Question

CRISPRseek: summary file

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Julie Zhu ★ 4.3k

@julie-zhu-3596

Last seen 5 months ago

United States

Dear Marius,

Thanks for the feedback!

Usually, for every gRNA, one of the top hits (score 100) in the offtarget file <font size="2">is the target sequence. That is why it is not included in the topN </font>offtarget<font size="2"> score calculation. Could you please post the gRNA sequence and the code you used for searching for offtargets so that I can check whether the one not included is the target site or not? Thanks!</font>

Best regards,

Julie

On 7/2/15 8:11 AM, "Marius Garmhausen" <marius.garmhausen@uni-koeln.de> wrote:

Hello Julie,

I might have come across a little bug in how the scores from the

offtarget-analysis are summed into the summary file. It seems that the

first offtarget for every gRNA is not included. If you have 1 offtarget

you get NA if you have two or more you get the sum from the second to

the last. Maybe there is good reason behind this and I haven't checked

the sourcecode, but I thought this might be of interest for you.

Best Regards,

Marius Garmhausen

PhD Student

Beyer Lab

Systems Biology

University of Cologne

Germany

CRISPRseek: summary file topN offtarget score • 893 views

ADD COMMENT • link updated 8.8 years ago by marius.garmhausen • 0 • written 8.8 years ago by Julie Zhu ★ 4.3k

score 0 · Answer 1 · 2015-07-06

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marius.garmhausen • 0

@mariusgarmhausen-8338

Last seen 7.3 years ago

Germany

Dear Julie,

thanks for the quick reply. My problem is/was partially related to the orientation of the sequence in the fasta file. The sequences I am using are based on the sequence from Ensembl Biomart. If you download a sequence at Biomart and its on the reverse strand, the order is reversed. After taking the reverse complement of this sequence, the results actually had the right 100% overlapping locations.

In my first attempt I was trying to limit the gRNAs discovered to the top-50 closest to the gene start. To do that I loaded the discovered gRNAs that were discovered by:

res <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE, REpatternFile = REpatternFile, findPairedgRNAOnly = FALSE, BSgenomeName = BSgenomeName, txdb = txdb, orgAnn = orgAnn, chromToSearch = "", annotateExon=FALSE, max.mismatch = 3,overlap.gRNA.positions = c(17, 18), outputDir = outputDir,overwrite = TRUE)

and stored them in a new fasta file. This I used as input for a new run without gRNA discovery. Here also the identification of the original target region failed. Maybe there is a better way to do so?

ADD COMMENT • link 8.8 years ago marius.garmhausen • 0

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Dear Marius, It should be able to find the target site even it is in reverse strand. Did you use the right Bsgenome? Could you please post the sequences you are trying to analyze? Also please post your complete code and session information. Thanks! Best regards, Julie From: "marius.garmhausen [bioc]" <noreply@bioconductor.org<mailto:noreply@bioconductor.org>> Reply-To: "reply+41111b5d+code@bioconductor.org<mailto:reply+41111b5d+code@bioconductor.org>" <reply+41111b5d+code@bioconductor.org<mailto:reply+41111b5d+code@bioconductor.org>> Date: Monday, July 6, 2015 11:12 AM To: Lihua Julie Zhu <julie.zhu@umassmed.edu<mailto:julie.zhu@umassmed.edu>> Subject: [bioc] A: CRISPRseek: summary file Activity on a post you are following on support.bioconductor.org<https: support.bioconductor.org=""> User marius.garmhausen<https: support.bioconductor.org="" u="" 8338=""/> wrote Answer: CRISPRseek: summary file<https: support.bioconductor.org="" p="" 69419="" #69525="">: Dear Julie, thanks for the quick reply. My problem is/was partially related to the orientation of the sequence in the fasta file. The sequences I am using are based on the sequence from Ensembl Biomart. If you download a sequence at Biomart and its on the reverse strand, the order is reversed. After taking the reverse complement of this sequence, the results actually had the right 100% overlapping locations. In my first attempt I was trying to limit the gRNAs discovered to the top-50 closest to the gene start. To do that I loaded the discovered gRNAs that were discovered by: res <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE, REpatternFile = REpatternFile, findPairedgRNAOnly = FALSE, BSgenomeName = BSgenomeName, txdb = txdb, orgAnn = orgAnn, chromToSearch = "", annotateExon=FALSE, max.mismatch = 3,overlap.gRNA.positions = c(17, 18), outputDir = outputDir,overwrite = TRUE) and stored them in a new fasta file. This I used as input for a new run without gRNA discovery. Here also the identification of the original target region failed. Maybe there is a better way to do so? ________________________________ Post tags: CRISPRseek: summary file topN offtarget score You may reply via email or visit A: CRISPRseek: summary file

ADD REPLY • link 8.8 years ago Julie Zhu ★ 4.3k