How to choose the best reference for retention time alignment

Automation has always been an important aspect of our Progenesis analysis software. Apart from freeing up your valuable time it also improves the objectivity and reproducibility of your results compared to using a more manual approach.

Automatic alignment processingStarting with the release of Progenesis LC-MS v4.1 earlier this year we’ve been improving the automation of some of the key analysis steps, such as reference run selection and retention time alignment.

In this post I’m going to talk about how you can set up the automatic processing to help find the best reference run for alignment. If you’re new to Progenesis, it might be worth a quick read of the alignment overview first.

The first choice you’ll be faced with when setting up the alignment processing is: “How do you want to choose your alignment reference?”

select an alignment reference

You can read a summary of the three options in the FAQ. I want to focus on the second method: Use the most suitable run from candidates that I select.

So, what does it mean and when would you want to choose it?

What does it mean?

The first (default) method of choosing the alignment reference considers every run in your experiment to be a potential reference. To find the best one it compares each run to every other run. In the absence of any other information about your experiment it will normally choose a good alignment reference for you.

The second method works in the same way, but finds the best reference from a subset of the runs (chosen by you). This time, only the runs in this subset need to be compared to every other run in order to choose the reference. The search space is reduced.

In order to understand why it might be appropriate to do this we first need to think about what makes a good reference: we want a run that has a good representation of the key features in the experiment. Now, you might have some prior knowledge about the design of the experiment which makes some runs more likely to contain these key features than others. By choosing this option you can provide this knowledge so that the software can pick the best reference from a set of runs without wasting time considering any runs you know to be less suitable reference candidates.

In the next section I’ll describe a couple of situations we’ve come across where choosing a subset of the runs for reference selection makes sense. Can you think of any others?

When should I choose this option?

I have some pooled QC runs in my experiment

There are plenty of benefits to incorporating pooled samples into your experiment. Running them at various points throughout your experiment is a good way of keeping an eye on the chromatography, and including them in a PCA plot can provide a useful QC check on the experiment design.

These runs make ideal candidates for the alignment reference because, by their very nature, they should contain a good representation of all of the key features in the experiment. Here’s a quick tip: depending on how you name your runs, you may be able to quickly select your pooled samples by using the search box to filter the list…

filtering the pools

Runs from the start or end of my experiment might not be good reference candidates

Before we introduced the automatic reference selection step we noticed a common practice was to choose a run from the middle of the experiment for the alignment reference. This is because the quality of the chromatography might not be as good at the start or end of the experiment.

In theory, you shouldn’t need to worry about this. Just sticking with the defaults will normally produce a good alignment reference regardless of where it is in the experiment. Again, though, if you want to reduce the process time a bit you can select a group of runs from the middle of your experiment. It will give a more objective result than just picking one run from the middle.


So that’s just two examples where choosing a subset of runs for reference selection is helpful. I hope it’s helped you see where it might be useful in your experiments. Think of it as giving the algorithm any additional knowledge you might have about your experiment that determines where the best alignment reference is likely to come from.

Please get in touch if you have any questions or suggestions on this or any other aspect of the software.