CRISPRseed gRNA.pattern is not producing expected result
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prp291 • 0
@prp291-9622
Last seen 6.1 years ago

Hi I am using the latest version of CRISPRseek (CRISPRseek_1.15.1). I used following script to search the gRNA without any off-target analysis

results <- offTargetAnalysis(
inputFilePath,
findgRNAs=TRUE,
gRNA.name.prefix="gRNA",
PAM.size=3,
gRNA.size=20,
PAM="NGG",
gRNA.pattern = "^G", #gRNA start with G only
#gRNA.pattern= "^(?:(?!T{3,}).)+$",  # three consecutive uracils in any position of a gRNA
#gRNA.pattern= "[ACG]{4,}.{3}$” , #avoid uracil in the last 4 positions of the guide sequence
min.score=0,
topN=1000,
findgRNAsWithREcutOnly = TRUE,
REpatternFile=system.file("extdata", "NEBenzymes.fa",package="CRISPRseek"),
minREpatternSize=6,
findPairedgRNAOnly = FALSE,
chromToSearch = "",
outputDir = outputDir,
overwrite = TRUE,
gRNAoutputName = "seq1gRNAs",
format="fasta",
exportAllgRNAs="fasta",     #c("all", "fasta", "genbank", "no"),
fetchSequence=TRUE,
upstream=200,
downstream=200,
#upstream.search=0,    
#downstream.search=0,
#baseBeforegRNA=4,    
#baseAfterPAM=3,    
useScore=TRUE,
overlap.gRNA.positions=c(17, 18),    
useEfficacyFromInputSeq=TRUE,      
outputUniqueREs=TRUE)

 

I used three different gRNA.pattern search but none of them produce the expected result. For example, gRNA.pattern = "^G" is also giving the gRNA starts with other than G. Thanks

 

 

crisprseek • 1.4k views
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Julie Zhu ★ 4.3k
@julie-zhu-3596
Last seen 4 months ago
United States
Could you please post the example input sequences for testing? Thanks! Best, Julie From: "prp291 [bioc]" <noreply@bioconductor.org<mailto:noreply@bioconductor.org>> Reply-To: "reply+c41de0c2+code@bioconductor.org<mailto:reply+c41de0c2+code@bioconductor.org>" <reply+c41de0c2+code@bioconductor.org<mailto:reply+c41de0c2+code@bioconductor.org>> Date: Tuesday, March 21, 2017 8:50 AM To: Lihua Julie Zhu <julie.zhu@umassmed.edu<mailto:julie.zhu@umassmed.edu>> Subject: [bioc] CRISPRseed gRNA.pattern is not producing expected result Activity on a post you are following on support.bioconductor.org<https: support.bioconductor.org=""> User prp291<https: support.bioconductor.org="" u="" 9622=""/> wrote Question: CRISPRseed gRNA.pattern is not producing expected result<https: support.bioconductor.org="" p="" 94062=""/>: Hi I am using the latest version of CRISPRseek (CRISPRseek_1.15.1). I used following script to search the gRNA without any off-target analysis results <- offTargetAnalysis( inputFilePath, findgRNAs=TRUE, gRNA.name.prefix="gRNA", PAM.size=3, gRNA.size=20, PAM="NGG", gRNA.pattern = "^G", #gRNA start with G only #gRNA.pattern= "^(?:(?!T{3,}).)+$", # three consecutive uracils in any position of a gRNA #gRNA.pattern= "[ACG]{4,}.{3}$� , #avoid uracil in the last 4 positions of the guide sequence min.score=0, topN=1000, findgRNAsWithREcutOnly = TRUE, REpatternFile=system.file("extdata", "NEBenzymes.fa",package="CRISPRseek"), minREpatternSize=6, findPairedgRNAOnly = FALSE, chromToSearch = "", outputDir = outputDir, overwrite = TRUE, gRNAoutputName = "seq1gRNAs", format="fasta", exportAllgRNAs="fasta", #c("all", "fasta", "genbank", "no"), fetchSequence=TRUE, upstream=200, downstream=200, #upstream.search=0, #downstream.search=0, #baseBeforegRNA=4, #baseAfterPAM=3, useScore=TRUE, overlap.gRNA.positions=c(17, 18), useEfficacyFromInputSeq=TRUE, outputUniqueREs=TRUE) I used three different gRNA.pattern search but none of them produce the expected result. For example, gRNA.pattern = "^G" is also giving the gRNA starts with other than G. Thanks ________________________________ Post tags: crisprseek You may reply via email or visit CRISPRseed gRNA.pattern is not producing expected result
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Thanks. I am using this FASTA sequence to search the gRNA.

