CRISPRseek with genome not available in BSgenome
2
0
Entering edit mode
@millepattesintersites-12592
Last seen 7.8 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
ADD COMMENT •
0
Entering edit mode
Julie Zhu ★ 4.3k
@julie-zhu-3596
Last seen 14 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
ADD COMMENT •
0
Entering edit mode

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

ADD REPLY •
0
Entering edit mode

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

ADD REPLY •
0
Entering edit mode

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

 

ADD REPLY •
0
Entering edit mode
Julie Zhu ★ 4.3k
@julie-zhu-3596
Last seen 14 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
ADD COMMENT •
1
Entering edit mode

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 

ADD REPLY •
0
Entering edit mode

Thanks Julie, that works exactly as I needed.

Dawid

ADD REPLY •
0
Entering edit mode
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
ADD REPLY •

Login before adding your answer.

Similar Posts
Loading Similar Posts
Traffic: 561 users visited in the last hour
Content Search
Users
Tags
Badges
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6