Hardware and Software requirements
I have a 32-bit version of Windows, can I run EaSeq?
Yes. Use EaSeq32bit.exe. Due to memory limitations EaSeq can only access app. 1.3GB of RAM in 32-bit Windows. This limits the number of datasets you can load and might result in crashed analyses, if EaSeq runs out of memory. We therefore strongly recommend that you use 64-bit Windows and have sufficient memory installed (>4GB is recommended for most applications).
I have a monitor with a resolution that is lower than 1920×1080 pixels, can I run EaSeq?
Yes, but it will be quite messy, include a lot of scrolling, and the tutorials will not work as intended. Frankly, we prioritized developing core functionality over a user interface that adapts to monitor resolutions. Most inexpensive monitors sold today have this resolution or better, and a higher resolution might be useful for other work as well.
I use a Mac, can I run EaSeq?
Yes. You will need to install a virtual machine allowing you to run Windows (and its programs) from your Mac OS desktop. Parallels and WMWare Fusion are among the most widely used. Please let us know about your experiences.
I use Linux, can I run EaSeq?
Yes. You will need to install a virtual machine allowing you to run Windows (and its programs) from your Linux desktop. A member of a nearby group runs EaSeq from an Oracle VM Virtualbox on his Ubuntu machine, and EaSeq performs fine in his environment.
Will you release native versions of EaSeq for other operating systems?
We would love to, but it is considerable work to do. It would also make more sense to undertake after the program has matured further, so do not expect a Mac version in the near future.
How to avoid viewing the licence form each time I start EaSeq?
Two types of issues are known to cause this. EaSeq is executed directly from 1) the compressed zip-file, or 2) a network folder. Make sure that it is extracted into its own folder on the machine that you are working on before it is started. Also make sure that there is plenty of available space and write permissions. EaSeq will need write files some temporary and permanent files – sometimes several GB.
How to see the entire interface on a 1920×1080 pixels monitor?
EaSeq needs a monitor capable of at least a resolution of 1920×1080 pixels. However, some users are unable to see the entire interface even on such a monitor. The most common reasons for this are: 1) the monitor magnifies the displayed material, or 2) it is set up to run a suboptimal resolution. To check for this, select the ‘Screen Settings’ from the ‘Control Panel’ in Windows. Here you can change the size of the displayed items. If your monitor has a resolution of 1920×1080 pixels, then make sure that this is set to ‘100%’ or ‘smallest’ depending on which version of windows that you use. Next, click on the ‘Change display settings’ menu option, and make sure that the resolution is set to 1920×1080 pixels.
Which format should my ChIP-seq data be in?
EaSeq imports mapped single-read datasets from .bed, .bam, and .aln files as well as coverage/intensity data from .bg or .wig files. EaSeq will automatically extract Gzip compressed files, and it can generally import single reads from most column separated text files. We have noticed that some formats are not as standardized as one would like, so data from some programs might not be imported smoothly even though that they are in one of the listed formats.
Can I use EaSeq for other data types than ChIP-seq data?
Yes, EaSeq can load data from many different library preparation methods as long as they are single reads, e.g. RNA-seq, DIP-seq, RIP-seq, CAGE-seq etc. EaSeq does not yet have a dedicated analysis pipeline for RNA-seq data, but RNA-seq data can easily be visualized along with ChIP-seq data.
How to I map (align) my reads?
Generally there are three options: 1) Often your core facility / sequencing company can provide your data as mapped reads, if you instruct which reference genome to use. 2) If you are familiar with Linux, then there are several mappers, such as ‘bowtie’. 3) If not, then you can setup mapping from a web browser using the free environment Galaxy.
I would like to integrate data from GEO / ENA in my analyses, how do I do that?
Raw reads deposited at these sites are generally unmapped, so you will need to map reads first. The options are as described above.
Do files need to be sorted?
No, EaSeq can do that, but it takes longer, and it might be a good idea to export sorted data from EaSeq or save data in a session to save time in the future.
Should I filter for unique reads?
EaSeq offers you to remove multiple reads that map to identical positions. When working on large genomes that is usually a good idea, because the probabilities that two reads from a typical library are at the same coordinates are quite small. Therefore reads that map to the same position are frequently PCR-artifacts skewing your analyses. In smaller genomes, this probability increases, and it might be best not to remove reads mapped to the same position.
Which fragment lengths should I choose? And does it matter?
It is not crucial to get the fragment length correct, and a fixed fragment length is anyway an approximation considering that the fragments in a ChIP-seq library have varying lengths. Using the standard is fine for most applications, and EaSeq normalize signal strength both to read count and fragment sizes by default. You might gain resolution by testing it with the ‘Phantom’-tool or by setting fragment lengths based on experimentally measured fragment sizes. For RNA-seq data we generally use a fragment length corresponding to the read size.
Data import takes quite some time, can I speed it up?
Yes. Saving data as a session accelerates loading a lot. We generally save a session just containing datasets after import.
Can I use paired end sequencing data?
Currently, EaSeq does not have any in-build support for paired end sequencing. Those are features that we would like to develop, but we haven’t had much use for it yet and generally things we work on ourselves are given the highest priority.
I have a peak-set made using MACS (or another peak-caller), can I use those?
Yes. It generally works smoothly to import them as a regionset from a text-file. If you encounter problems, then please contact us.
Will EaSeq know which reference genome I use?
No. EaSeq does not keep track of that, and it even allows you to mix data from different species. So you will have to be aware of that yourself.
Licensing and user rights
What are the costs of using EaSeq?
EaSeq is free for all users.
If EaSeq is free, why do I need to accept an End User License Agreement (EULA)?
Primarily to assure: 1) that you have the rights to all analyses and output that you make using EaSeq, 2) that parts of EaSeq are not put into commercial alternatives without our consent, 3) that you do not sue us for data loss etc., and 4) that you are informed about the statistics on usage that are transferred to our server.
Why do EaSeq transfer data about my usage?
For two reasons: 1) To learn how we can improve the user interface. 2) So we can base future decisions and funding on the extent of actual users. Data shared with third parties will be anonymized.
What data are transferred?
Mainly hardware and software information as well as information on the functions used in EaSeq. Absolutely no information about the nature or names of your data are transfered. You can see all data that are transfered by clicking ‘About’ and selecting the ‘Data Transfer’ tab page. You can also instruct EaSeq to anonymize the transferred information from there.
I would like to learn more, where can I find additional information?
Our manuscript, which can be found here, covers the general aspects of EaSeq. We also integrated a ‘wizard’ and tutorials in EaSeq, which will instruct you on how to do various analyses and visualizations. As EaSeq is quite comprehensive, they will most likely not cover all of you needs or questions. We therefore also integrated a user forum where people can post questions and answers (named ‘Squeaks’ in EaSeq). It is available by clicking on the upper vertical bar in the right side of EaSeq. Please contact us if you need further instructions, documentation, or would like to attend a future course or workshop.