Pan-evolutionary and regulatory genome architecture delineated by an integrated macro- and microsynteny approach

ABSTRACT The forthcoming massive genome data generated by the Earth BioGenome Project will open up a new era of comparative genomics, for which genome synteny analysis provides an important

framework. Profiling genome synteny represents an essential step in elucidating genome architecture, regulatory blocks/elements and their evolutionary history. Here we describe PanSyn,

(https://github.com/yhw320/PanSyn), the most comprehensive and up-to-date genome synteny pipeline, providing step-by-step instructions and application examples to demonstrate its usage.

PanSyn inherits both basic and advanced functions from existing popular tools, offering a user-friendly, highly customized approach for genome macrosynteny analysis and integrated

pan-evolutionary and regulatory analysis of genome architecture, which are not yet available in public synteny software or tools. The advantages of PanSyn include: (i) advanced microsynteny

analysis by functional profiling of microsynteny genes and associated regulatory elements; (ii) comprehensive macrosynteny analysis, including the inference of karyotype evolution from

ancestors to extant species; and (iii) functional integration of microsynteny and macrosynteny for pan-evolutionary profiling of genome architecture and regulatory blocks, as well as

integration with external functional genomics datasets from three- or four-dimensional genome and ENCODE projects. PanSyn requires basic knowledge of the Linux environment and Perl

programming language and the ability to access a computer cluster, especially for large-scale genomic comparisons. Our protocol can be easily implemented by a competent graduate student or

postdoc and takes several days to weeks to execute for dozens to hundreds of genomes. PanSyn provides yet the most comprehensive and powerful tool for integrated evolutionary and functional

genomics. KEY POINTS * PanSyn is a user-friendly pipeline that integrates popular and customized micro- and macrosynteny tools and provides access to external functional genomics datasets

for comparative genomic studies. * Compared with alternative methods, PanSyn allows advanced microsynteny analysis of regulatory blocks, comprehensive macrosynteny analysis of karyotype

evolution and integrated analysis of micro- and macrosynteny for the pan-evolutionary and functional investigation of genome architecture. Access through your institution Buy or subscribe

This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution Access Nature and 54 other Nature Portfolio journals Get Nature+, our

best-value online-access subscription $29.99 / 30 days cancel any time Learn more Subscribe to this journal Receive 12 print issues and online access $259.00 per year only $21.58 per issue

Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL

ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A COMPARATIVE ANALYSIS OF PLANARIAN

GENOMES REVEALS REGULATORY CONSERVATION IN THE FACE OF RAPID STRUCTURAL DIVERGENCE Article Open access 19 September 2024 DETECTION OF COLINEAR BLOCKS AND SYNTENY AND EVOLUTIONARY ANALYSES

BASED ON UTILIZATION OF MCSCANX Article 15 March 2024 RECONSTRUCTION OF HUNDREDS OF REFERENCE ANCESTRAL GENOMES ACROSS THE EUKARYOTIC KINGDOM Article Open access 16 January 2023 DATA

AVAILABILITY All data analyzed within this protocol are publicly available. Demo datasets used in the procedure section and expected results are included in the PanSyn package, which are

accessible at _Zenodo_ (https://zenodo.org/records/10115240). The accession numbers for the demo datasets used in the PanSyn procedure are listed in Supplementary Table 1. Source data are

provided with this paper. CODE AVAILABILITY All PanSyn source codes are publicly available at the GitHub website (https://github.com/yhw320/PanSyn/tree/main/scripts) and are provided in the

Supplementary Code. REFERENCES * Lewin, H. A. et al. The Earth BioGenome Project 2020: starting the clock. _Proc. Natl Acad. Sci. USA_ 119, e2115635118 (2022). Article  CAS  PubMed  PubMed

Central  Google Scholar  * ENCODE Project Consortium. Expanded encyclopaedias of DNA elements in the human and mouse genomes. _Nature_ 583, 699–710 (2020). Article  Google Scholar  * Dekker,

