CRISPRseek prediction of truncated Nme sgRNA and getting error message
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Daniel • 0
@daniel-9847
Last seen 7.0 years ago
US/Worcester/UMass Med

I had tried to predict the off-target sites of truncated Nme sgRNA up to 19nt (wild type length of spacer is 24 nt). I start getting the warning message shown below:

 

Warning messages:

1: In fromXStringViewsToStringSet(x, out.of.limits = "warning", use.names = use.names) :

  trimming "out of limits" views

2: In fromXStringViewsToStringSet(x, out.of.limits = "warning", use.names = use.names) :

  trimming "out of limits" views

OR

Warning messages:

2017-03-07 11:10:09.741 R[2576:1123339] IMKInputSession presentFunctionRowItemTextInputViewWithEndpoint:completionHandler: : *NO* NSRemoteViewController to client, NSError=Error Domain=NSCocoaErrorDomain Code=4099 "The connection from pid 0 was invalidated from this process." UserInfo={NSDebugDescription=The connection from pid 0 was invalidated from this process.}, com.apple.inputmethod.EmojiFunctionRowItem

1: In fromXStringViewsToStringSet(x, out.of.limits = "warning", use.names = use.names) :

  trimming "out of limits" views

2: In fromXStringViewsToStringSet(x, out.of.limits = "warning", use.names = use.names) :

  trimming "out of limits" views

Error in unlist(lapply(1:dim(mismatch.pos)[1], function(i) { : 

  error in evaluating the argument 'x' in selecting a method for function 'unlist': Error in subseq(subject[i], start = j, width = 1) : 

  error in evaluating the argument 'x' in selecting a method for function 'subseq': Error in get(seqname, envir = seqs_cache, inherits = FALSE) : 

  object 'chrUn_gl000249' not found

 

Even though sometimes there is a EXCEL datasheet showing all off-target sites, I want to check with you whether the data are still complete and valid. Besides, I am thinking to predict shorter truncated sgRNA up to 16nt. Would you mind taking some time to take a look. The Nme sgRNA sequence is GAGAAGGCCAGGGGTCACTCCAGGATT. Thanks!

 

 

 

 

crisprseek • 1.4k views
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Julie Zhu ★ 4.3k
@julie-zhu-3596
Last seen 4 months ago
United States
Daniel, Could you please post your R code and sessionInfo? Are you searching in human genome? Thanks! Best, Julie From: "Daniel [bioc]" <noreply@bioconductor.org<mailto:noreply@bioconductor.org>> Reply-To: "reply+0dbe2caa+code@bioconductor.org<mailto:reply+0dbe2caa+code@bioconductor.org>" <reply+0dbe2caa+code@bioconductor.org<mailto:reply+0dbe2caa+code@bioconductor.org>> Date: Tuesday, March 7, 2017 2:00 PM To: Lihua Julie Zhu <julie.zhu@umassmed.edu<mailto:julie.zhu@umassmed.edu>> Subject: [bioc] CRISPRseek prediction of truncated Nme sgRNA and getting error message Activity on a post you are following on support.bioconductor.org<https: support.bioconductor.org=""> User Daniel<https: support.bioconductor.org="" u="" 9847=""/> wrote Question: CRISPRseek prediction of truncated Nme sgRNA and getting error message<https: support.bioconductor.org="" p="" 93486=""/>: I had tried to predict the off-target sites of truncated Nme sgRNA up to 19nt (wild type length of spacer is 24 nt). I start getting the warning message shown below: Warning messages: 1: In fromXStringViewsToStringSet(x, out.of.limits = "warning", use.names = use.names) : trimming "out of limits" views 2: In fromXStringViewsToStringSet(x, out.of.limits = "warning", use.names = use.names) : trimming "out of limits" views OR Warning messages: 2017-03-07 11:10:09.741 R[2576:1123339] IMKInputSession presentFunctionRowItemTextInputViewWithEndpoint:completionHandler: : *NO* NSRemoteViewController to client, NSError=Error Domain=NSCocoaErrorDomain Code=4099 "The connection from pid 0 was invalidated from this process." UserInfo={NSDebugDescription=The connection from pid 0 was invalidated from this process.}, com.apple.inputmethod.EmojiFunctionRowItem 1: In fromXStringViewsToStringSet(x, out.of.limits = "warning", use.names = use.names) : trimming "out of limits" views 2: In fromXStringViewsToStringSet(x, out.of.limits = "warning", use.names = use.names) : trimming "out of limits" views Error in unlist(lapply(1:dim(mismatch.pos)[1], function(i) { : error in evaluating the argument 'x' in selecting a method for function 'unlist': Error in subseq(subject[i], start = j, width = 1) : error in evaluating the argument 'x' in selecting a method for function 'subseq': Error in get(seqname, envir = seqs_cache, inherits = FALSE) : object 'chrUn_gl000249' not found Even though sometimes there is a EXCEL datasheet showing all off-target sites, I want to check with you whether the data are still complete and valid. Besides, I am thinking to predict shorter truncated sgRNA up to 16nt. Would you mind taking some time to take a look. The Nme sgRNA sequence is GAGAAGGCCAGGGGTCACTCCAGGATT. Thanks! ________________________________ Post tags: crisprseek You may reply via email or visit CRISPRseek prediction of truncated Nme sgRNA and getting error message
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Hi Julie,

