Hi folks,
 
I am a new post-doc at Washington State University.  I don’t have a whole lot of bioinformatics experience, nevertheless I am trying to use CRISPRseek on the Arabidopsis genome.
 
Using a small test set (two genes), I can identify gRNA candidates, but there is a problem with the OfftargetAnalysis.xls in that the exon location is not being annotated and neither is the gene id.
 
What mistake am I making?
 
> library(CRISPRseek)
> library(BSgenome.Athaliana.TAIR.TAIR9)
> library(TxDb.Athaliana.BioMart.plantsmart22)
> library(org.At.tair.db)
> outputDir <- getwd()
> inputFilePath <- system.file('extdata', 'TAIR10_cds_20110103_representative_gene_model_updated.fa', package = 'CRISPRseek')
> REpatternFile <- system.file('extdata', 'NEBenzymes.fa', package = 'CRISPRseek')
> results <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE, REpatternFile = REpatternFile, findPairedgRNAOnly = FALSE, BSgenomeName = Athaliana, chromToSearch = "all", txdb = TxDb.Athaliana.BioMart.plantsmart22, orgAnn = org.At.tair.db, max.mismatch = 4, outputDir = outputDir, overwrite = TRUE)
The main thing I want to see is whether the off-target is exonic and the Arabidopsis locus ID (i.e. something looking like AT1G11110) for each off-target hit.
 
 
Thank you for any insight you can give me.

Jeremy,

It turns out that the <font size="2">TxDb.Athaliana.BioMart.plantsmart22 has chromosome labeled as 1, 2, 3 </font>…<font size="2"> instead of Chr1, Chr2, Chr3, …</font>

<font size="2">Please create your own TxDb object as following. I tested it and it worked.</font>

# the gff file can be downloaded at ftp://ftp.arabidopsis.org/home/tair/Genes/TAIR10_genome_release/TAIR10_gff3 

txdb <- makeTranscriptDbFromGFF("TAIR10_GFF3_genes.gff",format="gff")

Please remember to set txdb = txdb in the offTargetAnalysis call, as highlighted in the following code. In addition, for final output, you probably want to set max.mismatch to 3 instead of 1. For testing, 1 is good.

setwd("~/test/")
outputDir <- getwd()
inputFilePath <- 'test.fa'

results <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE,  findPairedgRNAOnly = FALSE, BSgenomeName = Athaliana, chromToSearch = "all", txdb = txdb, orgAnn = org.At.tairSYMBOL, max.mismatch = 1, outputDir = outputDir, overwrite = TRUE)

Please let me know how it works out for you. Thanks!

Best regards,

Julie

Jeremy,

Could you please change orgAnn = org.At.tairSYMBOL? Thanks!

 results <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE, REpatternFile = REpatternFile, findPairedgRNAOnly = FALSE, BSgenomeName = Athaliana, chromToSearch = "all", txdb = TxDb.Athaliana.BioMart.plantsmart22, orgAnn = org.At.tairSYMBOL, max.mismatch = 4, outputDir = outputDir, overwrite = TRUE)

Best regards,

Julie

Could you please try the most recent version CRISPRseek_1.5.5.tgz at http://bioconductor.org/packages/devel/bioc/html/CRISPRseek.html? If you still encounter problems, please send me the fasta file and code? Thanks!

Best,

Julie

Jeremy,

I noticed that in your code, you set the inputFilePath to the test file included in the package, which is different from the test.fa you intended to use for your analysis.

Instead of set inputFilePath as the following,

inputFilePath <- system.file('extdata', 'test.fa', package = 'CRISPRseek')

You would first set your working directory to the file path where your test.fa is located, for example ~/testCRISPRseek/. 

setwd("the file path where your test.fa is located")

Then set inputFilePath as

inputFilePath <- 'test.fa'

Best,

Julie