for genomes are also available through the Bowtie webpage.
FASTA file for homology evaluation
Transcriptome FASTA file to evaluate the homology of the designs using Blast (HOMOLOGY option):
agambiae_transcript.zip (14MB)
Design criteria
Start of program
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perl nextrnai.pl -i agambiae_exons_split.fa -s 2500 -r d -d agambiae_transcript -e NO -o options.txt -n AgamP3.5 |
Descriptions for start parameters used are available here.
Options file
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DESIGNWINDOW=80,250 DESIGNNUM=50 OUTPUTNUM=1 SIRNALENGTH=19 EFFICIENCY=SIR,0 REDESIGN=ON BOWTIE=/usr/bin/ TARGETGROUPS=agambiae_targetgroups.tab PRIMER3=/usr/bin/ GENOMEBOWTIE=agambiae_genome GFF=GFF3 GBROWSEBASE=http://www.dkfz.de/signaling/cgi-bin/gbrowse_img/agambiae/ GBROWSETRACK=GENE+TXN AFF=YES LOWCOMPEVAL=/usr/bin/ CANEVAL=6 HOMOLOGY=/usr/bin/,agambiae_transcript.fa,1e-10 RANKD=SPEC |
Descriptions for all options used are available here.