Here are the info.

sessionInfo()

R version 3.2.0 (2015-04-16)
Platform: x86_64-apple-darwin13.4.0 (64-bit)
Running under: OS X 10.12.3 (unknown)

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] stats4    parallel  stats     graphics  grDevices utils     datasets  methods  
[9] base     

other attached packages:
 [1] org.Hs.eg.db_3.1.2                      RSQLite_1.0.0                          
 [3] DBI_0.3.1                               TxDb.Hsapiens.UCSC.hg19.knownGene_3.1.2
 [5] GenomicFeatures_1.20.1                  AnnotationDbi_1.30.1                   
 [7] Biobase_2.28.0                          BSgenome.Hsapiens.UCSC.hg19_1.4.0      
 [9] CRISPRseek_1.9.1                        seqinr_3.1-3                           
[11] ade4_1.7-2                              BSgenome_1.36.1                        
[13] rtracklayer_1.28.5                      GenomicRanges_1.20.5                   
[15] GenomeInfoDb_1.4.1                      Biostrings_2.36.1                      
[17] XVector_0.8.0                           IRanges_2.2.4                          
[19] S4Vectors_0.6.0                         BiocGenerics_0.14.0                    

loaded via a namespace (and not attached):
 [1] zlibbioc_1.14.0         GenomicAlignments_1.4.1 BiocParallel_1.2.5     
 [4] tools_3.2.0             lambda.r_1.1.7          futile.logger_1.4.1    
 [7] futile.options_1.0.0    bitops_1.0-6            biomaRt_2.24.0         
[10] RCurl_1.95-4.6          Rsamtools_1.20.4        XML_3.98-1.2           

R code:

Results2 <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE, findPairedgRNAOnly = FALSE, gRNA.size = 19, min.score=0, BSgenomeName = Hsapiens, chromToSearch = "all",  PAM = "NNNNGATT", PAM.size=8, PAM.pattern="NNNNGNNN$",weights = c(0, 0.014, 0, 0, 0.395, 0.317, 0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804, 0.685, 0.583), txdb = TxDb.Hsapiens.UCSC.hg19.knownGene, orgAnn = org.Hs.egSYMBOL, max.mismatch = 6, outputDir = outputDir, allowed.mismatch.PAM = 7,overwrite = TRUE)

19mer sgRNA sequence: GAGAAGGCCAGGGGTCACT(PAM:CCAGGATT)

Thanks!

