Background: Since 2009, numerous tools have been developed to detect structural variants using short read technologies. Insertions >50 bp are one of the hardest type to discover and are drastically underrepresented in gold standard variant callsets. The advent of long read technologies has completely changed the situation. In 2019, two independent cross technologies studies have published the most complete variant callsets with sequence resolved insertions in human individuals. Among the reported insertions, only 17 to 28% could be discovered with short-read based tools.
Results: In this work, we performed an in-depth analysis of these unprecedented insertion callsets in order to investigate the causes of such failures. We have first established a precise classification of insertion variants according to four layers of characterization: the nature and size of the inserted sequence, the genomic context of the insertion site and the breakpoint junction complexity. Because these levels are intertwined, we then used simulations to characterize the impact of each complexity factor on the recall of several structural variant callers. We showed that most reported insertions exhibited characteristics that may interfere with their discovery: 63% were tandem repeat expansions, 38% contained homology larger than 10 bp within their breakpoint junctions and 70% were located in simple repeats. Consequently, the recall of short-read based variant callers was significantly lower for such insertions (6% for tandem repeats vs 56% for mobile element insertions). Simulations showed that the most impacting factor was the insertion type rather than the genomic context, with various difficulties being handled differently among the tested structural variant callers, and they highlighted the lack of sequence resolution for most insertion calls.
Conclusions: Our results explain the low recall by pointing out several difficulty factors among the observed insertion features and provide avenues for improving SV caller algorithms and their combinations.
Keywords: Insertions; Short reads; Structural variants; Variant calling.