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!
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$",weights = c(0, 0.014, 0, 0, 0.395, 0.317, 0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804, 0.685, 0.583), txdb = TxDb.Hsapiens.UCSC.hg19.knownGene, orgAnn = org.Hs.egSYMBOL, max.mismatch = 6, outputDir = outputDir, allowed.mismatch.PAM = 7,overwrite = TRUE)
19mer sgRNA sequence: GAGAAGGCCAGGGGTCACT(PAM:CCAGGATT)
Thanks!
Hi Julie,
I updated R to the current version 3.3.3 and this allows me to update my CRISPRseek to 1.14.0 checked by sessionInfo().
I downloaded all the packages and libraries such as
library(CRISPRseek)
library("BSgenome.Hsapiens.UCSC.hg19")
library(TxDb.Hsapiens.UCSC.hg19.knownGene)
library(org.Hs.eg.db).
However,
After typing in:
Results2 <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE, findPairedgRNAOnly = FALSE, gRNA.size = 19, min.score=0, BSgenomeName = Hsapiens, chromToSearch = "all", PAM = "NNNNGATT", PAM.size=8, PAM.pattern="NNNNGNNN$",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