Dear All,
I am analysing data produced from Zymo's Targeted Bisulfite sequencing approach which uses a Fluidigm's array to simultaneously amplify 48 samples using 48 specific genetic loci. I have a heterogenous mix of samples but have started comparing cancers with normal tissue.
I am having some trouble using the BSseq package with these samples: I am following a protocol 'Analysing WGBS with the bsseq package' after going through the protocol with the example. I realise this may not be the most suitable for my data as I have used a targeted approach but I thought the general procedure would be very similar.
I get to the point in the protocol where removal of CpGs with little or no methylation data is necessary and then get an error message:
> keepLoci.ex <- which(rowSums(BS.cov[, bsseq.data$Type == "SSA-"] >= 2) >= 2 & rowSums(BS.cov[, bsseq.data$Type == "normal-"] >= 2) >= 2)
Error in which(rowSums(BS.cov[, bsseq.data$Type == "SSA-"] >= 2) >= 2 & : error in evaluating the argument 'x' in selecting a method for function 'which': Error in rowSums(BS.cov[, bsseq.data$Type == "SSA-"] >= 2) : 'x' must be an array of at least two dimensions
When I look at the number of CpGs that are covered by at least 1 read in all samples I get around 500 which seems quite low.
In addition I get the following warning messages when I use read.bismark:
Warning messages: 1: In .Seqinfo.mergexy(x, y) : Each of the 2 combined objects has sequence levels not in the other: - in 'x': chrX - in 'y': chrMT Make sure to always combine/compare objects based on the same reference genome (use suppressWarnings() to suppress this warning). 2: In .Seqinfo.mergexy(x, y) : Each of the 2 combined objects has sequence levels not in the other: - in 'x': chrX, chrMT - in 'y': chr8, chr21 Make sure to always combine/compare objects based on the same reference genome (use suppressWarnings() to suppress this warning). 3: In .Seqinfo.mergexy(x, y) : Each of the 2 combined objects has sequence levels not in the other: - in 'x': chrX, chr1, chr8, chr21 - in 'y': chr9 Make sure to always combine/compare objects based on the same reference genome (use suppressWarnings() to suppress this warning). 4: In .Seqinfo.mergexy(x, y) : Each of the 2 combined objects has sequence levels not in the other: - in 'x': chr16, chrMT, chr8, chr21, chr9 - in 'y': chr13 Make sure to always combine/compare objects based on the same reference genome (use suppressWarnings() to suppress this warning). 5: In .Seqinfo.mergexy(x, y) : Each of the 2 combined objects has sequence levels not in the other: - in 'x': chr15, chrX, chrMT, chr8, chr21, chr9, chr13 - in 'y': chr14 Make sure to always combine/compare objects based on the same reference genome (use suppressWarnings() to suppress this warning)
Please could you let me know what might be wrong and how I should proceed.
Many thanks in advance!