J. et al. The 4D nucleome project. _Nature_ 549, 219–226 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Darwin Tree of Life Project Consortium. Sequence locally, think

globally: the Darwin tree of life project. _Proc. Natl Acad. Sci. USA_ 119, e2115642118 (2022). Article  Google Scholar  * Meyer, A. & Schartl, M. Gene and genome duplications in

vertebrates: the one-to-four (-to-eight in fish) rule and the evolution of novel gene functions. _Curr. Opin. Cell Biol._ 11, 699–704 (1999). Article  CAS  PubMed  Google Scholar  * Simakov,

O. et al. Deeply conserved synteny resolves early events in vertebrate evolution. _Nat. Ecol. Evol._ 4, 820–830 (2020). Article  PubMed  PubMed Central  Google Scholar  * Putnam, N. H. et

al. The amphioxus genome and the evolution of the chordate karyotype. _Nature_ 453, 1064–1071 (2008). Article  CAS  PubMed  Google Scholar  * Simakov, O. et al. Deeply conserved synteny and

the evolution of metazoan chromosomes. _Sci. Adv._ 8, eabi5884 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Nguyen, N. T. T., Vincens, P., Dufayard, J. F., Roest Crollius,

H. & Louis, A. Genomicus in 2022: comparative tools for thousands of genomes and reconstructed ancestors. _Nucleic Acids Res._ 50, D1025–D1031 (2022). Article  CAS  PubMed  Google

Scholar  * Lemons, D. & McGinnis, W. Genomic evolution of Hox gene clusters. _Science_ 313, 1918–1922 (2006). Article  CAS  PubMed  Google Scholar  * Wang, S. et al. Scallop genome

provides insights into evolution of bilaterian karyotype and development. _Nat. Ecol. Evol._ 1, 120 (2017). Article  PubMed  Google Scholar  * Wilson, M. A. & Makova, K. D. Genomic

analyses of sex chromosome evolution. _Annu. Rev. Genomics Hum. Genet._ 10, 333–354 (2009). Article  CAS  PubMed  Google Scholar  * Guo, L. et al. Island-specific evolution of a sex-primed

autosome in a sexual planarian. _Nature_ 606, 329–334 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Han, W. et al. Ancient homomorphy of molluscan sex chromosomes sustained

by reversible sex-biased genes and sex determiner translocation. _Nat. Ecol. Evol._ 6, 1891–1906 (2022). Article  PubMed  Google Scholar  * Dunning, L. T. et al. Lateral transfers of large

DNA fragments spread functional genes among grasses. _Proc. Natl Acad. Sci. USA_ 116, 4416–4425 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Simion, P. et al.

Chromosome-level genome assembly reveals homologous chromosomes and recombination in asexual rotifer _Adineta vaga_. _Sci. Adv._ 7, eabg4216 (2021). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Harmston, N. et al. Topologically associating domains are ancient features that coincide with Metazoan clusters of extreme noncoding conservation. _Nat. Commun._ 8, 441

(2017). Article  PubMed  PubMed Central  Google Scholar  * Schmidbaur, H. et al. Emergence of novel cephalopod gene regulation and expression through large-scale genome reorganization. _Nat.

Commun._ 13, 2172 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Méteignier, L. V., Nützmann, H. W., Papon, N., Osbourn, A. & Courdavault, V. Emerging mechanistic

insights into the regulation of specialized metabolism in plants. _Nat. Plants_ 9, 22–30 (2023). Article  PubMed  Google Scholar  * Zimmermann, B., Robert, N. S. M., Technau, U. &

