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Long-read mapping to repetitive reference sequences using Winnowmap2


Approximately 5–10% of the human genome remains inaccessible due to the presence of repetitive sequences such as segmental duplications and tandem repeat arrays. We show that existing long-read mappers often yield incorrect alignments and variant calls within long, near-identical repeats, as they remain vulnerable to allelic bias. In the presence of a nonreference allele within a repeat, a read sampled from that region could be mapped to an incorrect repeat copy. To address this limitation, we developed a new long-read mapping method, Winnowmap2, by using minimal confidently alignable substrings. Winnowmap2 computes each read mapping through a collection of confident subalignments. This approach is more tolerant of structural variation and more sensitive to paralog-specific variants within repeats. Our experiments highlight that Winnowmap2 successfully addresses the issue of allelic bias, enabling more accurate downstream variant calls in repetitive sequences.

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Fig. 1: Illustration of allelic bias in near-identical genomic repeats.
Fig. 2: Visualization of alignment pileup near the mutated bases of chromosome 8 by using IGV tool47.
Fig. 3: A comparison of false-negative and false-positive rates of SV calls using four read mapping methods.
Fig. 4: Wall-clock time and memory usage of four mapping methods.
Fig. 5: Comparison of Winnowmap2 and minimap2 using GIAB SV benchmark set defined for HG002 human sample.
Fig. 6: The left plots indicate the size distribution of SVs computed by Winnowmap2-Sniffles pipeline using HG004 and HG007 samples.

Data availability

This study used publicly available data for evaluation. Complete gapless genome assembly of CHM13 human cell line (v.1.0), chromosome 8 (v.9) and chromosome X (v.0.7) can be accessed from GitHub at ONT and PacBio HiFi sequencing data for HG002, HG003 and HG004 samples are available at E.coli K12 nanopore sequencing data used for training NanoSim simulator are available at the European Nucleotide Archive (PRJEB36648). The GIAB SV call set (v.0.6) for HG002 human sample is available at

Code availability

Winnowmap2 code is available at


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We thank K. Shafin, A. Mikheenko, M. Kirsche and S. Nurk for providing useful feedback regarding Winnowmap2. We also acknowledge H. Li for responding to our queries regarding minimap2 code. Winnowmap2 and Winnowmap were developed on top of minimap2 code. This research was supported in part by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health and funding from the Indian Institute of Science.

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Authors and Affiliations



C.J. designed, implemented and tested the Winnowmap2 algorithm. A.R., S.K., N.H. and A.P. provided valuable feedback while designing the algorithm and benchmark. C.J. prepared an initial draft of the manuscript. N.H. and A.P. edited the manuscript.

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Correspondence to Chirag Jain.

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Nature Methods thanks Fritz Sedlazeck, Rayan Chikhi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling editor: Lin Tang, in collaboration with the Nature Methods team.

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Jain, C., Rhie, A., Hansen, N.F. et al. Long-read mapping to repetitive reference sequences using Winnowmap2. Nat Methods 19, 705–710 (2022).

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