BSseq package for Targeted DNA bisulfite sequencing for DMR analysis
2
0
Entering edit mode
parker • 0
@parker-7456
Last seen 6.7 years ago
Switzerland

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!

 

 

 

 

methylation DMR analysis • 1.6k views
ADD COMMENT
0
Entering edit mode
@katja-hebestreit-6495
Last seen 3.7 years ago
United States
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
ADD COMMENT
0
Entering edit mode
parker • 0
@parker-7456
Last seen 6.7 years ago
Switzerland

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 readBismark(files, colData) : 
  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...

ADD COMMENT

Login before adding your answer.

Traffic: 812 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6