Frequently Asked Questions

This site will be continuously updated

This FAQ section has been updated on 2010-04-05.

General information and purpose

What is CyMATE?
For which purposes can I use CyMATE?
Which features are included in CyMATE?
What are the advantages of using CyMATE?
Who is behind CyMATE?
Who should be cited when using results from CyMATE in published work?

Using CyMATE: access and availability

How can I use CyMATE?
Do I have to become a registered user to use CyMATE?
Is there a limit to the number of requests per user?
Which software has to be installed on my computer?
Which operating system is required to use CyMATE?
How much does using CyMATE cost?
Is there a stand-alone version of CyMATE?

Using CyMATE: getting started

What kind of input is required for the analysis?
What is a clone sequence? What is the master sequence?
How do I get started with the analysis?
Which settings should I choose when I generate my multiple sequence alignment via the web-based version of ClustalW?
How do I use the web-interface?

Using CyMATE: double strand analysis

What does the input for double strand analysis have to look like?
What kind of hairpin linker sequence format is accepted?

General information and purpose

What is CyMATE?

CyMATE is a software platform to perform DNA methylation analysis in silico. It is suitable for analyses of sequence data obtained with bisulfite genomic sequencing and hairpin-bisulfite sequencing. It has been designed as a universal tool for quick, comprehensive and automated analysis of bisulfite sequencing data, providing detailed qualitative and quantitative results. Analysis with CyMATE is simple, very fast, platform-independent and the most detailed of computational analysis methods.

CyMATE is freely available via a web-interface. To use CyMATE, visit the website www.cymate.org

See also:
For which purposes can I use CyMATE?
Using CyMATE: access and availability

For which purposes can I use CyMATE?

CyMATE analyses prealigned sequence data from bisulfite genomic sequencing and hairpin-bisulfite genomic sequencing. It analyses any number of bisulfite sequences to obtain their individual methylation profile, i.e., the distribution of methylated an unmethylated cytosines within the sequence.

See also:
Using CyMATE: getting started
Which features are included in CyMATE?

Which features are included in CyMATE?

CyMATE has been designed as a universal tool for analysis if bisulfite sequence data. The following features are currently implemented:

support for FASTA, CLUSTAL and NEXUS file input formats
statistics output (per sequence, per methylation site, and global)
scalable vector graphics output (PDF)
analysis of CpG- and non-CpG methylation
support for single-strand and double-strand data
support for mutation analysis
detection of sequence artefacts

What are the advantages of using CyMATE?

CyMATE has been designed as a fast, easy-to use tool for universal methylation analysis. It can handle any kind of bisulfite data, regardless of its origin: plant or mammal, single-strand or double strand.

See also:
Which features are included in CyMATE?
Using CyMATE: access and availability

Who is behind CyMATE?

CyMATE is a joint initiative of work groups at the Gregor Mendel Institute (GMI) of Molecular Plant Biology and the Vienna University of Technology.

Who should be cited when using results from CyMATE in published work?

If you want to include results obtained with CyMATE in your published work, please cite:

Hetzl J, Foerster AM, Raidl G, Mittelsten Scheid O (2007) CyMATE: a new tool for methylation analysis of plant genomic DNA after bisulfite sequencing. Plant J 51(3):526–536

Please check this FAQ from time to time for updated bibliographic information.

Using CyMATE: access and availability

How can I use CyMATE?

CyMATE is freely available via a web-interface. Visit the website www.cymate.org to use CyMATE. To submit your request for analysis, follow the link to the “Perform Analysis” section.

Do I have to become a registered user to use CyMATE?

Currently, there is no user registration required.

We are reserving the right to introduce technical measures to enforce a fair use policy and equal access for all prospective users.

Is there a limit to the number of requests per user?

Currently, there is no limit to the number of requests that can be submitted for analysis with CyMATE.

We are reserving the right to introduce technical measures to enforce a fair use policy and equal access for all prospective users.

Which software has to be installed on my computer?

A web-browser is necessary to access and to use the web-interface of CyMATE interactively. The interface has been successfully tested with Firefox, Internet Explorer, Opera and Safari.

A valid email address and an email client are also required, because results from CyMATE are delivered exclusively by email. Results from CyMATE include

ASCII text and
PDF

files. The text file can be read with any text editor, whereas the PDF file requires a PDF viewer.

See also:
Which operating system is required to use CyMATE?
What are the results I get from analysis with CyMATE?

Which operating system is required to use CyMATE?

CyMATE is provided via a web-interface (www.cymate.org) which can be accessed from any work station with a network connection.

The use of CyMATE is platform-independent and no specific operating system is required.

See also:
Which software has to be installed on my computer?

How much does using CyMATE cost?

Currently, using the web-interface of CyMATE for academic and scientific purposes is free of charge. We are reserving the right to introduce technical measures to enforce a fair use policy and equal access for all prospective users.

