CRISPRseek with genome not available in BSgenome
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@millepattesintersites-12592
Last seen 7.7 years ago

Hello,

I would like to use CRISPRSeek to design gRNA targeting a genome which is not available in BSgenome database. This concerns the bacterial genome of Streptomyces ambofaciens ATCC 23877 (NCBI Reference Sequence: NZ_CP012382.1).

Is there any tutorial explaining how to use CRISPRSeek in this case. I found some indications in a supplementary information (article published in PloSOne, 2014), but I did not clearly understand how to create the file (with the genome, and with the target sequence) as well as how I should modify the instructions.

 

I thank you very much for your help.

Best regards

Stéphanie.
bsgenome crisprseek • 2.0k views
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Julie Zhu ★ 4.3k
@julie-zhu-3596
Last seen 13 months ago
United States
Stephanie, FYI, to search for gRNAs without performing off target analysis, please set chromToSearch = "" and there is no need to set the BSgenomeName https://www.bioconductor.org/packages/devel/bioc/vignettes/CRISPRseek/inst/doc/CRISPRseek.pdf (2.6 Scenario 6. Quick gRNA finding without target or off-target analysis). To perform off target analysis, you do need to set BSgenomeName. Here is the instruction on how to forge a BSgenome https://www.bioconductor.org/packages/devel/bioc/vignettes/BSgenome/inst/doc/BSgenomeForge.pdf If it is too much work to forge a genome, you could try the function offTargetAnalysisOfPeakRegions (http://www.bioconductor.org/packages/release/bioc/manuals/GUIDEseq/man/GUIDEseq.pdf) in the GUIDEseq package http://www.bioconductor.org/packages/release/bioc/html/GUIDEseq.html. Here is an example assuming gRNAFile contains the gRNA(s) in fasta format and fastaSequenceFile contains the genome sequence of your interest in fasta format. offTargets <- offTargetAnalysisOfPeakRegions(gRNA = gRNAFile, peaks = fastaSequenceFile, format="fasta", peaks.withHeader = FALSE, upstream = 0, downstream =0, PAM.size = 3, gRNA.size = 20, PAM = "NGG", PAM.pattern = "N[A|G]G$", max.mismatch = 3, outputDir = outputDir, orderOfftargetsBy = "predicted_cleavage_score", allowed.mismatch.PAM = 1, overwrite = TRUE ) Best, Julie From: "millepattes.intersites [bioc]" <noreply@bioconductor.org<mailto:noreply@bioconductor.org>> Reply-To: "reply+d57e7bd1+code@bioconductor.org<mailto:reply+d57e7bd1+code@bioconductor.org>" <reply+d57e7bd1+code@bioconductor.org<mailto:reply+d57e7bd1+code@bioconductor.org>> Date: Tuesday, March 14, 2017 3:27 PM To: Lihua Julie Zhu <julie.zhu@umassmed.edu<mailto:julie.zhu@umassmed.edu>> Subject: [bioc] CRISPRseek with genome not available in BSgenome Activity on a post you are following on support.bioconductor.org<https: support.bioconductor.org=""> User millepattes.intersites<https: support.bioconductor.org="" u="" 12592=""/> wrote Question: CRISPRseek with genome not available in BSgenome<https: support.bioconductor.org="" p="" 93835=""/>: Hello, I would like to use CRISPRSeek to design gRNA targeting a genome which is not available in BSgenome database. This concerns the bacterial genome of Streptomyces ambofaciens ATCC 23877 (NCBI Reference Sequence: NZ_CP012382.1). Is there any tutorial explaining how to use CRISPRSeek in this case. I found some indications in a supplementary information (article published in PloSOne, 2014), but I did not clearly understand how to create the file (with the genome, and with the target sequence) as well as how I should modify the instructions. I thank you very much for your help. Best regards St�phanie. ________________________________ Post tags: bsgenome, crisprseek You may reply via email or visit CRISPRseek with genome not available in BSgenome
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Hi Julie,

I am not sure if I understand this correctly. Can you test a guide(s) (in FASTA) on any type of sequence (not necessarily any genome sequence but i.e. "artificial" sequence of your interest)?

I tried to run a code as you suggested above with guide and sequence of my interest (both in FASTA format) and get this error:

Error in if (format[2] == "bed") { : 
  missing value where TRUE/FALSE needed

Best reagrds,

Dawid

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Dawid,

Could you please try to set the parameter format = c("fasta" "fasta")? The default is format = c("fasta", "bed"). You might want to tune other parameters such as  max.mismatch (default  6). For the detailed parameter list, please take a look at https://www.rdocumentation.org/packages/GUIDEseq/versions/1.2.1/topics/offTargetAnalysisOfPeakRegions . Thanks!

Best regards,

Julie

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Julie,

After updating to  format = c("fasta", "fasta") and changing some parameters I get:

Error in offTargetAnalysisOfPeakRegions(gRNA = gRNAFile, peaks = fastaSequenceFile,  : 

  only bed file with at least 4 columns are supported

Best,
Dawid

 

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Julie Zhu ★ 4.3k
@julie-zhu-3596
Last seen 13 months ago
United States

Dawid, could you please post your code, a sample gRNA, sample genome sequences and session information using sesssionInfo()? Thanks!

Best regards,

Julie
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David,  

Sorry that the function does not support fasta format for peak input yet. I think another function in CRISPRseek package fits your need better.

Here is an example that you can modify to fit your needs. You can tune the parameters according to https://github.com/Bioconductor-mirror/CRISPRseek/blob/master/man/compare2Sequences.Rd.

 

library(CRISPRseek)
  inputFile1Path <- system.file("extdata", "rs362331T.fa",
  package = "CRISPRseek")
  inputFile2Path <- system.file("extdata", "rs362331C.fa",
  package = "CRISPRseek")
   
  seqs <- compare2Sequences(inputFile1Path, inputFile2Path,
  outputDir = getwd(), searchDirection = "1to2", findgRNAs = c(TRUE, FALSE), 
overwrite = TRUE)

 

Best regards,

Julie 

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Thanks Julie, that works exactly as I needed.

Dawid

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Dawid, Great! Thanks for letting me know! Best regards, Julie On Jul 7, 2017, at 9:53 PM, Dawid G. Nowak [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 Dawid G. Nowak<https: support.bioconductor.org="" u="" 6790=""/> wrote Comment: CRISPRseek with genome not available in BSgenome<https: support.bioconductor.org="" p="" 93835="" #97868="">: Thanks Julie, that works exactly as I needed. Dawid ________________________________ Post tags: bsgenome, crisprseek You may reply via email or visit C: CRISPRseek with genome not available in BSgenome
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