Error in CummeRbund while creating database

[Richard Barker]

Hello

I'm trying to investigate my Tophat 2.05 Bowtie 2.0 aligned BAM file, plus BAM bai file and cuffdiff 2.02 outputs using the latest version of cummeRbund in R version 2.15.2.

While generating the database using the > cuff <- readCufflinks(rebuild=T) command i encounted an error. Does anyone know what could have caused it?

Any feedback greatfully received

[dr_richard_barker@vm142-21 cuffdiff_out]$ R

R version 2.15.2 (2012-10-26) -- "Trick or Treat"
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ISBN 3-900051-07-0
Platform: x86_64-redhat-linux-gnu (64-bit)

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> library (cummeRbund)
Loading required package: BiocGenerics
Attaching package: 'BiocGenerics'
The following object(s) are masked from 'package:stats':
xtabs
The following object(s) are masked from 'package:base':
Filter, Find, Map, Position, Reduce, anyDuplicated, cbind,
colnames, duplicated, eval, get, intersect, lapply, mapply, mget,
order, paste, pmax, pmax.int, pmin, pmin.int, rbind, rep.int,
rownames, sapply, setdiff, table, tapply, union, unique

Loading required package: RSQLite
Loading required package: DBI
Loading required package: ggplot2
Loading required package: reshape2
Loading required package: fastcluster

Attaching package: 'fastcluster'

The following object(s) are masked from 'package:stats':
hclust

Loading required package: rtracklayer
Loading required package: GenomicRanges
Loading required package: IRanges
Loading required package: Gviz
Loading required package: grid
> cuff <- readCufflinks()
> cuff <- readCufflinks(rebuild=T)
Creating database /mydata2/0_GS_atmosphere_test/cuffdiff_out/cuffData.db
Reading Run Info File /mydata2/0_GS_atmosphere_test/cuffdiff_out/run.info
Writing runInfo Table
Reading Read Group Info /mydata2/0_GS_atmosphere_test/cuffdiff_out/read_groups.info
Writing replicates Table
Error in sqliteExecStatement(con, statement, bind.data) :
RS-DBI driver: (RS_SQLite_exec: could not execute: column rep_name is not unique)
>

[TorontoHeather]

Hi Richard,

I am having the exact same problem, but from searching Google it doesn't seem to be a common one.

Did you figure out what was going on? Any advice would be very much appreciated!

Thanks,

Heather

[Richard Barker]

Dear Heather

I didn't solve that issue in the end so found a couple of ways around the issue. The first thing i did was set up a free account with the iPlant collaborative (http://www.iplantcollaborative.org/). They have intergrated the Tuxedo protocol into a graphical user interface (GUI) that makes the whole system really easy to use. It runs on some super computers in Texas and/or Arazona so its also really fast. You have to up load your data on to their cloud computing system, but their data stores are limitless and their security tight. If you want to use the Tuxedo protocol i would strongly recormend their system.

After analyzing my samples with Tuxedo i wasn't happy with my results (one of my biological repeats had double the reads of the others and i don't think the normalisation methods within Tuxedo are the greatest). So i investigated some other packages. I used the Max Planck's RobiNA package (http://mapman.gabipd.org/web/guest/robin) and was very impressed! It can take your data as raw sequencing files, BAM files or even as counts per gene. Its got a really easy to use GUI and best of all incorporates 2 normalisation packages DESeq and EdgeR which gave me some really interesting lists of genes to chase up...

I hope this helps...

Best wishes, Richard

[jp.]

TorontoHeather and Rechard
re-run cufflinks/cuff_diff, change sample names (sample1...control....treatment...).

[TorontoHeather]

Dear Richard and JP,

Thank you both for your responses. I've been trying to get the iPlant Cloud services working but was encountering a different error when I tried running my data there.

I just recently heard from someone who was able to solve both errors. The one I posted on here was solved by replacing "-" with "neg" and "+" with "pos" in the sample names. I guess re-running cufflinks/cuffdiff and renaming the samples would have the same effect!

Now I am working through another error: the cummeRbund "getsig" does not return anything even though I can see that there are many that are significant. If I do not get it sorted soon I will write a new post with more details.

Thanks again!

Heather