SMRT

The truthset for the SMRT dataset from NA24385 does not contain variants annotated as inversions, so Fig. 3 (SV 1001–10,000 bp) and S6 (SV 10,001–100,000 bp) only contain three panels each. We can see immediately in Fig. 3 that the bars generated from the reference set are much closer to the height of the bars from the VCFs, and this is not affected by the use of only the 15 kbp insert set. There is one notable exception with regard to duplications. As with the nanopore dataset, an NGMLR alignment contains the most duplications.

However, these duplications are far in excess of what is present in the reference set. This is notable, as the reference set has been carefully validated. What was missed, versus what is a false positive, is not resolvable in this experiment and likely not resolvable without wet lab bench work or orthogonal validation.

The largest SV show a complex picture (S6). NGMLR calls the most and the largest deletions and no tools do well for large insertions in absolute terms or relative to the truthset. However, Minimap2 and NGMLR preserve no breakpoints called as insertions. For duplications 10,001–20,000 bp, all tools except LRA resolve many more than are present in the reference. NGMLR continues to include many more duplications than the other alignments, and more than the reference.

The disparity between the number of large variants greater 10,001 bp in the NA24385 truthset versus the NA12878 is striking. This, along with the general lack of reference standards for all publicly-available genomes, highlights the need for better, more comprehensive reference sets for all publicly-available resources. Inability to resolve large duplications may yield false negative results for conditions where structural variation is a recognized etiology, such as DMD or FSHD, where variants range between the tens and hundreds of thousands of kbp (Barseghyan et al., 2017; Sharim et al., 2019), beyond the size of variants accurately detected in this study. It is also a critical issue when the technology is used for broad exploratory surveys seeking to identify new etiology in low-complexity regions of the genome where long-read sequencing should shine.