Abstract
The natural occurrence of 2,6-diaminopurine (Z) as a substitute for adenine (A) in certain bacteriophage genomes has profound evolutionary implications and promising biotechnological potential. Progress in this field, however, has been stymied by the absence of reliable methods to detect dZ-DNA, particularly in mixed samples, and to distinguish A from Z at the single-nucleotide level. Here, we introduce Z-Calling, a machine learning-based tool designed to identify dZ-DNA and discriminate A/Z bases directly from PacBio Circular Consensus Sequencing (CCS) reads without additional processing. By analyzing sequence context-dependent kinetic signal changes induced by Z/A substitution, Z-Calling achieves exceptional sensitivity, reliably detecting dZ-DNA even in samples with as little as ~1% dZ-DNA content. Its A/Z base-calling module demonstrates robust performance, with AUC scores of 0.942–0.952 across diverse DNA sequence contexts. Z-Calling represents a significant advancement in accessible and accurate dZ-DNA sequencing, paving the way for its broader application in biotechnology. Z-Calling is freely available at https://github.com/xiaochuanle/Z-Calling .
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Data availability
Pacbio CCS BAMs (containing kinetics signals) sequenced by our lab have been deposited in Genome Sequence Archive of China National Center for Bioinformation in project ID PRJCA031439 under GSA accessions CRA020168, CRA019888, and CRA020191. Human Sequel Ⅱ(HG002) and Revio (HG00106) datasets were acquired from the study of Baid et al.42 and Human Pangenome Reference Consortium34, which are available at https://console.cloud.google.com/storage/browser/details/brain-genomics-public/research/deepconsensus/publication/sequencing/hg00215kb/m64008201124002822.subreads.bam?pageState=(%22StorageObjectListData%22:(%22f%22:%22%255B%255D%22))&walkthrough%20id=panels--storage--bucket and https://s3-us-west-2.amazonaws.com/human-pangenomics/working/HPRC/HG00106/raw_data/PacBio_HiFi/m84081_231112_034048_s4.hifi_reads.bc2070.bam. The plasmids pRS426-ApPurZ-ApdATPase and pRS425-ApDUF550 generated during the current study are available from the corresponding author on reasonable request under a standard Material Transfer Agreement. Source data for the graphs and charts in this study are available in the Figshare repository (https://doi.org/10.6084/m9.figshare.31281748)43.
Code availability
All codes written and used by this study have been deposited in our github repository (https://github.com/xiaochuanle/Z-Calling) and in Zenodo (https://doi.org/10.5281/zenodo.17840213)41. Partial command lines used in data analysis are described in Supplementary Notes.
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Acknowledgements
We acknowledge financial support from the National Key R&D Program of China (2022YFF1201900 to C.-L.X.), the National Natural Science Foundation of China (no. 32270713, 62350004 to C.-L.X. and no. 32522004 and 32200051 to Y.Zhou); Guangdong Basic and Applied Basic Research Foundation (2020B1515020057 to C.-L.X.); Distinguished Young Scholars of China (no. 32125002 to Y.Zhang); the New Cornerstone Science Foundation (NCI2002321 to Y.Zhang); Natural Science Foundation of Jiangsu Province (BK20220591 to Y.Zhou); Key Project Fund of National Natural Science Foundation (no. 82230031 to W.C.); the Regional Innovation and Development Joint Fund of the National Natural Science Foundation of China (U24A20706 to W.C.); the Key Special Project of ‘Cutting-Edge Biotechnology’ in the National Key Research and Development Program of China (2024YFC3406200 to W.C.); Sanming Project of Medicine in Shenzhen (No. SZSM202411007 to W.C.); Guangdong Basic and Applied Basic Research Foundation Regional Joint Fund Key Program (2023B1515120051).
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C.-L.X., Y.Zhang, W.C., and S.Z. conceived the study. B.W., Y.C., C.-L.X., and H.-X.C. implemented the algorithms of Z-Calling. H.-X.C., Y.C., and B.W. wrote the codes of Z-Calling. L.N., Y.Zhou, and Y.L. carried out experiments. B.W., Y.Zhou, and J.-Y.Z. carried out data analysis. B.W., Y.Zhang, Y.C., Y.Zhou, L.N., and H.-X.C. wrote the manuscript. S.Z., W.C, C.-L.X., J.-Y.Z., and Y.L. modified and improved the manuscript. All authors read and approved the final version of the manuscript.
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Wu, B., Chen, Y., Zhou, Y. et al. Z-Calling: a tool for A/Z (2,6-diaminopurine) base calling and dZ-DNA detection using PacBio HiFi reads. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09849-8
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DOI: https://doi.org/10.1038/s42003-026-09849-8
