RNAi experiments in mammalian systems require the application of either in vitro diced long dsRNAs (esiRNAs) or synthetic siRNAs. NEXT-RNAi was used to design a genome-wide siRNA library targeting all human genes annotated by the NCBI RefSeq database (release 40). To this end regions common to all RefSeq transcripts belonging to the same gene were computed for the complete human genome (37,627 regions). Further, sequences of low-complexity were filtered using mdust and remaining sequences longer 100 nt were splitted into two sequences to obtain a higher number of potential target sites for NEXT-RNAi reagent designs. Overall this resulted in 100,270 input sequences used as input for NEXT-RNAi.
NEXT-RNAi HTML ouputs are available here
Overall 100,264 designs were obtained, covering 99.9% of the genome. 83.4% of all genes are covered by at least one design that does not show homology of 19 nt to any other gene. 97% of all genes are additionally covered by at least one second, independent design.
Input files and settings used
Input FASTA file
human.rnaMOD.COMMON_mdust_split_crsplit.zip (20MB) containing target sequences as input file (-i input).
>LOC729774_cr1:1 CCAAGCCTGCAGCAGGGAGAGCAACAAGCCCTGGCCCTCAGAGCTCAGCCGGATGAGCGCAGCCCAGAG ACAGCAGCTTCTCGAGGAAGGAAGGACCCGGTTTCAGGAGCTGCTGTCCAGTCCGGCCTACAGAGCCAG CACCCTGGTGGCCATCGGGCAGACGCTGGCCCGGCAGATGCAGCTGGAAGATGGCGGCCAGCTCTGA >LOC100128610_cr3:1 ATGAGGCTGAGTCTTATCCCTCGGAACACGGGCACCCCACAGAGGGTCCTGCCTCCTGTGGTCTGGAGC TCCCCCTCAAGGAAGAAACCCTTGCTGTCTGCTTGCAACTCCATGATGTTTGGACACCTCAGCCCCGTG AGGATCCCTTATCTCAGAGGCAAGTTTAAC >RNF185_cr2:1 AAGTCCCTCCGAGAGGGGCGGCTCCGCGTCATGTGACTGGAGTCCGCGTAGGAGGGGTCGGAGGTCTTA CCCAACAGATTGACGCGGCGTTAGTATTGGCCGTGTACCCGAAAAACTGATTGACTGGGCTGGCGTTAA CTGTGCGGAGG
Targetgroup file (tab-delimited)
TargetGroups_GeneID.tab (1.3MB) defining which RefSeq transcripts belong to the same gene (headers Target and TargetGroup) (TARGETGROUPS option)
Target TargetGroup TargetGroup2 NM_001012993.2 C9orf152 401546 NM_001015.3 RPS11 6205 XM_002348062.1 LOC100291269 100291269 NM_182764.1 ELMO2 63916 NM_133171.3 ELMO2 63916 NM_052854.2 CREB3L1 90993 NM_006029.4 PNMA1 9240 NM_004530.4 MMP2 4313 NM_001127891.1 MMP2 4313
Bowtie database/index for off-target evaluation
Bowtie database/index containing annotated RefSeq transcripts (release 40) for specificity calculations (-d input):
Feature file with UTR and SNP locations
Tab-delimited feature file containing mappings of UTRs and SNPs (from NCBI dbSNP) to chromosomes that is used to calculate UTR and SNP 'contents' (FEATURE option) of designed reagents: Hs_UTR_SNP.tar.gz (484MB)
ID FeatureName FeatureLoc FeatureStart FeatureEnd UTR_1 UTR 11 11643172 11643172 UTR_2 UTR 11 6741799 6741799 UTR_3 UTR 11 127219 127219 UTR_4 UTR 11 2662385 2662385 UTR_5 UTR 11 45952962 45952962 rs80303196 SNP 6 29985811 29985811 rs80303196 SNP 6 32995534 32995534 rs80303196 SNP 6 29080247 29080247 rs80303196 SNP 6 30038873 30038873 rs80303196 SNP 6 32181031 32181031
FASTA file for homology evaluation
Transcriptome FASTA file to evaluate the homology of the designs using Blast (HOMOLOGY and TXNFASTA options):
Bowtie database/index for seed match evaluation
To compute the number of siRNA seed matches (seed complement frequency) a Bowtie database/index containing all annotated 3'-UTR sequences (RefSeq release 40) was generated to be used with the SEEDMATCH option (Hs_3UTR.tar.gz (26MB)).
Start of program
perl nextrnai.pl -i human.rnaMOD.COMMON_mdust_split_crsplit.fa -s 7500 -r s -d human.rnaMOD -e NO -o options.txt -n Hs_RefSeq40
Descriptions for start parameters used are available here.
SIRNALENGTH=19 EFFICIENCY=SIR,63 BOWTIE=/usr/bin/ TARGETGROUPS=TargetGroups_GeneID.tab SOURCE=CDS BLAT=/usr/bin/ BLATPROGRAM=gfClient BLATHOST=b110-cellarray3 BLATPORT=3500 TXNFASTA=human.rnaMOD.fna GFF=GFF3 GBROWSEBASE=http://www.dkfz.de/signaling/cgi-bin/gbrowse_img/hsrefseq/ GBROWSETRACK=GENE+TXN+ENSEMBLGENESPAN+ENSEMBLGENE AFF=YES CANEVAL=6 HOMOLOGY=/usr/bin/,human.rnaMOD.fna,0.1 FEATURE=Hs_UTR_SNP.tab SEEDMATCH=6,2500,Hs_3UTR
Descriptions for all options used are available here.