Introduction

Coffalyser.Net is free MLPA data analysis software made and supported by MRC Holland. This article provides a brief overview of the steps required to get started with your first MLPA data analyses using Coffalyser.Net.

For a broader overview of the application and benefits of Coffalyser.Net, see this page.

Step 1: Preparation

Download and install

If you have not yet done so, download Coffalyser.Net. Detailed information about installation and configuration can be found in the Installation Manual. You can also read more about advanced (network) setups in multi-user environments.

Activate with a license

A newly installed Coffalyser.Net server must be activated with a free license that can be found on the download page. The license must be renewed periodically, but all licenses are free and will remain free – read more about why we use a licensing system.

Prepare for your first analysis

When you first log into a new installation of Coffalyser.Net, a little preparation is required. On an existing Coffalyser.Net server you can likely skip these steps.

• Download and install a sheet library update as described in the Reference Manual. The sheet library update contains the latest product definitions and analysis sheet information from MRC Holland.

• Add a definition for the capillary electrophoresis (CE) device(s) used in your lab as described in this article and shown in the video below.

  

Create your first project and consider data organisation

Before you are ready to import your first experiment, you must create at least one project as described in the Reference Manual. A project is a way to organise your experiments, with each project able to hold multiple experiments.

This is the perfect time to think about data organisation. For example, you could create a project per year or month, application or operator. If you require a higher level of data organisation, you can also add one or more organisations to your database, each of which can hold multiple projects. You can even use multiple separate databases, which can each hold multiple organisations. A clear data organisation is particularly useful in a multi-user environment, as this helps maintain a clean work environment and offers the possibility of access control. Read more about the options.

General tips

Some dialogs in Coffalyser.Net can seem overwhelming due to a large number of options. However, it is best to leave analysis settings at their default values. When Coffalyser.Net is used in a diagnostic setting or in combination with probemixes registered for in vitro diagnostic use, this is even mandatory.

A lot of functionality in Coffalyser.Net is accessible through right-click context menus. If you're unsure how to proceed, try right-clicking to see what options are available.

Add and analyse your first experiment

You are now ready to add and analyse your first experiment. Watch the videos below for an overview of the entire proces. If you do not yet have MLPA data available, you can use a demo data set.

Add and configure the experiment

Add an experiment as described in the Reference Manual and shown in the videos above. After opening the experiment, you first see the details tab. This is where you configure the basic setup of your experiment and your capillary electrophoresis run. In particular:

• Select the correct experiment type: DNA/MLPA or DNA/MS-MLPA for MLPA and MS-MLPA experiments, respectively.

• Select your CE device. If you see no CE devices, add one first (see above).

• Select the size marker you used in the capillary electrophoresis run.

• Select the analysis sheet corresponding to the probemix version and lot used for the experiment. If you do not see an appropriate analysis sheet, first create one from MRC Holland's product definitions as described in this article and shown in the video below.

  

Import raw data

Switch to the fragment analysis tab to import the capillary electrophoresis data of all samples in the experiment. These are usually FSA or ESD files. Next, select the appropriate sample type for each sample (tip: this process can be automated).

Fragment analysis

The first stage of data analysis is the fragment analysis, in which peaks are size-called and assigned to MLPA fragments. During this process, Coffalyser.Net creates 'bins' to indicate the expected size ranges of every MLPA fragment. You should inspect the results of this automatic process, make manual corrections as required, and store the result in a manual bin set that can be reused as long as your electrophoresis conditions remain constant. For details, see this article and the video below.

Following the fragment analysis, several quality scores are available to assess the quality of your data. The most important ones are:

• FRSS (Fragment Run Separation Score): indicates the quality of fragment separation and size-calling.

• FMRS (Fragment MLPA Reaction Score): indicates the quality of the peak pattern of MLPA probes and the MLPA reaction itself.

At this point, you can open the fragment analysis results of individual samples (right-click) to inspect the data, view analysis steps, or dig deeper into the origin of quality warnings. Where to find information on the interpretation of quality scores is discussed below.

Comparative analysis

Switch to the comparative analysis tab to continue with the second stage of analysis, where samples are compared to each other to arrive at probe ratios. At this point, select the samples you want to include in the comparative analysis. These would generally only be samples with four green bars for both the FRSS and FMRS.

Following the comparative analysis, there are again several quality scores to help you evaluate the quality of the results. The most important score is:

• CAS (Coffalyser Analysis Score): an aggregate of all other important scores, including the quality of the sample (FMRS), the quality of the reference samples, and the quality and reproducibility of the comparative analysis.

For reliable result interpretation, the CAS score should have four green bars as well. Like after the fragment analysis, you can open individual samples to view more details about quality checks.

View results

After the comparative analysis it is time to look at results (right-click a sample). You can either look at individual samples or at the experiment as a whole. In each of these views, you have the possibility to generate PDF reports or to export results (e.g. as images or CSV files). The easiest way to access these options is through the right-click context menu or via the toolbar.

Result interpretation, quality control and troubleshooting

Results in Coffalyser.Net are reported as final ratios or intra ratio percentages. To learn more about these, read this article. To read more about the styles and colors used to report these results, read more about reporting styles.

The quality checks performed by Coffalyser.Net are described in detail in an appendix of the Reference Manual together with basic troubleshooting information. This is particularly useful to review when you are faced with unfamiliar quality warnings.

If you have not yet done so, we recommend reviewing the steps about quality control and result interpretation in the getting started with MLPA guide, which explain the underlying principles in more detail.

More information and additional resources

The following key resources might be useful to review as well:

• Read the Reference Manual: as mentioned several times, this is a great resource for detailed step-by-step instructions, some more advanced processes, and background information about the analysis and quality checks.

• Learn how to back up your data: all data is stored within your own network or on your own computer, depending on your setup. Coffalyser.Net is not a cloud application, and MRC Holland cannot access your data in any way. You are responsible for your own backups. We recommend regular backups of your entire database as well as retaining the raw data files so that experiments can be recreated if necessary.

• View our collection of tips & tricks for Coffalyser.Net for more advanced tips.

Get in touch

If you would like to know more or prefer learning in a more classical setting, you can join one of our (virtual) workshops or webinars. More information about the contents and schedule of our workshops is available in our events overview.

You are also welcome to contact us. We are always happy to hear from you.