 

 

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Daniel, Please update CRISPRseek to the current released version 1.14.0 at http://bioconductor.org/packages/release/bioc/html/CRISPRseek.html. Also please change allowed.mismatch.PAM = 3 from 7 with the updated version. The allowed.mismatch.PAM specifies maximum allowed edit distance between PAM.pattern (NNNNGNNN) and PAM (NNNNGATT), which is 3 in your case. Best, Julie From: "Daniel [bioc]" <noreply@bioconductor.org<mailto:noreply@bioconductor.org>> Reply-To: "reply+7ed41389+code@bioconductor.org<mailto:reply+7ed41389+code@bioconductor.org>" <reply+7ed41389+code@bioconductor.org<mailto:reply+7ed41389+code@bioconductor.org>> Date: Tuesday, March 7, 2017 4:57 PM To: Lihua Julie Zhu <julie.zhu@umassmed.edu<mailto:julie.zhu@umassmed.edu>> Subject: [bioc] C: CRISPRseek prediction of truncated Nme sgRNA and getting error message Activity on a post you are following on support.bioconductor.org<https: support.bioconductor.org=""> User Daniel<https: support.bioconductor.org="" u="" 9847=""/> wrote Comment: CRISPRseek prediction of truncated Nme sgRNA and getting error message<https: support.bioconductor.org="" p="" 93486="" #93504="">: Hi Julie, Here are the info. sessionInfo() R version 3.2.0 (2015-04-16) Platform: x86_64-apple-darwin13.4.0 (64-bit) Running under: OS X 10.12.3 (unknown) locale: [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 attached base packages: [1] stats4 parallel stats graphics grDevices utils datasets methods [9] base other attached packages: [1] org.Hs.eg.db_3.1.2 RSQLite_1.0.0 [3] DBI_0.3.1 TxDb.Hsapiens.UCSC.hg19.knownGene_3.1.2 [5] GenomicFeatures_1.20.1 AnnotationDbi_1.30.1 [7] Biobase_2.28.0 BSgenome.Hsapiens.UCSC.hg19_1.4.0 [9] CRISPRseek_1.9.1 seqinr_3.1-3 [11] ade4_1.7-2 BSgenome_1.36.1 [13] rtracklayer_1.28.5 GenomicRanges_1.20.5 [15] GenomeInfoDb_1.4.1 Biostrings_2.36.1 [17] XVector_0.8.0 IRanges_2.2.4 [19] S4Vectors_0.6.0 BiocGenerics_0.14.0 loaded via a namespace (and not attached): [1] zlibbioc_1.14.0 GenomicAlignments_1.4.1 BiocParallel_1.2.5 [4] tools_3.2.0 lambda.r_1.1.7 futile.logger_1.4.1 [7] futile.options_1.0.0 bitops_1.0-6 biomaRt_2.24.0 [10] RCurl_1.95-4.6 Rsamtools_1.20.4 XML_3.98-1.2 R code: Results2 <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE, findPairedgRNAOnly = FALSE, gRNA.size = 19, min.score=0, BSgenomeName = Hsapiens, chromToSearch = "all", PAM = "NNNNGATT", PAM.size=8, PAM.pattern="NNNNGNNN$",weights = c(0, 0.014, 0, 0, 0.395, 0.317, 0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804, 0.685, 0.583), txdb = TxDb.Hsapiens.UCSC.hg19.knownGene, orgAnn = org.Hs.egSYMBOL, max.mismatch = 6, outputDir = outputDir, allowed.mismatch.PAM = 7,overwrite = TRUE) 19mer sgRNA sequence: GAGAAGGCCAGGGGTCACT(PAM:CCAGGATT) Thanks! ________________________________ Post tags: crisprseek You may reply via email or visit C: CRISPRseek prediction of truncated Nme sgRNA and getting error message
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Hi Julie,

I updated R to the current version 3.3.3 and this allows me to update my CRISPRseek to 1.14.0 checked by sessionInfo(). 

I downloaded all the packages and libraries such as 

library(CRISPRseek)

library("BSgenome.Hsapiens.UCSC.hg19")

library(TxDb.Hsapiens.UCSC.hg19.knownGene)

library(org.Hs.eg.db).