>test
ATGACGGAAAATTCAAAAGTATAACAATATAATTTTCAGGAAAATCATACTAATGATGTTCAAGGATTATTGAGTTTGTATGAAGCAGCACATATGAGAGTGCACGACGAGGAAATTCTAGAAGAAGCTCTTATCTTTACCACGACTCATCTCGCCATCGTAATTCGCGAATTTTGGAGTCCGTGATCCCGAATTTGAGCAACTCGCTTAAGGTACAAGTTACTGAAGCCTTAAGCCATCCTATTCGCAAAGCTATACCAAGGGTGGGAGCAAGGAAATACATACACATATATGAAAACATTGGAACACATAATGATTTACTTTTGAAATTTGCAAAGTTGGACTTCAACATGTTACAAAAGCTTCATCGAAAAGAGCTTAACGAGCTAACAAGGTACATCTACTATTCTTGTCATCTTCATAAGAGAGATGTGTACGCAGATCTTACCCTACCTTATAGAGGTAGAAATGTTGTTCCCGATTTATGGTACAATCAGACCTCTCTATAAAACACATCCTTTATAACAACAGTTCACTATAACGGTCAAGTTTTCTTTAAAATCAATCTTTTATGTTACCAAATATTTTTGAAAGAAACGTGACTATTATAGAGAGGTTTGACTGTAACTCGCGCTAATTAATAACACCTAAAGTTTAAGTATGTTAATGTTGTTATGATATCTATAGCTGGTGGAAAGATTTGGATCGTGCAAACAAATATGGATACACAAATCCCAAATCCAAAGTTAAAGACTTGATTGCTTCGTTGTTTGTCGAATCTGT

 

