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Question: GUIDESeq analysis - handling biological and technical replicates
0
gravatar for aamir.mir
17 months ago by
aamir.mir0
aamir.mir0 wrote:

I am relatively new to GUIDEseq analysis, and was wondering how to handle multiple biological replicates. Currently, I have 3 biological replicates of a sample and they are differentiated by the P5 index. For every biological replicate, there are 8 libraries (P7 index is different), 4 for + strand and 4 for the - strand. How should I analyze my data so that I have one output file with off targets for my particular gRNA? It seems like GUIDEseAnalysis only takes two files at a time. 
Any help would be appreciated.

Thanks in advance.

guideseq • 524 views
ADD COMMENTlink modified 17 months ago by Julie Zhu3.9k • written 17 months ago by aamir.mir0
Answer: GUIDESeq analysis - handling biological and technical replicates
2
gravatar for Julie Zhu
17 months ago by
Julie Zhu3.9k
United States
Julie Zhu3.9k wrote:
Aamir, Yes. You can analyze multiple replicates together by specifying alignment.inputfile = c(replicate1.bam, replicate2.bam, replicate3.bam), umi.inputfile = c(replicate1.umi.txt, replicate2.bam.umi.txt, replicate3.umi.txt) in GUIDEseqAnalysis<https: rdrr.io="" bioc="" guideseq="" man="" guideseqanalysis.html="">, then follow the Example 5 at https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-017-3746-y For technical replicates, I recommend merge the fastq files and treat it as a single experiment. Best, Julie Sent from my iPhone On Aug 27, 2017, at 12:28 AM, aamir.mir [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 aamir.mir<https: support.bioconductor.org="" u="" 13831=""/> wrote Question: GUIDESeq analysis - handling biological and technical replicates<https: support.bioconductor.org="" p="" 99746=""/>: I am relatively new to GUIDEseq analysis, and was wondering how to handle multiple biological replicates. Currently, I have 3 biological replicates of a sample and they are differentiated by the P5 index. For every biological replicate, there are 8 libraries (P7 index is different), 4 for + strand and 4 for the - strand. How should I analyze my data so that I have one output file with off targets for my particular gRNA? It seems like GUIDEseAnalysis only takes two files at a time. Any help would be appreciated. Thanks in advance. ________________________________ Post tags: guideseq You may reply via email or visit GUIDESeq analysis - handling biological and technical replicates
ADD COMMENTlink written 17 months ago by Julie Zhu3.9k

I was able to find off-targets for my samples. However, when I try to remove the peaks shared with the control sample, I keep getting an error message: "Error in fix.by(by.y, y) : 'by' must specify uniquely valid columns"

My code looks like this: 
offtargetfolder <- c("NTS42-output/", "neg-NTS42-output/")
offtargets.hotspots.removed <- combineOfftargets(offtarget.folder = offtargetfolder, 
                                                 sample.name = c("NTS42", "Cas9-only"),
                                                 control.sample.name = "Cas9-only",
                                                 outputFileName = "NTS42-offtarget-hotspots-removed.xls")

Do I have rename the columns in the output that I get from GuideseqAnalysis function?

ADD REPLYlink written 17 months ago by aamir.mir0
Aamir, You should not need to rename the column or the output file. Best, Julie On Aug 28, 2017, at 2:43 PM, aamir.mir [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 aamir.mir<https: support.bioconductor.org="" u="" 13831=""/> wrote Comment: GUIDESeq analysis - handling biological and technical replicates<https: support.bioconductor.org="" p="" 99746="" #99765="">: I was able to find off-targets for my samples. However, when I try to remove the peaks shared with the control sample, I keep getting an error message: "Error in fix.by(by.y, y) : 'by' must specify uniquely valid columns" My code looks like this: offtargetfolder <- c("NTS42-output/", "neg-NTS42-output/") offtargets.hotspots.removed <- combineOfftargets(offtarget.folder = offtargetfolder, sample.name = c("NTS42", "Cas9-only"), control.sample.name = "Cas9-only", outputFileName = "NTS42-offtarget-hotspots-removed.xls") Do I have rename the columns in the output that I get from GuideseqAnalysis function? ________________________________ Post tags: guideseq You may reply via email or visit C: GUIDESeq analysis - handling biological and technical replicates
ADD REPLYlink written 17 months ago by Julie Zhu3.9k

So why do you think I am getting that error message?

ADD REPLYlink written 17 months ago by aamir.mir0
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