Error in edgeR analysis: library size of zero detected when executing DGEList function

[BioinfoHub-PeiQinNg]

I have checked the following places for your error:

• nf-core website: troubleshooting
• nf-core/smrnaseq pipeline documentation

Description of the bug

I have tried executing nf-core/smrnna workflow, up until the edgeR step the following error shows up
<Command exit status:
1

Command output:
$mature
[1] "SAGCFN_22_00196.mature.stats" "SAGCFN_22_00194.mature.stats"
[3] "SAGCFN_22_00188.mature.stats" "SAGCFN_22_00190.mature.stats"
[5] "SAGCFN_22_00189.mature.stats" "SAGCFN_22_00193.mature.stats"
[7] "SAGCFN_22_00186.mature.stats" "SAGCFN_22_00187.mature.stats"
[9] "SAGCFN_22_00197.mature.stats" "SAGCFN_22_00191.mature.stats"
[11] "SAGCFN_22_00195.mature.stats" "SAGCFN_22_00192.mature.stats"

$hairpin
[1] "SAGCFN_22_00187.hairpin.stats" "SAGCFN_22_00192.hairpin.stats"
[3] "SAGCFN_22_00191.hairpin.stats" "SAGCFN_22_00188.hairpin.stats"
[5] "SAGCFN_22_00186.hairpin.stats" "SAGCFN_22_00195.hairpin.stats"
[7] "SAGCFN_22_00189.hairpin.stats" "SAGCFN_22_00197.hairpin.stats"
[9] "SAGCFN_22_00196.hairpin.stats" "SAGCFN_22_00190.hairpin.stats"
[11] "SAGCFN_22_00193.hairpin.stats" "SAGCFN_22_00194.hairpin.stats"

Command error:
Loading required package: limma
Loading required package: edgeR
Loading required package: statmod
Loading required package: data.table
Loading required package: gplots

Attaching package: ‘gplots’

The following object is masked from ‘package:stats’:

  lowess

Error in cpm.default(y$counts, lib.size = lib.size, log = log, prior.count = prior.count) :
library sizes should be finite and non-negative
Calls: cpm -> cpm.DGEList -> cpm.default
In addition: Warning message:
In DGEList(counts = data, genes = rownames(data)) :
library size of zero detected
Execution halted

Work dir:
/cancer/storage/SAGC/projects/SAGCQA0237-RainerHaberberger/raw_data/work/9a/6eedaca4a80898bec9761a56b5a926

Tip: when you have fixed the problem you can continue the execution adding the option -resume to the run command line>

Steps to reproduce

Steps to reproduce the behaviour:

1. Command line:
   'nextflow run nf-core/smrnaseq --input '*.fastq.gz' --genome GRCh38 -profile sahmri --custom_config_base 'https://raw.githubusercontent.com/sagc-bioinformatics/configs/sahmri' --mature ../miRBase/mature.fa.gz --hairpin ../miRBase/hairpin.fa.gz -resume -r 1.1.0'

2.. See error:
< Error in cpm.default(y$counts, lib.size = lib.size, log = log, prior.count = prior.count) :
library sizes should be finite and non-negative
Calls: cpm -> cpm.DGEList -> cpm.default
In addition: Warning message:
In DGEList(counts = data, genes = rownames(data)) :
library size of zero detected
Execution halted>

Expected behaviour

Log files

Have you provided the following extra information/files:

• The command used to run the pipeline
• The .nextflow.log file

System

• Hardware:
• Executor:

Nextflow Installation

• Version:

Container engine

• Engine:
• Image tag: <nf-core 1.1.0>

[apeltzer]

Can you check if this is also the case for dev - the soon new released version?
You can simply run that with -r dev and providing a samplesheet for your samples.

[apeltzer]

@lpantano any idea what might be causing this? A bit clueless here, as we see that same behaviour for dev too...

[apeltzer]

@BioinfoHub-PeiQinNg - you may give -r fix-issue-134 a try - we're running some tests to see if this fixes the issue permanently (I believe it does, filtering out samples with entirely zero expression across all genes...)

[lpantano]

I believe @apeltzer is right, this was reproduced by some people in the past and that should fixed it.

[apeltzer]

Fixed in #136

[rrashford]

Hello,

I am running into this problem, but am using RStudio instead of the command line because I'm pretty new at bioinformatics. I am trying to obtain normalization factors from my alignment files using the edgeR package. When I run regionCounts that my takes in my alignment bam files (pe argument = list of all .bam files in my working directory), the consensus peakset for my experiment, and my parameters, then feed the result of that into asDGEList to get avgLogCPM, I get the following error:

> peak.counts <- regionCounts(pe.bams, all.peaks.GRanges, param=param)
> peak.abundances <- aveLogCPM(asDGEList(peak.counts)) 
Warning message:
In (function (counts = matrix(0, 0, 0), lib.size = colSums(counts),  :
  library size of zero detected

Is there some way to fix this by running some code within RStudio so that peak.counts picks up counts from the reads? Any suggestions greatly appreciated!

> sessionInfo
function (package = NULL) 
{
    z <- list()
    z$R.version <- R.Version()
    z$platform <- z$R.version$platform
    if (nzchar(.Platform$r_arch)) 
        z$platform <- paste(z$platform, .Platform$r_arch, sep = "/")
    z$platform <- paste0(z$platform, " (", 8 * .Machine$sizeof.pointer, 
        "-bit)")
    z$locale <- Sys.getlocale()
    z$running <- osVersion
    z$RNGkind <- RNGkind()
    if (is.null(package)) {
        package <- grep("^package:", search(), value = TRUE)
        keep <- vapply(package, function(x) x == "package:base" || 
            !is.null(attr(as.environment(x), "path")), NA)
        package <- .rmpkg(package[keep])
    }
    pkgDesc <- lapply(package, packageDescription, encoding = NA)
    if (length(package) == 0) 
        stop("no valid packages were specified")
    basePkgs <- sapply(pkgDesc, function(x) !is.null(x$Priority) && 
        x$Priority == "base")
    z$basePkgs <- package[basePkgs]
    if (any(!basePkgs)) {
        z$otherPkgs <- pkgDesc[!basePkgs]
        names(z$otherPkgs) <- package[!basePkgs]
    }
    loadedOnly <- loadedNamespaces()
    loadedOnly <- loadedOnly[!(loadedOnly %in% package)]
    if (length(loadedOnly)) {
        names(loadedOnly) <- loadedOnly
        pkgDesc <- c(pkgDesc, lapply(loadedOnly, packageDescription))
        z$loadedOnly <- pkgDesc[loadedOnly]
    }
    z$matprod <- as.character(options("matprod"))
    es <- extSoftVersion()
    z$BLAS <- as.character(es["BLAS"])
    z$LAPACK <- La_library()
    l10n <- l10n_info()
    if (!is.null(l10n["system.codepage"])) 
        z$system.codepage <- as.character(l10n["system.codepage"])
    if (!is.null(l10n["codepage"])) 
        z$codepage <- as.character(l10n["codepage"])
    class(z) <- "sessionInfo"
    z
}
<bytecode: 0x000002353acde460>
<environment: namespace:utils>