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Thank you very much for the bug report! Yes, a bug was introduced when a section of code was moved to a function. I have fixed the bug in CRISPRseek_1.15.2, which can be download using svn svn co --username readonly --password readonly https://hedgehog.fhcrc.org/bioconductor/trunk/madman/Rpacks/CRISPRseek Best regards, Julie Here is the list of gRNAs with findgRNAsWithREcutOnly = TRUE A DNAStringSet instance of length 6 width seq names [1] 23 GAATTTGAGCAACTCGCTTAAGG NA_gR207f [2] 23 GATCTTACCCTACCTTATAGAGG NA_gR457f [3] 23 GAAATGTTGTTCCCGATTTATGG NA_gR482f [4] 23 GGTCTGATTGTACCATAAATCGG NA_gR484r [5] 23 GTCTGATTGTACCATAAATCGGG NA_gR483r [6] 23 GTTGTTATGATATCTATAGCTGG NA_gR685f A DNAStringSet instance of length 6 width seq names Here is the list of gRNAs with findgRNAsWithREcutOnly = FALSE [1] 23 GAATTTGAGCAACTCGCTTAAGG NA_gR207f [2] 23 GATCTTACCCTACCTTATAGAGG NA_gR457f [3] 23 GAAATGTTGTTCCCGATTTATGG NA_gR482f [4] 23 GAAACGTGACTATTATAGAGAGG NA_gR610f [5] 23 GTTGTTATGATATCTATAGCTGG NA_gR685f [6] 23 GTTATGATATCTATAGCTGGTGG NA_gR688f [7] 23 GGTCTGATTGTACCATAAATCGG NA_gR484r [8] 23 GTCTGATTGTACCATAAATCGGG NA_gR483r [9] 23 GTATAGCTTTGCGAATAGGATGG NA_gR242r [10] 23 GTTGCTCAAATTCGGGATCACGG NA_gR187r From: "prp291 [bioc]" <noreply@bioconductor.org<mailto:noreply@bioconductor.org>> Reply-To: "reply+cbae2f4f+code@bioconductor.org<mailto:reply+cbae2f4f+code@bioconductor.org>" <reply+cbae2f4f+code@bioconductor.org<mailto:reply+cbae2f4f+code@bioconductor.org>> Date: Tuesday, March 21, 2017 10:31 AM To: Lihua Julie Zhu <julie.zhu@umassmed.edu<mailto:julie.zhu@umassmed.edu>> Subject: [bioc] C: CRISPRseed gRNA.pattern is not producing expected result Activity on a post you are following on support.bioconductor.org<https: support.bioconductor.org=""> User prp291<https: support.bioconductor.org="" u="" 9622=""/> wrote Comment: CRISPRseed gRNA.pattern is not producing expected result<https: support.bioconductor.org="" p="" 94062="" #94069="">: Thanks. I am using this FASTA sequence to search the gRNA. >test ATGACGGAAAATTCAAAAGTATAACAATATAATTTTCAGGAAAATCATACTAATGATGTTCAAGGATTATTGAGTTTGTATGAAGCAGCACATATGAGAGTGCACGACGAGGAAATTCTAGAAGAAGCTCTTATCTTTACCACGACTCATCTCGCCATCGTAATTCGCGAATTTTGGAGTCCGTGATCCCGAATTTGAGCAACTCGCTTAAGGTACAAGTTACTGAAGCCTTAAGCCATCCTATTCGCAAAGCTATACCAAGGGTGGGAGCAAGGAAATACATACACATATATGAAAACATTGGAACACATAATGATTTACTTTTGAAATTTGCAAAGTTGGACTTCAACATGTTACAAAAGCTTCATCGAAAAGAGCTTAACGAGCTAACAAGGTACATCTACTATTCTTGTCATCTTCATAAGAGAGATGTGTACGCAGATCTTACCCTACCTTATAGAGGTAGAAATGTTGTTCCCGATTTATGGTACAATCAGACCTCTCTATAAAACACATCCTTTATAACAACAGTTCACTATAACGGTCAAGTTTTCTTTAAAATCAATCTTTTATGTTACCAAATATTTTTGAAAGAAACGTGACTATTATAGAGAGGTTTGACTGTAACTCGCGCTAATTAATAACACCTAAAGTTTAAGTATGTTAATGTTGTTATGATATCTATAGCTGGTGGAAAGATTTGGATCGTGCAAACAAATATGGATACACAAATCCCAAATCCAAAGTTAAAGACTTGATTGCTTCGTTGTTTGTCGAATCTGT ________________________________ Post tags: crisprseek You may reply via email or visit C: CRISPRseed gRNA.pattern is not producing expected result
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Thanks for help. I just modified my script like this to get the minimum free en-ergy of the secondary structure of gRNA with gRNA backbone but I didn't get anything. Actually Summary file is also not generated in the end. Can you check whether it is also a kind of bug.

