Duplicated Events in EventDetection and EventPointer_RNASeq

[machalen]

Hello

I used EventPointer to analyze RNA-seq data. I followed the tutorial "EventPointer.Rmd" in vignettes:

SG_RNASeq<-PrepareBam_EP(Samples=Samples, SamplePath=PathToSamples, Ref_Transc="GTF", fileTransc=PathToGTF, cores=6) AllEvents_RNASeq<-EventDetection(Input=SG_RNASeq,cores=6,Path=resultsDir)

Events <- EventPointer_RNASeq(Events = AllEvents_RNASeq, Design=Dmatrix, Contrast=Cmatrix, Statistic="LogFC",PSI=TRUE)

When looking at AllEvents_RNASeq and Events objects and EventsFound_RNASeq.txt I found each event is duplicated. Column Gene is different because one has the annotation and the other no, but the other columns are the same. I used "Homo_sapiens.GRCh37.75.gtf" annotation file. I don't know if somebody had the same issue, any help will be welcome!

`sessionInfo() R version 3.4.0 (2017-04-21) Platform: x86_64-pc-linux-gnu (64-bit) Running under: Debian GNU/Linux 9 (stretch)

Matrix products: default BLAS: /usr/lib/libblas/libblas.so.3.7.0 LAPACK: /usr/lib/lapack/liblapack.so.3.7.0

locale: [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
[3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
[5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
[7] LC_PAPER=en_US.UTF-8 LC_NAME=C
[9] LC_ADDRESS=C LC_TELEPHONE=C
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C

attached base packages: [1] stats4 parallel stats graphics grDevices utils datasets [8] methods base

other attached packages: [1] EventPointer_1.0.0 Matrix_1.2-10
[3] SGSeq_1.10.0 SummarizedExperiment_1.6.3 [5] DelayedArray_0.2.7 matrixStats_0.52.2
[7] Biobase_2.36.2 Rsamtools_1.28.0
[9] Biostrings_2.44.2 XVector_0.16.0
[11] GenomicRanges_1.28.4 GenomeInfoDb_1.12.2
[13] IRanges_2.10.2 S4Vectors_0.14.3
[15] BiocGenerics_0.22.0 nnls_1.4
[17] limma_3.32.5

loaded via a namespace (and not attached): [1] Rcpp_0.12.12 compiler_3.4.0 iterators_1.0.8
[4] GenomicFeatures_1.28.4 bitops_1.0-6 tools_3.4.0
[7] zlibbioc_1.22.0 biomaRt_2.32.1 digest_0.6.12
[10] bit_1.1-12 RSQLite_2.0 memoise_1.1.0
[13] tibble_1.3.4 lattice_0.20-35 pkgconfig_2.0.1
[16] rlang_0.1.2 foreach_1.4.3 graph_1.54.0
[19] igraph_1.1.2 DBI_0.7 prodlim_1.6.1
[22] GenomeInfoDbData_0.99.0 stringr_1.2.0 affxparser_1.48.0
[25] rtracklayer_1.36.4 bit64_0.9-7 grid_3.4.0
[28] AnnotationDbi_1.38.2 survival_2.41-3 RBGL_1.52.0
[31] XML_3.98-1.5 BiocParallel_1.10.1 lava_1.5
[34] magrittr_1.5 blob_1.1.0 splines_3.4.0
[37] codetools_0.2-15 MASS_7.3-47 GenomicAlignments_1.12.2 [40] RUnit_0.4.31 stringi_1.1.5 doParallel_1.0.10
[43] RCurl_1.95-4.8
`

[jpromeror]

Hi @machalen,

Sorry for the delay in the answer.

Could you paste an example of a duplicated Event from EventsFound_RNASeq.txt

I will check this issue as quickly as possible.

[machalen]

Hi @jpromeror

The BAM files I was using didn't include the XS-flag, so maybe the error is due to that. In the vignette you specify BAM files should include the XS-flag, but since I didn't get an error with the "PrepareBam_EP" function (it was a warning), and the object "SG_RNASeq" was generated, I decided to continue with the analysis, not a good choice by the way. I will let you know if the problem is solved with the new alignment.

Sorry for inconvenience

[jpromeror]

Hi @machalen

Were you able to solve the problem after adding the XS-flag? If not, I think it could be related on how SGSeq deals with the information when building the splicing graphs. I will run some tests to see if I can replicate those duplicates.

Please let me know of anything else!

[machalen]

Hi @jpromeror

Unfortunately we have problems in our cluster and I couldn't perform the analysis yet. I will let you know in the next weeks if I get the same duplicates. Could you please let us know if you can replicate the error with the XS-flag?

Thanks a lot

[machalen]

I did the analysis again with the XS-flag and now the results are ok. So I guess the problem was due to not including the flag in the BAM files. I hope this issue will be helpful for other users.

Thanks for the support @jpromeror,

Magda