Simakov, O. Ancient animal genome architecture reflects cell type identities. _Nat. Ecol. Evol._ 3, 1289–1293 (2019). Article  PubMed  Google Scholar  * Wong, E. S. et al. Deep conservation

of the enhancer regulatory code in animals. _Science_ 370, eaax8137 (2020). Article  CAS  PubMed  Google Scholar  * Wang, Y. et al. MCScanX: a toolkit for detection and evolutionary analysis

of gene synteny and collinearity. _Nucleic Acids Res._ 40, e49 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Haas, B. J. et al. DAGchainer: a tool for mining segmental

genome duplications and synteny. _Bioinformatics_ 20, 3643–3646 (2004). Article  CAS  PubMed  Google Scholar  * Soderlund, C., Bomhoff, M. & Nelson, W. M. SyMAP v3.4: a turnkey synteny

system with application to plant genomes. _Nucleic Acids Res._ 39, e68 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wei, J. et al. EDomics: a comprehensive and comparative

multi-omics database for animal evo–devo. _Nucleic Acids Res._ 51, D913–D923 (2023). Article  CAS  PubMed  Google Scholar  * Xiao, Z. & Lam, H. M. ShinySyn: a Shiny/R application for

the interactive visualization and integration of macro- and micro-synteny data. _Bioinformatics_ 38, 4406–4408 (2022). Article  CAS  PubMed  Google Scholar  * Robert, N. S. M., Sarigol, F.,

Zieger, E. & Simakov, O. SYNPHONI: scale-free and phylogeny-aware reconstruction of synteny conservation and transformation across animal genomes. _Bioinformatics_ 38, 5434–5436 (2022).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Bao, Y. et al. Genomic insights into the origin and evolution of molluscan red-bloodedness in the blood clam _Tegillarca granosa_.

_Mol. Biol. Evol._ 38, 2351–2365 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Li, Y. et al. Adaptive bird-like genome miniaturization during the evolution of scallop

swimming lifestyle. _Genomics Proteom. Bioinforma._ 20, 1066–1077 (2022). Article  CAS  Google Scholar  * Wang, S. et al. Construction of a high-resolution genetic linkage map and

comparative genome analysis for the reef-building coral _Acropora millepora_. _Genome Biol._ 10, R126 (2009). Article  PubMed  PubMed Central  Google Scholar  * Liu, Z. et al. The channel

catfish genome sequence provides insights into the evolution of scale formation in teleosts. _Nat. Commun._ 7, 11757 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Li, Y. et

al. Scallop genome reveals molecular adaptations to semi-sessile life and neurotoxins. _Nat. Commun._ 8, 1721 (2017). Article  PubMed  PubMed Central  Google Scholar  * Li, Y. et al. Sea

cucumber genome provides insights into saponin biosynthesis and aestivation regulation. _Cell Discov._ 4, 29 (2018). Article  PubMed  PubMed Central  Google Scholar  * Liu, F. et al.

MolluscDB: an integrated functional and evolutionary genomics database for the hyper-diverse animal phylum Mollusca. _Nucleic Acids Res._ 49, D988–D997 (2021). Article  CAS  PubMed  Google

Scholar  * Zeng, Q. et al. High-quality reannotation of the king scallop genome reveals no ‘gene-rich’ feature and evolution of toxin resistance. _Comput. Struct. Biotechnol. J._ 19,

4954–4960 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ye, N. et al. The role of zinc in the adaptive evolution of polar phytoplankton. _Nat. Ecol. Evol._ 6, 965–978

(2022). Article  PubMed  Google Scholar  * Bao, L., Zhong, X., Yang, Y. & Yang, L. Starfish infers signatures of complex genomic rearrangements across human cancers. _Nat. Cancer_ 3,

1247–1259 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Jiao, Y. & Paterson, A. H. Polyploidy-associated genome modifications during land plant evolution. _Philos.

