roryk / bcbio.rnaseq Goto Github PK

Hi roryk,
I want to run bcbio-rnaseq to generate bcbio-nextgen report and differential expression analysis using DESeq2. My bcbio-nextgen has been successfully finished. When I use the the following command:
bcbio-rnaseq summarize ../config/seqc.yaml -f "~batch+panel" -d -o human -s
I got the output as below:

WARNING: update already refers to: #'clojure.core/update in namespace: incanter.core, being replaced by: #'incanter.core/update
Running DESeq2.
Running DEXSeq, this will take a long time.
Setting up DEXSeq template.
Setting up Sleuth template.
Setting up pathway analysis template.
Summary report can be found here /app/datamart/RNAseq-example/subset-test/final/../config/summary/qc-summary.Rmd

The running lasted only a few second and in the /config/summary/ directory I found only a qc-summary.Rmd file and a few tmp files that looks like R scripts.

How to make bcbio-rnaseq correctly run? Thanks a lot.

Hi @roryk ,

I would like the tool to compare different groups using deseq, edgeR,.. What is the field in the config file that assigns samples into different groups ?

Thanks !!!

Hello!

I am trying to subset a bcbiornaseq object to include fewer samples and then run deseq to do the differential analysis. I am having problems with (I assume) the DESeqDataSet object not understanding that I have subset the data and still looking for all the samples in the design. Is there a way to get around this? I have included my code and error message below.

If I run this on the whole bcb dataset and not the subset then I have no error message.

bcb <- bcbioRNASeq(
uploadDir = "final/",
interestingGroups = c("phenotype"),
organism = "Homo sapiens"
)
bcb_samples <- colnames(bcb)[c(7:9, 13:15)]
bcb_subset <- bcb[, bcb_samples]
dds <- as(bcb_subset, "DESeqDataSet")
design(dds) <- ~phenotype
dds <- DESeq(dds)

Error in designAndArgChecker(object, betaPrior) : factors in design formula must have samples for each level. this error can arise when subsetting a DESeqDataSet, in which all the samples for one or more levels of a factor in the design were removed. if this was intentional, use droplevels() to remove these levels, e.g.: dds$condition <- droplevels(dds$condition)

hi Rory:

make
...
Including stax-api-1.0.1.jar
Including tigris-0.1.1.jar
Including edn-java-0.4.4.jar
Including xalan-2.6.0.jar
Including xml-apis-ext-1.3.04.jar
Including xml-apis-1.3.04.jar
Created /raid/sonpham/bcbio/bcbio.rnaseq/bcbio.rnaseq-0.1.1a-SNAPSHOT-standalone.jar
cat bin/bcbio-rnaseq.template target/-standalone.jar > bin/bcbio-rnaseq
cat: target/-standalone.jar: No such file or directory
make: *** [all] Error 1

Hi

re: this piece of code in the function resources/comparisons/qPCR_foldchange.template
plot_FC = function(df) {
df = df[, c("id", "logFC", "algorithm")]
colnames(df) = c("id", "logFC", "method")
qpcr_foldchange = foldchange[, c("id", "logFC")]
df = merge(df, qpcr_foldchange, by="id")
colnames(df) = c("id", "seq_logFC", "method", "qPCR_logFC")
p = ggplot(df, aes(seq_logFC, qPCR_logFC)) + geom_point() + facet_grid(. ~ method) +
xlab(expression(paste(log["2"], " fold change via RNA-seq"))) +
ylab(expression(paste(log["2"], " fold change via qPCR")))
ggsave(filename=FC_file, plot=p)
}

I understand this piece of code plots the measured logFC from RNA-seq, versus the expected logFC obtained from the qPCR reference file.
I'm wondering however how a given gene is mapped between the RNA-seq file (e.g.,
deseq_HBRR_vs_UHRR.tsv) and the qPCR reference file (qpcr_HBRR_vs_UHRR.tidy).
the list of gene is different between both files, so I'm wondering how the mapping works between the 2.
example of genes from qpcr_HBRR_vs_UHRR.tidy
'18S'
'ABC1'
'ABCA1'
'ABCA2'
'ABCB1'
'ABCB11'
'ABCB6'
'ABCC1'
'ABCC11'
'ABCC2'
'ABCC4'
example of genes from deseq_HBRR_vs_UHRR.tsv
'ENSG00000223972'
'ENSG00000227232'
'ENSG00000243485'
'ENSG00000237613'
'ENSG00000268020'
'ENSG00000240361'
'ENSG00000186092'
'ENSG00000238009'
'ENSG00000239945'
'ENSG00000233750'
'ENSG00000237683'
'ENSG00000268903'
'ENSG00000269981'

Thanks!
Severine

Hi author,
After run the bcbio-nextgen, I have the final results :