Is there a stand-alone version of CyMATE?

Currently, we have no plans to introduce a desktop version of CyMATE. We are providing CyMATE as a web-based service to ensure equal access for all current and prospective users.

Using CyMATE: getting started

What kind of input is required for the analysis?

CyMATE reads pre-aligned sequence data, i.e. multiple sequence alignment files in either sequential (standard FASTA) or interleaved (standard CLUSTAL) format. The alignment can be prepared with any software, e.g. ClustalW2.

Please make sure that the input file is named correctly. The following file extensions are supported:

myFile.fa, myFile.fas, myFile.fasta
myFile.aln
myFile.afa

The input must contain a reference sequence (the master) and can contain any number of samples obtained with bisulfite sequencing (the clones).

See also:
How do I get started with the analysis?
What is a clone sequence? What is a master sequence?

What is a clone sequence? What is the master sequence?

A sequence obtained with bisulfite sequencing techniques is designated a clone sequence. It represents a sample sequence which can be evaluated with respect to a reference sequence. The clone sequence is analysed with CyMATE to obtain its methylation profile, i.e. the distribution of methylated and unmetylated cytosines within the sequence.

The reference sequence is called a master sequence. It does not result from bisulfite sequencing, but represents the untreated genomic sequence of the target sequence. Thus, any sequence from sequence databases may serve as a master sequence, whereas clone sequences can be obtained from individual bench work.

The clones and the master sequence must be combined in a multiple sequence alignment to analyse them.

How do I get started with the analysis?

In silico analysis starts after bisulfite sequencing of the clones to be analysed. The following protocol indicates how to proceed after bench work:

Create a blunt-ended multiple sequence alignment (MSA) with appropriate software (e.g., ClustalW2. Blunt-ended indicates that all sequences in the alignment are of the same length, and that some of them may contain leading or trailing gaps. Make sure that the reference sequence (the master, or original sequence, e.g. from sequence database) is on top, i.e. the first sequence in the alignment. For example, choose the option “input” with ClustalW.

The master sequence can be followed by any number of different clones, i.e. bisulfite-treated sequences, in any desired order.
NB:There are no restrictions in the number and length of sequences.

Save the alignment with blunt ends only. If necessary, restrict the sequence to the region of interest and remove any leading or trailing nucleotides which are irrelevant for the analysis. Save the alignment either in FASTA or CLUSTAL format.

See also:
Which settings should I choose when I create a multiple sequence alignment with ClustalW?

Which settings should I choose when I generate my multiple sequence alignment via the web-based version of ClustalW?

ClustalW (a multiple sequence alignment tool) is available via http://www.ebi.ac.uk/clustalw2.

Make sure to choose the proper options under OUTPUT:
For output order, please select "input". This option ensures that when you submit the alignment, the master sequence is on top (required). As of now, CyMATE does not support user-specific selection of the master sequence as reference. For output format, alignments can be generated either with or without character count ("aln wo/numbers" or "aln w/numbers" ).

How do I use the web-interface?

Using CyMATE is very easy und intuitive. There are only a few steps necessary to analyse bisulfite date:

Visit the website www.cymate.org and follow the link to the “Perform Analysis” section
Fill in the form
Indicate your email address
Upload your MSA file
Choose “Methylation Analysis Mode” (default selection)
Choose optional parameters for arguments
Click “Analyse this!”

Within a short period of time (usually a few seconds, depending on your network connection and the number of other CyMATE users using the service simultaneously), CyMATE will deliver the results by email.

Using CyMATE: double strand analysis

What does the input for double strand analysis have to look like?

The input file and its format should look the same as for single strand analysis, however, the sequences in the file must be linearised double strand sequences in either of the following order:

top strand — hairpin linker sequence — bottom strand
bottom strand — hairpin linker sequence — top strand

Make sure to check the checkbox labeled "Bottomfirst" for the latter option.

See also:
What kind of input is required for the analysis?
What kind of hairpin linker sequence format is accepted?

What kind of hairpin linker sequence format is accepted?

The hairpin linker (HPL) sequence has to be entered in the field labeled "Hair-Pin-Linker:" after checking the "Double strand" radio button in the analysis form. The HPL consists of a conserved stem sequence, i.e. two short complementary sequences, and an individual loop sequence, usually indicated by a succession of non-C characters represented by the ambiguous symbol for a nucleotide base, D.

The HPL sequence may contain gaps, as represented by a dash character ("-"), as a result from sequence alignment. Please make sure to check your input data accordingly. If the HPL is to contain gap characters, we strongly recommend to remove those clones causing the gaps to be introduced from the alignment, re-align the sequences, and re-submit the request for analysis. Please make sure that the gaps are included in the HPL sequence if you want to include the clones causing the gaps to occur in the analysis.