results <- offTargetAnalysis(
inputFilePath,
findgRNAs=TRUE,
gRNA.name.prefix="gRNA",
PAM.size=3,
gRNA.size=20,
PAM="NGG",
#gRNA.pattern = "^G",
gRNA.pattern= "^G(?:(?!T{3,}).)+$",  # Start with G and avoid three consecutive uracils in any position of a gRNA
#gRNA.pattern= “[ACG]{4,}.{3}$”, #avoid uracil in the last 4 positions of the guide sequence
min.score=0,
topN=1000,
findgRNAsWithREcutOnly = TRUE,
REpatternFile=system.file("extdata", "NEBenzymes.fa",package="CRISPRseek"),
minREpatternSize=6,
findPairedgRNAOnly = FALSE,
chromToSearch = "",
outputDir = outputDir,
overwrite = TRUE,
gRNAoutputName = "seq1gRNAs",
format="fasta",
exportAllgRNAs="fasta",     #c("all", "fasta", "genbank", "no"),
fetchSequence=TRUE,
upstream=200,
downstream=200,
#upstream.search=0,    
#downstream.search=0,
#baseBeforegRNA=4,    
#baseAfterPAM=3,    
useScore=TRUE,
overlap.gRNA.positions=c(17, 18),    
useEfficacyFromInputSeq=TRUE,      
outputUniqueREs=TRUE,
foldgRNAs=TRUE, gRNA.backbone="GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU",
temperature=37,   
scoring.method="CFDscore",
subPAM.activity = hash( AA = 0,
                                    AC = 0,
                                    AG = 0.259259259,
                                    AT = 0,
                                    CA = 0,
                                    CC = 0,
                                    CG = 0.107142857,
                                    CT = 0,
                                    GA = 0.069444444,
                                    GC = 0.022222222,
                                    GG = 1,
                                    GT = 0.016129032,
                                    TA = 0,
                                    TC = 0,
                                    TG = 0.038961039,
                                    TT = 0))

 

 

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If GeneRfold and GeneR are installed (http://bioconductor.case.edu/bioconductor/2.8/bioc/html/GeneRfold.html, http://bioc.ism.ac.jp/packages/2.8/bioc/html/GeneR.html), then the minimum free energy and bracket notation of secondary structure of gRNA and gRNA backbone constant region will be included in the summary file. Unfortunately these packages are not included in the current release due to lack of maintenance. Best, Julie On Mar 21, 2017, at 1:45 PM, prp291 [bioc] <noreply@bioconductor.org<mailto:noreply@bioconductor.org>> wrote: Activity on a post you are following on support.bioconductor.org<https: support.bioconductor.org=""> User prp291<https: support.bioconductor.org="" u="" 9622=""/> wrote Comment: CRISPRseed gRNA.pattern is not producing expected result<https: support.bioconductor.org="" p="" 94062="" #94080="">: Thanks for help. I just modified my script like this to get the minimum free en-ergy of the secondary structure of gRNA with gRNA backbone but I didn't get anything. Actually Summary file is also not generated in the end. Can you check whether it is also a kind of bug. results <- offTargetAnalysis( inputFilePath, findgRNAs=TRUE, gRNA.name.prefix="gRNA", PAM.size=3, gRNA.size=20, PAM="NGG", #gRNA.pattern = "^G", gRNA.pattern= "^G(?:(?!T{3,}).)+$", # Start with G and avoid three consecutive uracils in any position of a gRNA #gRNA.pattern= �[ACG]{4,}.{3}$�, #avoid uracil in the last 4 positions of the guide sequence min.score=0, topN=1000, findgRNAsWithREcutOnly = TRUE, REpatternFile=system.file("extdata", "NEBenzymes.fa",package="CRISPRseek"), minREpatternSize=6, findPairedgRNAOnly = FALSE, chromToSearch = "", outputDir = outputDir, overwrite = TRUE, gRNAoutputName = "seq1gRNAs", format="fasta", exportAllgRNAs="fasta", #c("all", "fasta", "genbank", "no"), fetchSequence=TRUE, upstream=200, downstream=200, #upstream.search=0, #downstream.search=0, #baseBeforegRNA=4, #baseAfterPAM=3, useScore=TRUE, overlap.gRNA.positions=c(17, 18), useEfficacyFromInputSeq=TRUE, outputUniqueREs=TRUE, foldgRNAs=TRUE, gRNA.backbone="GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU", temperature=37, scoring.method="CFDscore", subPAM.activity = hash( AA = 0, AC = 0, AG = 0.259259259, AT = 0, CA = 0, CC = 0, CG = 0.107142857, CT = 0, GA = 0.069444444, GC = 0.022222222, GG = 1, GT = 0.016129032, TA = 0, TC = 0, TG = 0.038961039, TT = 0)) ________________________________ Post tags: crisprseek You may reply via email or visit C: CRISPRseed gRNA.pattern is not producing expected result
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Thanks for your help.

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