Contigs assembled from the second generation sequencing short reads may contain misassemblies, and thus complicate downstream analysis or even lead to incorrect analysis results. Fortunately, with more and more sequenced species available, it becomes possible to use the reference genome of a closely related species to detect misassemblies. In addition, long reads of the third generation sequencing technology have been more and more widely used, and can also help detect misassemblies.


Here, we introduce ReMILO, a reference assisted misassembly detection algorithm that uses both short reads and PacBio SMRT long reads. ReMILO aligns the initial short reads to both the contigs and reference genome, and then constructs a novel data structure called red-black multipositional de Bruijn graph to detect misassemblies. In addition, ReMILO also aligns the contigs to long reads and find their differences from the long reads to detect more misassemblies. In our performance test on short read assemblies of human chromosome 14 data, ReMILO can detect 41.8–77.9% extensive misassemblies and 33.6–54.5% local misassemblies. On hybrid short and long read assemblies of S.pastorianus data, ReMILO can also detect 60.6–70.9% extensive misassemblies and 28.6–54.0% local misassemblies.

Availability and implementation

The ReMILO software can be downloaded for free under Artistic License 2.0 from this site:

Supplementary information

Supplementary data are available at Bioinformatics online.

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