How to use RIPSeeker?

Question

[xiaocong3333]

Hi, is there anyone very familiar with RIPSeeker? I have tested it on my RIPseq data, I found some problems which I can't solve. My code as below:

library("RIPSeeker")
library("biomaRt")
library("curl")
library("org.Dm.eg.db")

outDir <- file.path(getwd(), "TEST")
# Parameters setting
binSize <- NULL     
minBinSize <- 11000 
maxBinSize <- 12000
multicore <- TRUE
strandType <- "-"   

biomart <- "ensembl"
biomaRt_dataset <- "dmelanogaster_gene_ensembl"
goAnno <- "org.Dm.eg.db"                    

seekOut.PRC2 <- ripSeek(bamPath = "/home/.../RIPSEQ/test", cNAME = 
                        "Non-...sorted.bam",
                        reverseComplement = TRUE, paired = TRUE,
                        strandType = strandType,
                        uniqueHit = TRUE, assignMultihits = TRUE,
                        rerunWithDisambiguatedMultihits = TRUE,
                        binSize=binSize, minBinSize = minBinSize,
                        maxBinSize = maxBinSize,
                        biomart = biomart,
                        biomaRt_dataset = biomaRt_dataset,
                        multicore=multicore, outDir=outDir)

I run the test data, the result is good, but for the ripRPKM, it reported errors with package “curl” .

When I run my RIPseq data, there are some errors. My library is a directional strand library from fly. I used Hisat2 (an subsitition for Tophat)for mapping, I tried both Dm3/BDGP6 as ref, I changed the strandType with “+”, “-”, “*”, and reverseComplement as TRUE or FALSE.

There are always some problems. When I set strandType = "-/+", reverseComplement as "TRUE/FALSE", the error is: Error in (function (...) : all elements in '...' must be GRanges objects Calls: ripSeek ... as -> asMethod -> do.call -> do.call -> <anonymous> I set the genomeBiuld as 'dm', 'dm3', "dm6", or just delete it, the final result seems no difference.

But when I delete the paired = TRUE and goAnno( If I keep goAnno, it will report less than 2 gene for GO enrichment), but my libraries are paired ends, it seems work. But when I check the RIPregions_annotated.txt, there are only a few information, these are all the results from RIPregions_annotated.txt:

chr2L   22423991        22435020        11030   -       NA      NA      NA      16018   1452.22121486854        379.155319281055        6.45394687429009e-18    6.45394687429009e-18    10573   958.56754306437 -379.422603380833       6.45394687429009e-18    1       NA      NA      NA      NA      NA      NA      NA      NA
chr2R   10239301        10250310        11010   -       NA      NA      NA      597589  54276.9300635786        804.930196349324        9.57741037608529e-33    9.57741037608529e-33    108069  9815.53133514986
        -683.951264684174       9.57741037608529e-33    2       NA      NA      NA      NA      NA      NA      NA      NA
chr2RHet        684171  695205  11035   -       NA      NA      NA      2324    210.602628001812        700.935428402131        8.94887332351163e-15    1.78977466470233e-14    4903    444.313547802447        -699.260953254821       8.94887332351163e-15    3       NA      NA      NA      NA      NA      NA      NA      NA
chr3LHet        2391341 2402360 11020   -       NA      NA      NA      12510   1135.20871143376        7.0982117815557 0.442349878910107       0.442349878910107       9353    848.729582577133        3.01016459563286        0.442349878910107       4       NA      NA      NA      NA      NA      NA      NA      NA
chr3RHet        2470841 2481920 11080   -       NA      NA      NA      5928    535.018050541516        1.33358189119612        0.0200600377053582      0.0200600377053582      7284    657.400722021661        5.73707063985334        0.0200600377053582      5       NA      NA      NA      NA      NA      NA      NA      NA
chrUextra       28523001        28534000        11000   -       NA      NA      NA      9862    896.545454545455        7.23892616977548        0.304860511433238       0.304860511433238       7010    637.272727272727        16.7024751279322        0.304860511433238       6       NA      NA      NA      NA      NA      NA      NA      NA
chrX    7826941 7837995 11055   -       FBgn0029962     CG1402  NA      11171   1010.49298959747        702.925367593253        2.28797076005909e-06    2.36746089331267e-06    9042    817.910447761194        -702.771393425973       2.28797076005909e-06    7       FBgn0029962     7826197 7842679 +       inside  744     744     NearestLocation
chrX    17555341        17566395        11055   -       FBgn0263706     CG43658 NA      11266   1019.08638625057        701.768520799361        2.36746089331267e-06    2.36746089331267e-06    13053   1180.73270013569        -701.847435810435       2.36746089331267e-06    8       FBgn0263706     17456169        17561180        -       overlapStart    5839    5215    NearestLocation
chrXHet 34636   46180   11545   -       NA      NA      NA      833     72.1524469467302        0.896358105384567       0.898471262092674       0.898471262092674       1176    101.862278042443        2.00304273493262        0.898471262092674       9       NA      NA      NA      NA      NA      NA      NA      NA

Thanks in advance!

[A. Domingues]

Not an answer but unless you really need to use RIPseeker, I would advise to look for alternatives. As far as I can tell development as been stagnating fro a long time now, with the last commit in github being 2 years ago, and pull requests and issues going unanswered from around that time. I also ran into several issues when dealing with non-human genome data and eventually choose to go the DEseq way, with my own custom functions for annotation.

[xiaocong3333]

Thank you Domingues. I used DEseq2 to analyze my RNAseq data. Is it OK just use it for RIPseq data? Did you make any modifications?

[A. Domingues]

No modification at all, I simply did a IP / input comparison and it worked well - at least that is what my colleagues which did the experimental validation told me.