RIPSeeker Bioconductor package, insights?

Question

[CrisMar]

Hi, Has anyone used the RIPSeeker package from Bioconductor? I am analyzing RNA Immunoprecipitation (RIP)-seq data. I cannot find any documents online with examples of the commands used for RIP-seq analysis. I only found the paper and a few documents defining the commands.

It would help greatly to actually see the commands being used for a RIPseq analysis.

I have triplicates for my pulldown as well as INPUT and IgG antibody libraries.

Thanks for any insight.

[ximena.garate]

Hi CrisMar,

I'm analyzing some RIP-seq data too using the RIPSeeker package. Did you have any luck running the function Ripseek? I keep getting the following error and I don't know what I should change to make it work...

This is how I set all parameters:

cNAME <- "Aligned.out_1B_sort"
outDir <- file.path(getwd(), "RIPSeeker_analysis")
binSize <- NULL     
minBinSize <- NULL          
maxBinSize <- NULL        
multicore <- TRUE                 
strandType <- "+"                 
biomart <- "ensembl"           
biomaRt_dataset <- "hsapiens_gene_ensembl" 
goAnno <- "org.Hs.eg.db"                    

seekOut.iPS_1 <- ripSeek(bamPath = "~/Desktop/AGO_IP/iPSrep1", cNAME = cNAME, reverseComplement = TRUE, 
                genomeBuild = "hg38", strandType = strandType, uniqueHit = TRUE, assignMultihits = TRUE, 
                rerunWithDisambiguatedMultihits = TRUE, binSize=binSize, minBinSize = minBinSize, maxBinSize = maxBinSize, 
                biomart=biomart, goAnno = goAnno, biomaRt_dataset = biomaRt_dataset, multicore=multicore, outDir=outDir)

And the error message I got is:

Error in mclapply(as.list(split(alignGR, seqnames(alignGR))), mainSeekSingleChrom,  : 
  'mc.cores' must be >= 1

Did you have any problems like this? Any suggestion will be much appreciated. Thanks Ximena

[Friederike]

are you using a Windows machine? mclapply does not work on Windows. Apart from that, what happens when you set multicore <- FALSE?

[CrisMar]

Hi ximena. Did Friderike advise help? I can post what worked for me ( I am running R on a mac or Linux server).

[rew_27]

Hello everyone,

I am using RIPSeeker for the first time and have a few questions. I have 7 samples, each sample has 3 biological replicates. (So total 21 samples). The bological replicates correspond to, 1. Total Input 2. Knockdown of desired RBP 3. IgG negative control.

My questions are:

1. Should I be performing a separate analysis for each triplet of Input, knockdown and IgG?
2. For now, I am working with one such triplet, and considering that the BAM files are about 2GB each, is it possible that the program runs for more than 24 hours? I am using the multicore option.

Any help/pointers would be greatly appreciated!

[GenoMax]

Please post this as a new question. This is unrelated to the original question in this thread.

[CrisMar]

The vignette indicates that technical replicates should be combined but no biological ones before analysis. It did take my runs about ~16-20 hrs.

Answer 1

[CrisMar]

Setting the variables was:

# specify control name
cNAME <- "SRR039214"

 # output file directory
outDir <- file.path(getwd(), "RIPSeeker_vigenette_example_PRC2")

# Parameters setting
binSize <- NULL      # set to NULL to automatically determine bin size

minBinSize <- 10000  # min bin size in automatic bin size selection

maxBinSize <- 10100

multicore <- TRUE

 strandType <- "-"

biomart <- "ENSEMBL_MART_ENSEMBL"

biomaRt_dataset <- "mmusculus_gene_ensembl" # mouse dataset id name

host <- "dec2011.archive.ensembl.org"       # use ensembl 65 for annotation f

goAnno <- "org.Mm.eg.db"                    # GO annotation database

[Friederike]

thanks for posting what ended up working for you!

[CrisMar]

Hi Freiderike. I ended up getting it to work after all! I had many errors where I was assigning incorrect bin size and also I had align my bam files with the a different genome release in which I was trying to annotate. After re-reading the package instructions, vignette and paper etc I am more familiar with how it works. But indeed there are still many bugs that relate to package updates and defunct functions. Currently I am stuck with the viewRIP function, but it wasn't as important as the ripSeek function (will post this soon).

After running ripSeek on each biological triplicate I combined the triplicates but cannot get statistical outputs with the combine so I am looking at each output for each biological triplicate.

Thank you for the advise!

Answer 2

[Friederike]

The vignette didn't do it for you? Do you run into errors?

[CrisMar]

Thanks Friederike, I had confused the file with another file. Regardless everything worked well until I ran ripSeek command. I got the following error:

seekOut.PRC2 <- ripSeek( bamPath = bamFiles, cNAME = cNAME, reverseComplement = TRUE, genomeBuild = "mm9", strandType = strandType, uniqueHit = TRUE, assignMultihits = TRUE, rerunWithDisambiguatedMultihits = TRUE, binSize=binSize, minBinSize = minBinSize, maxBinSize = maxBinSize, biomart=biomart, host=host, biomaRt_dataset = biomaRt_dataset, goAnno = goAnno, multicore=multicore, outDir=outDir)

Error: 1: Entity 'nbsp' not defined 2: Entity 'hellip' not defined 3: Entity 'hellip' not defined 4: Entity 'nbsp' not defined 5: Entity 'raquo' not defined 6: Entity 'hellip' not defined 7: Entity 'hellip' not defined 8: Entity 'hellip' not defined 9: Entity 'hellip' not defined 10: Entity 'hellip' not defined 11: Opening and ending tag mismatch: img line 68 and li 12: Opening and ending tag mismatch: li line 68 and ul 13: Opening and ending tag mismatch: ul line 67 and div 14: expected '>' 15: Entity 'copy' not defined 16: AttValue: " or ' expected 17: attributes construct error 18: Couldn't find end of Start Tag input line 258 19: Opening and ending tag mismatch: div line 18 and body 20: Opening and ending tag mismatch: body line 16 and html 21: Premature end of data in tag html line 2 In addition: Warning messages: 1: package ‘VennDiagram’ was built under R version 3.2.5 2: package ‘futile.logger’ was built under R version 3.2.5 3: package ‘RSQLite’ was built under R version 3.2.5

I think it might have something to do with updating R and the rest of the packages?

I was also confused as to when I could combine my replicates, before or after processing my alignments?

[Friederike]

I don't recall this exact error message, but I do remember that it was definitely a buggy package. I think, in my case I definitely had to define the size of the bin (not by max and min, but with binSize) in order to get it to run -- since you seem very sure that the bin size should be around 10,000 bp you could try it with specifying it.

That being said, I did a quick search in the package's source code on github and it doesn't seem as if the errors are directly related to functions in that package (e.g., nbsp, hellip etc.). My best bet would be the retrieval of the annotation that is throwing the error. In my notes from when I used RIPseeker, I simply defined it so: genomeBuild = "hg38". I recommend you try to dig a bit deeper there.

Did you run the example code from the vignette without running into errors?

[Friederike]

actually, it seems as if this is a common problem for RIPseeker which has been reported on the bioconductor website. See, for example here and here.

Now that I think about it, I did not use any annotation of RIPseeker, probably because it simply kept on failing. You can just generate the peak regions and do the annotation yourself, either in R or in the command line, e.g. using HOMER