Question: BSseq package for Targeted DNA bisulfite sequencing for DMR analysis
0
4.4 years ago by
parker0
Switzerland
parker0 wrote:

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.

methylation dmr analysis • 772 views
modified 4.4 years ago • written 4.4 years ago by parker0
Answer: BSseq package for Targeted DNA bisulfite sequencing for DMR analysis
0
4.4 years ago by
United States
Katja Hebestreit110 wrote:
Hello! We developed an R/Bioconductor package particularly for targeted BS data, which is called BiSeq. Here is the article: http://www.ncbi.nlm.nih.gov/pubmed/23658421 And here the package: http://bioconductor.org/packages/release/bioc/html/BiSeq.html You might want to give this one a try. Cheers, Katja ----- Original Message ----- > From: "parker [bioc]" <noreply@bioconductor.org> > To: katjah@stanford.edu > Sent: Wednesday, March 18, 2015 8:01:44 AM > Subject: [bioc] BSseq package for Targeted DNA bisulfite sequencing for DMR > analysis > Activity on a post you are following on support.bioconductor.org > User parker wrote Question: BSseq package for Targeted DNA bisulfite > sequencing for DMR analysis : > 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! > You may reply via email or visit BSseq package for Targeted DNA bisulfite sequencing for DMR analysis
Answer: BSseq package for Targeted DNA bisulfite sequencing for DMR analysis
0
4.4 years ago by
parker0
Switzerland
parker0 wrote:

Thank you very much for your answer - I have looked into this package and it looks more appropriate for targeted analysis. I am following your protocol and I want to try and create the BSraw object with the Bismark output files. However, when I run readBismark(files, colData) I get an error:

error in evaluating the argument 'files' in selecting a method for function 'readBismark': Error: object 'files' not found

Do you know how to overcome this?

I have previously for the BSseq just imported the cov. files and this worked fine as far as I know...