Hi there Julie

When I run the following code:

library(CRISPRseek)
library(BSgenome.Hsapiens.UCSC.hg19)
library(TxDb.Hsapiens.UCSC.hg19.knownGene) 
outputDir <- getwd() 
inputFilePath <- DNAStringSet("CCTTCCAGGAATTCTTTGGCCTGAATAATTGCAGTAGCTCTAACAGGTTGGACCAAGCTATGCAGGTGACAGAGACTCTTGGGATGACGC")
results <- offTargetAnalysis(inputFilePath, format="fasta",findgRNAs=TRUE, findgRNAsWithREcutOnly=FALSE, findPairedgRNAOnly=TRUE, annotatePaired=TRUE, min.gap=0, max.gap=100, gRNA.name.prefix="gRNA", PAM.size=6, gRNA.size=21, PAM="NNG[A|G][A|G]T", BSgenomeName=Hsapiens, chromToSearch="all", max.mismatch=4, PAM.pattern = "NNG[A|G][A|G]N$", gRNA.pattern="^G", min.score=0, topN=1000, topN.OfftargetTotalScore=10, annotateExon=TRUE, txdb=TxDb.Hsapiens.UCSC.hg19.knownGene, outputDir=outputDir, fetchSequence=TRUE, upstream=200, downstream=200, weights=c(0, 0, 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), overwrite=TRUE)

I get the error message:

Error in offTargetAnalysis(inputFilePath, format = "fasta", findgRNAs = TRUE,  : 
  Please enter a name for the gRNA ouput file name!

Could you please advise as to how I name the gRNA output file as I am unable to do this.

Thanks for your time

Kind regard

Britt

Britt,

Please try the following code! I tested it with CRISPRseek 1.14.0. CRISPRseek 1.14.0 (http://bioconductor.org/packages/release/bioc/html/CRISPRseek.html). Please note that besides setting gRNAoutputName = "seq1gRNAs", you need to set  min.gap = -2 and  PAM="NNGRRT". No paired gRNAs were found with the default min.gap = 0.

Best regards,

Julie

inputFilePath <-DNAStringSet("ATTCCCAGCGCACTGCTGCATGTTTTGGCCTGAATAATCAGACTCTTGGGATGAAGGTCAAGCTGCATGGACCTTCCAGGAAGCTCTAATATGCAGGTGA")

names(inputFilePath) <- "seq1"

outputDir = "seq1CRISPRseekOutput"

library(BSgenome.Hsapiens.UCSC.hg19)

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

library(CRISPRseek)

results <- offTargetAnalysis(inputFilePath, gRNAoutputName = "seq1gRNAs", format="fasta", findgRNAs=TRUE, exportAllgRNAs=c("all", "fasta", "genbank", "no"), findgRNAsWithREcutOnly=FALSE, findPairedgRNAOnly=TRUE, min.gap=-2, max.gap=20, gRNA.name.prefix="gRNA", PAM.size=6, gRNA.size=21, PAM="NNGRRT", BSgenomeName=Hsapiens, chromToSearch="all", max.mismatch=4, PAM.pattern = "NNG[A|G][A|G]N$", gRNA.pattern="^G", min.score=0, topN=1000, topN.OfftargetTotalScore=10, annotateExon=TRUE, txdb=TxDb.Hsapiens.UCSC.hg19.knownGene, outputDir=outputDir, fetchSequence=TRUE, upstream=200, downstream=200, weights=c(0, 0, 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), overwrite=TRUE)

On 25 Oct 2016 5:54 PM, "Zhu, Lihua (Julie)" <Julie.Zhu@umassmed.edu> wrote:

Britt,

You are correct that currently, it does not have paired off target search capability. You could pair yourself afterwards.

Yes, please post all your response on the forum moving forward! Thanks!

Best,

Julie

From: Britt Hanson <bhanson1806@gmail.com>
Date: Tuesday, October 25, 2016 11:52 AM
To: Lihua Julie Zhu <julie.zhu@umassmed.edu>
Subject: Re: CRISPR/Cas9 and GUIDE-Seq

Hi Julie

Thanks - I actually put in a random sequence on the forum (thinking back I realise it was silly) so I performed it with the correct sequence on my own. One thing I've realised looking at my results now is that I have been given off target sites for each sgRNA alone but not as a pair... Did I make a mistake in the code somewhere for the output to show paired off target sites not individual guides? Would you prefer I asked the question on the forum?

Thanks again 
Britt

On 25 Oct 2016 5:47 PM, "Zhu, Lihua (Julie)" <Julie.Zhu@umassmed.edu> wrote:

Britt,

You are welcome!

I just want to let you know that  blat your sequence to hg19 did not have 100% alignment in case you have different version of the genome or something. 

https://genome.ucsc.edu/cgi-bin/hgBlat

Best,

Julie

Britt, 

Please remember to change min.gap and max.gap in the code with your real sequence to reflect the requirements of the distance between the paired gRNAs. I will add paired off target search in the new feature list. Thanks!

Best,

Julie