2015-07-15_meiyanrnaseq2
Neuron31A_0h-701-2-ATTACT_ATTACT_L002_R1_001
Neuron31A_1h-702-2-TCCGGA_TCCGGA_L002_R1_001
Neuron31A_6h-703-2-CGCTCA_CGCTCA_L002_R1_001
Neuron32B_0h-704-2-GAGATT_GAGATT_L002_R1_001

Then , I ran the "bcbio-rnaseq compare" command, I got this error:

hoabo@son:~/example/bcbio_nextgen/meiyanrnaseq2/final$ bcbio-rnaseq compare

Usage: bcbio-rnaseq compare [options] project-file key

Options:
-h, --help
-n, --cores CORES 1 Number of cores
--counts-only Only run count-based analyses
--seqc Data is from a SEQC alignment

Arguments:
project-file A bcbio-nextgen project file
key Key in the metadata field to do pairwise comparisons

Thanks !!!

Thanks Roryk for the great pipeline. But it just yields exception when I run summarize command.
bcbio-rnaseq summarize 2015-07-15_rnaseq2/project-summary.yaml
Exception in thread "main" java.lang.ExceptionInInitializerError
at java.lang.Class.forName0(Native Method)
at java.lang.Class.forName(Class.java:274)
at clojure.lang.RT.loadClassForName(RT.java:2098)
at clojure.lang.RT.load(RT.java:430)
at clojure.lang.RT.load(RT.java:411)
at clojure.core$load$fn__5018.invoke(core.clj:5530)
at clojure.core$load.doInvoke(core.clj:5529)
at clojure.lang.RestFn.invoke(RestFn.java:408)
at clojure.core$load_one.invoke(core.clj:5336)
at clojure.core$load_lib$fn__4967.invoke(core.clj:5375)
at clojure.core$load_lib.doInvoke(core.clj:5374)
at clojure.lang.RestFn.applyTo(RestFn.java:142)
at clojure.core$apply.invoke(core.clj:619)
at clojure.core$load_libs.doInvoke(core.clj:5413)
at clojure.lang.RestFn.applyTo(RestFn.java:137)
at clojure.core$apply.invoke(core.clj:619)
at clojure.core$require.doInvoke(core.clj:5496)
at clojure.lang.RestFn.invoke(RestFn.java:703)
at bcbio.rnaseq.simulate$loading__4910__auto__.invoke(simulate.clj:1)
at bcbio.rnaseq.simulate__init.load(Unknown Source)
at bcbio.rnaseq.simulate__init.(Unknown Source)
at java.lang.Class.forName0(Native Method)
at java.lang.Class.forName(Class.java:274)
at clojure.lang.RT.loadClassForName(RT.java:2098)
at clojure.lang.RT.load(RT.java:430)
at clojure.lang.RT.load(RT.java:411)
at clojure.core$load$fn__5018.invoke(core.clj:5530)
at clojure.core$load.doInvoke(core.clj:5529)
at clojure.lang.RestFn.invoke(RestFn.java:408)
at clojure.core$load_one.invoke(core.clj:5336)
at clojure.core$load_lib$fn__4967.invoke(core.clj:5375)
at clojure.core$load_lib.doInvoke(core.clj:5374)
at clojure.lang.RestFn.applyTo(RestFn.java:142)
at clojure.core$apply.invoke(core.clj:619)
at clojure.core$load_libs.doInvoke(core.clj:5413)
at clojure.lang.RestFn.applyTo(RestFn.java:137)
at clojure.core$apply.invoke(core.clj:619)
at clojure.core$require.doInvoke(core.clj:5496)
at clojure.lang.RestFn.invoke(RestFn.java:482)
at bcbio.rnaseq.core$loading__4910__auto__.invoke(core.clj:1)
at bcbio.rnaseq.core__init.load(Unknown Source)
at bcbio.rnaseq.core__init.(Unknown Source)
at java.lang.Class.forName0(Native Method)
at java.lang.Class.forName(Class.java:274)
at clojure.lang.RT.loadClassForName(RT.java:2098)
at clojure.lang.RT.load(RT.java:430)
at clojure.lang.RT.load(RT.java:411)
at clojure.core$load$fn__5018.invoke(core.clj:5530)
at clojure.core$load.doInvoke(core.clj:5529)
at clojure.lang.RestFn.invoke(RestFn.java:408)
at clojure.lang.Var.invoke(Var.java:415)
at bcbio.rnaseq.core.(Unknown Source)
Caused by: java.io.FileNotFoundException: /bin/bcbio-rnaseq!/comparisons/baseprop.tsv (No such file or directory)
at java.io.FileInputStream.open(Native Method)
at java.io.FileInputStream.(FileInputStream.java:146)
at clojure.java.io$fn__8642.invoke(io.clj:242)
at clojure.java.io$fn__8577$G__8542__8584.invoke(io.clj:73)
at clojure.java.io$fn__8650.invoke(io.clj:260)
at clojure.java.io$fn__8577$G__8542__8584.invoke(io.clj:73)
at clojure.java.io$fn__8612.invoke(io.clj:169)
at clojure.java.io$fn__8551$G__8546__8558.invoke(io.clj:73)
at clojure.java.io$reader.doInvoke(io.clj:106)
at clojure.lang.RestFn.invoke(RestFn.java:410)
at incanter.io$read_dataset.doInvoke(io.clj:66)
at clojure.lang.RestFn.invoke(RestFn.java:439)
at bcbio.rnaseq.simulator__init.load(Unknown Source)
at bcbio.rnaseq.simulator__init.(Unknown Source)
... 52 more

