Merge overlapping pairs

Some paired end library preparation methods using relatively short fragment size will generate data with overlapping pairs. This type of data can be handled as standard paired-end data in the CLC Genomics Workbench, and it will work perfectly fine (see details for variant detection in Detailed information about overlapping paired reads).

However, in some situations it can be useful to merge the overlapping pair into one sequence read instead. The benefit is that you get longer reads, and that the quality improves (normally the quality drops towards the end of a read, and by overlapping the ends of two reads, the consensus read now reflects two read ends instead of just one).

In the CLC Genomics Workbench, there is a tool for merging overlapping reads:

        Toolbox | NGS Core Tools (Image ngsfolder) | Merge Overlapping Pairs (Image overlapping_pairs)

Select one or more sequence lists with paired end sequencing reads as input. Clicking Next allows you to set parameters as displayed in figure 23.24.

Image overlapping_pairs_step2
Figure 23.24: Setting parameters for merging overlapping pairs.

In order to understand how these parameters should be set, an explanation of the merging algorithm is needed: Because the fragment size is not an exact number of base pairs and is different from fragment to fragment, an alignment of the two reads has to be performed. If the alignment is good and long enough, the reads will be merged. Good enough in this context means that the alignment has to satisfy some user-specified score criteria (details below). Because of sequencing errors that typically are more abundant towards the end of the read, the alignment is not expected always to be perfect, and the user can decide how many errors are acceptable. Long enough in this context means that the overlap between the reads has to be non-coincidental. Merging two reads that do not really overlap, leads to errors in the downstream analysis, thus it is very important to make sure that the overlap is big enough. If only a few bases overlap was required, some read pairs will match by chance, so this has to be avoided.

The following parameters are used to define what is good enough and long enough

Click Next allows you to select a report to be produced as part of the output. The main result will be two sequence lists for each list in the input: one containing the merged reads (marked as single end reads), and one containing the reads that could not be merged (still marked as paired data). Since the CLC Genomics Workbench handles a mix of paired and unpaired data, both these sequence lists can be used in the further analysis. However, please note that one of the reasons for a pair not to be merged is low quality, so the list of reads that could not be paired is likely to contain more reads with errors than the one with the merged reads.