Question

GUIDESeq analysis - handling biological and technical replicates

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aamir.mir • 0

@aamirmir-13831

Last seen 6.7 years ago

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 • 1.5k views

ADD COMMENT • link updated 6.6 years ago by Julie Zhu ★ 4.3k • written 6.6 years ago by aamir.mir • 0

score 2 · Accepted Answer · 2017-08-27

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Julie Zhu ★ 4.3k

@julie-zhu-3596

Last seen 5 months ago

United States

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 COMMENT • link 6.6 years ago Julie Zhu ★ 4.3k

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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 REPLY • link 6.6 years ago aamir.mir • 0

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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