Hi Rory:
We used : lein run summarize /raid/final/project-summary.yaml -f "~ batch + panel"
and it says: Summary report can be found here /raid/final/summary/qc-summary.html
but in that founder, I just see 3 files:
deseq2-de.tmp
qc-summary.Rmd
qc-summary.tmp

Anything wrong?

Hi @roryk ,

When I run voom_limma_6_vs_7.R :

Rscript 2015-07-15_meiyanrnaseq2/de/voom_limma_6_vs_7.R

I got this error :

Thanks !!!

I missing this package ggdendro used in this line:

>p = ggheatmap(cor(counts))
Loading required package: ggdendro
Loading required package: grid
Error in ggheatmap(cor(counts)) : could not find function "dendro_data"
In addition: Warning message:
In library(package, lib.loc = lib.loc, character.only = TRUE, logical.return = TRUE,  :
  there is no package called ‘ggdendro’

Hi,

I followed the quick start guide, and have lein installed.

but when I run:
lein run simulate -s 5 -d /staging/bcbio.rnaseq/sim_test/
i see:
Running templates/deseq2.template.
but i don't see any results generated in the specified output directory.

Also, if the results of a bcbio-nextgen rnaseq are saved under /bcbio/seqc_test/work
how can I run
lein run compare to use that result directory?

Thanks
Severine

Hi @roryk ,

When i run :

Rscript 2015-07-15_meiyanrnaseq2/de/deseq2_6_vs_7.R

I got the error below :

Thanks !!!

Getting the following error on heatmap figures, but if you just run pheatmap on normalized counts, it works fine

heatmap_fn(cor(normalized_counts, method="spearman"))
Error in cut.default(a, breaks = 100) : 'breaks' are not unique
Called from: (function () 
{
    .rs.breakOnError(TRUE)
})()

Hi Roryk,

when I run bcbio-rnaseq summarize it seem to me the path of the quant.sf files is wrong :

sf_files = file.path("..", "..", rownames(summarydata), "sailfish",
rownames(summarydata), "quant.sf")

should be in my case

sf_files = file.path("..", "..", rownames(summarydata), "sailfish", "quant", "quant.sf")

is this correct?
Thanks!

Hi ,

When i run the command "bcbio-rnaseq summarize project-summary.yaml", it is Ok such as the image below

When i add the FORMULA ' bcbio-rnaseq summarize project-summary.yaml -f "~ batch" ', it is error such as the invalid parameters

@notes: I updated the BCBIO-RNASEQ.
Please help me. Thanks !!!

Is possible to run the DE for only two levels of a set of metadata-to-test-on?

I have a project file with 5 different metadata-to-test-on (two different set of control and 3 different set of treatment) all in duplicate. I was wondering if is possible to use one control and one treatment at the time for the bcbio_rnaseq compare.

Thanks!

deseq2 gives error:
Error in results(dds, independentFiltering = TRUE, addMLE = TRUE) :
unused argument (addMLE = TRUE)

Hi @roryk ,

When i run :

Rscript 2015-07-15_meiyanrnaseq2/de/deseq2_l6_vs_l7.R

I got the error below :

What is the reason ?

Thanks !!!

Hi Roryk,

Just a question is the --organism still working?
Thanks!
Alessandro

Hi Rory:
It's still unclear from the manual how to assign sample to groups for differential expression analysis. It seems that the project-summary.yaml doesn't contain this grouping (conditions) information.

Hello

i was able to run successfully:
bcbio-rnaseq compare --seqc project-summary2.yaml panel

to completion, except for DESeq2, which version it seems i need to upgrade.

This command line created a folder called 'de'.
i can see there is a R script that got copied in this directory (qPCR_foldchange.R) so i tried running it with:
Rscript qPCR_foldchange.R
but I got the following error:

Type 'citation("pROC")' for a citation.

Attaching package: \u2018pROC\u2019

The following objects are masked from \u2018package:stats\u2019:

cov, smooth, var

Loading required package: methods
Saving 7 x 7 in image
Error in data.frame(PANEL = panel, ROW = rows, COL = cols, base) :
arguments imply differing number of rows: 0, 1
Calls: plot_FC ... facet_train_layout.grid -> layout_grid -> data.frame
Execution halted

any idea what the problem is?
Thanks!
Severine