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We evaluated the Ambion (Silencer Select) and Qiagen (human druggable v3.0 and human whole genome supplement v1.0 sets) siRNA libraries containing 64,781 siRNAs and 70,308 siRNAs, respectively. For annotations NCBI RefSeq release 40 was used.



NEXT-RNAi results

NEXT-RNAi HTML ouputs for the library evaluations are available here:


Input files and settings used

Input FASTA file

The files contain siRNA sequences used as input for NEXT-RNAi (-i input).

>SI00077350
TCCCGTATAAGTATGTTCC
>SI00077336
GAGGACTATACCAACTAGT
>SI00077343
CAGACCAAACCGAGGACTA
>SI00077357
ACGCCATTATTCAGAACCT
>SI00147777
ACCCAGGATATCTCCACCA
>SI00147756
CCGGATCCTCAAGGTCTTC
>SI00147770
CTCCATATTCTTCTGCATC
>SI03092887
CTGAAGCGGCTCATCGAGA

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):
human.rnaMOD.tar.gz (114MB)

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):
human.rnaMOD.fna.zip (32MB)

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)).


Design criteria

Start of program

Ambion

perl nextrnai.pl -i Ambion_siRNA.fa -s 30000 -r s -d human.rnaMOD
-e OLIGO -o options.txt -n Ambion_RefSeq40

Qiagen

perl nextrnai.pl -i Qiagen_siRNA.fa -s 30000 -r s -d human.rnaMOD
-e OLIGO -o options.txt -n Qiagen_RefSeq40


Descriptions for start parameters used are available here.

Options file

SIRNALENGTH=19
EFFICIENCY=SIR,63
BOWTIE=/usr/bin/
SOURCE=CDS
TARGETGROUPS=TargetGroups_GeneID.tab
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
LOWCOMPEVAL=/usr/bin/
CANEVAL=6
HOMOLOGY=/usr/bin/,human.rnaMOD.fna,0.1
FEATURE=Hs_UTR_SNP.tab
SEEDMATCH=Hs_3UTR


Descriptions for all options used are available here.


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