Dear Aaron,

Hi! I have used csaw before and it worked beautifully. Recently I tried to use csaw from Rstudio. When I get to the normalisation step, things started to look a bit funny:

> require(csaw)
> pe.bam <- c("X_1.bam", "X_2.bam", "Y_1.bam", "Y_2.bam")
> pe.param <- readParam(minq=50, restrict=c("Pf3D7_01_v3", "Pf3D7_02_v3", "Pf3D7_03_v3","Pf3D7_04_v3","Pf3D7_05_v3","Pf3D7_06_v3","Pf3D7_07_v3","Pf3D7_08_v3","Pf3D7_09_v3","Pf3D7_10_v3","Pf3D7_11_v3","Pf3D7_12_v3","Pf3D7_13_v3","Pf3D7_14_v3"), max.frag=1150, pe="both")
> data <- windowCounts(pe.bam, param=pe.param, spacing=50, width=300)
> design <- model.matrix(~factor(c('X', 'X', 'Y', 'Y')))
> colnames(design) <- c("intercept", "condition")
> head(assay(data))
     [,1] [,2] [,3] [,4]
[1,]    7    2    2    6
[2,]    8    8    4    8
[3,]    8   10    5   11
[4,]    9   12    5   13
[5,]    9   17    5   14
[6,]    9   21    5   17
> head(rowRanges(data))
GRanges object with 6 ranges and 0 metadata columns:
         seqnames     ranges strand
            <Rle>  <IRanges>  <Rle>
  [1] Pf3D7_01_v3 [  1, 300]      *
  [2] Pf3D7_01_v3 [ 51, 350]      *
  [3] Pf3D7_01_v3 [101, 400]      *
  [4] Pf3D7_01_v3 [151, 450]      *
  [5] Pf3D7_01_v3 [201, 500]      *
  [6] Pf3D7_01_v3 [251, 550]      *
  -------
  seqinfo: 14 sequences from an unspecified genome
> data$totals
[1] 1593882 2422822 1685087 3548254
> require(edgeR)
> abundances <- aveLogCPM(asDGEList(data))
> summary(abundances)
   Min. 1st Qu.  Median    Mean 3rd Qu.    Max. 
 0.9018  4.0230  4.6160  4.5440  5.2340  7.8680 
> keep.simple <- abundances > 2
> filtered.data <- data[keep.simple,]
> summary(keep.simple)
   Mode   FALSE    TRUE    NA's 
logical   11260  446392       0 
> data <- data[keep.simple,]                                                                                                                                                                      > require(edgeR)
> binned <- windowCounts(pe.bam, param=pe.param, bin=TRUE, width=10000)
> normfacs <- normalize(binned)
> normfacs
class: RangedSummarizedExperiment 
dim: 2335 4 
metadata(6): spacing width ... param final.ext
assays(1): counts
rownames: NULL
rowData names(0):
colnames: NULL
colData names(5): bam.files totals ext rlen norm.factors
> y <- asDGEList(data, norm.factors=normfacs)
Error in (function (counts = matrix(0, 0, 0), lib.size = colSums(counts),  : 
  Length of 'norm.factors' must equal number of columns in 'counts'

I have never seen this the last time I used csaw. Could you please kindly inform me what could have gone wrong?

Thank you very much from your time!

Jingyi from Melbourne

Dear all, 

Hi! I know why! I have checked the latest user manual and realised that normalise function has been changed to normOffSsets function. I will need to re-read the updated user manual.

Cheers,

J