Error ID 2 - No peaks found

Error Description

You received this error because you used one of the peak calling pipelines, but no peaks were found.

There could be a variety of causes and solutions for this. Often this can be resolved by using less stringent parameters, but the risk here is that the identified peaks are more likely be false positives. Anothe potential cause (if you are using an input control) is your test and control are just not sufficiently different (so no peaks could be found). For example, this can happen if not enough antibody was used or the antibody was not very good. The worst case scenario is then having to do more experiments and sequence more data.

Solutions

Here are various solutions that can either resolve your issue or at least help you better understand it:

Solution 1 - less stringent parameters

The pipeline uses the MACS software and there are several parameters you can change. All are listed in the "More options" button on the analysis setup page:

You can try the following:

• P-value cutoff: you can change this to something higher like 1e-4 or 1e-3.
• MFOLD (lower): although a value of 5 is recommended, you can lower this to 4, 3, or even 2.

Solution 2 - check what is removed from blacklist filtering (ChIP-seq only)

A common step performed in ChIP-seq peak calling is blacklist filtering. The blacklist is a set of regions identified to commonly contain false positive peaks (you can read more about it here https://www.ncbi.nlm.nih.gov/pubmed/31249361). Hence, peaks overlapping blacklist regions are removed.

One thing you can check is to see if any peaks at all were called prior to blacklist filtering. You can look for a file ending in "macs2_peaks.xls" to view the raw set of peaks called. If you see peaks there then that means everything was removed with blacklist filtering. Therefore, this may indicate your sample is of poorer quality and you might either (1) try the suggestions in Solution 1 above, or (2) you can optionally turn off blacklist filtering (not recommended) if you strongly think some of the peaks in those regions are real. For (2), you can do this by changing the default parameter under "Change default options" when you setup your analysis. Set "Enable blacklist filtering" to False.

Solution 3 - compare IGV coverage tracks (if using input control)

If you are using an input control, use the "Genome Browser" to compare the coverage tracks. Doing this can reveal how "similar" your test and input control samples are on a global scale. If they appear very similar (in terms of coverage location and signal strength), then the workflow will unlikely to be able to find peaks. The causes of this could be varied depending on how your data was generated (e.g. not enough antibody was used in ChIP-seq). You can try using less string parameters as suggested in solution 1.

Solution 4 - contact us

If you still cannot resolve your issue, please don't hesitate to contact us by either:

• Creating a ticket here: http://support.basepairtech.com/support/tickets/new
• Message us using the chat icon in the lower right corner of your screen on the Basepair dashboard.