However, 

After typing in:

Results2 <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE, findPairedgRNAOnly = FALSE, gRNA.size = 19, min.score=0, BSgenomeName = Hsapiens, chromToSearch = "all",  PAM = "NNNNGATT", PAM.size=8, PAM.pattern="NNNNGNNN$",weights = c(0, 0.014, 0, 0, 0.395, 0.317, 0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804, 0.685, 0.583), txdb = TxDb.Hsapiens.UCSC.hg19.knownGene, orgAnn = org.Hs.egSYMBOL, max.mismatch = 6, outputDir = outputDir, allowed.mismatch.PAM = 3,overwrite = TRUE)

Error: could not find function "offTargetAnalysis"

I cannot figure it out. Did I miss something to download. Also, I remember the NmeCas9 system has PAM.size=8. The default allowed.mismatch.PAM=4 as you informed me before. I believe if searching something is NNNNGNNN$, the allowed.mismatch.PAM should be 7 but not 3.

Thanks!

Daniel

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Daniel, That is odd. I ran your example without problem. Could you please try to type CRISPRseek::offTargetAnalysis instead of offTargetAnalysis? You did right with the previous version, the current version has a new implementation and interpretation of the allowed.mismatch.PAM. It specifies the number of mismatches allowed in the offtarget PAM sequence comparing to the PAM. Since the PAM sequence has 4 N out of 8 already and allowed.mismatch.PAM only applies to the non-N bases, allowed.mismatch.PAM here will be between 0 and 4. Best regards, Julie From: "Daniel [bioc]" <noreply@bioconductor.org<mailto:noreply@bioconductor.org>> Reply-To: "reply+fb6f5912+code@bioconductor.org<mailto:reply+fb6f5912+code@bioconductor.org>" <reply+fb6f5912+code@bioconductor.org<mailto:reply+fb6f5912+code@bioconductor.org>> Date: Wednesday, March 8, 2017 7:52 AM To: Lihua Julie Zhu <julie.zhu@umassmed.edu<mailto:julie.zhu@umassmed.edu>> Subject: [bioc] C: CRISPRseek prediction of truncated Nme sgRNA and getting error message Activity on a post you are following on support.bioconductor.org<https: support.bioconductor.org=""> User Daniel<https: support.bioconductor.org="" u="" 9847=""/> wrote Comment: CRISPRseek prediction of truncated Nme sgRNA and getting error message<https: support.bioconductor.org="" p="" 93486="" #93540="">: Hi Julie, I updated R to the current version 3.3.3 and this allows me to update my CRISPRseek to 1.14.0 checked by sessionInfo(). I downloaded all the packages and libraries such as library(CRISPRseek) library("BSgenome.Hsapiens.UCSC.hg19") library(TxDb.Hsapiens.UCSC.hg19.knownGene) library(org.Hs.eg.db). However, After typing in: Results2 <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE, findPairedgRNAOnly = FALSE, gRNA.size = 19, min.score=0, BSgenomeName = Hsapiens, chromToSearch = "all", PAM = "NNNNGATT", PAM.size=8, PAM.pattern="NNNNGNNN$",weights = c(0, 0.014, 0, 0, 0.395, 0.317, 0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804, 0.685, 0.583), txdb = TxDb.Hsapiens.UCSC.hg19.knownGene, orgAnn = org.Hs.egSYMBOL, max.mismatch = 6, outputDir = outputDir, allowed.mismatch.PAM = 3,overwrite = TRUE) Error: could not find function "offTargetAnalysis" I cannot figure it out. Did I miss something to download. Also, I remember the NmeCas9 system has PAM.size=8. The default allowed.mismatch.PAM=4 as you informed me before. I believe if searching something is NNNNGNNN$, the allowed.mismatch.PAM should be 7 but not 3. Thanks! Daniel ________________________________ Post tags: crisprseek You may reply via email or visit C: CRISPRseek prediction of truncated Nme sgRNA and getting error message
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