Trans. R. Soc. Lond. B_ 369, 20130355 (2014). Article  Google Scholar  * Wu, S. et al. Genome sequences of two diploid wild relatives of cultivated sweetpotato reveal targets for genetic

improvement. _Nat. Commun._ 9, 4580 (2018). Article  PubMed  PubMed Central  Google Scholar  * Wu, H. et al. A high-quality _Actinidia chinensis_ (kiwifruit) genome. _Hortic. Res._ 6, 117

(2019). Article  PubMed  PubMed Central  Google Scholar  * Ma, D. et al. Chromosome-level reference genome assembly provides insights into aroma biosynthesis in passion fruit (_Passiflora

edulis_). _Mol. Ecol. Resour._ 21, 955–968 (2021). Article  CAS  PubMed  Google Scholar  * Yin, Y. et al. The chromosome-scale genome of _Magnolia officinalis_ provides insight into the

evolutionary position of magnoliids. _iScience_ 24, 102997 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Xu, Q. et al. Ancestral flowering plant chromosomes and gene orders

based on generalized adjacencies and chromosomal gene co-occurrences. _J. Comput. Biol._ 28, 1156–1179 (2021). Article  CAS  PubMed  Google Scholar  * Hong, S. et al. Genome-wide

comparative analysis of flowering-time genes: insights on the gene family expansion and evolutionary perspective. _Front. Plant Sci._ 12, 702243 (2021). Article  PubMed  PubMed Central 

Google Scholar  * Peng, R. et al. Evolutionary divergence of duplicated genomes in newly described allotetraploid cottons. _Proc. Natl Acad. Sci. USA_ 119, e2208496119 (2022). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Hoopes, G. et al. Phased, chromosome-scale genome assemblies of tetraploid potato reveal a complex genome, transcriptome, and predicted proteome

landscape underpinning genetic diversity. _Mol. Plant_ 15, 520–536 (2022). Article  CAS  PubMed  Google Scholar  * Luo, J. et al. From asymmetrical to balanced genomic diversification during

rediploidization subgenomic evolution in allotetraploid fish. _Sci. Adv._ 6, eaaz7677 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Blanc-Mathieu, R. et al. Hybridization

and polyploidy enable genomic plasticity without sex in the most devastating plant-parasitic nematodes. _PLoS Genet._ 13, e1006777 (2017). Article  PubMed  PubMed Central  Google Scholar  *

Zhao, T. & Schranz, M. E. Network-based microsynteny analysis identifies major differences and genomic outliers in mammalian and angiosperm genomes. _Proc. Natl Acad. Sci. USA_ 116,

2165–2174 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gamboa-Tuz, S. D., Pereira-Santana, A., Zhao, T. & Schranz, M. E. Applying synteny networks (SynNet) to study

genomic arrangements of protein–coding genes in plants. _Methods Mol. Biol._ 2512, 199–215 (2022). CAS  PubMed  Google Scholar  * Almeida-Silva, F., Zhao, T., Ullrich, K. K., Schranz, M. E.

& Van de Peer, Y. Syntenet: an R/Bioconductor package for the inference and analysis of synteny networks. _Bioinformatics_ 39, btac806 (2022). Article  PubMed Central  Google Scholar  *

Sun, P. et al. WGDI: a user-friendly toolkit for evolutionary analyses of whole-genome duplications and ancestral karyotypes. _Mol. Plant._ 15, 1841–1851 (2022). Article  CAS  PubMed  Google

Scholar  * Conover, J. et al. pSONIC: ploidy-aware syntenic orthologous networks identified via collinearity. _G3_ 11, jkab170 (2021). Article  PubMed  PubMed Central  Google Scholar  *

Luo, X. et al. 3D genome of macaque fetal brain reveals evolutionary innovations during primate corticogenesis. _Cell_ 184, 723–740 (2021). Article  CAS  PubMed  Google Scholar  * Lu, J.,

Huang, P., Sun, J. & Liu, J. DupScan: predicting and visualizing vertebrate genome duplication database. _Nucleic Acids Res._ 51, D906–D912 (2023). Article  CAS  PubMed  Google Scholar 

* Wang, Y. et al. Comparative genome anatomy reveals evolutionary insights into a unique amphitriploid fish. _Nat. Ecol. Evol._ 6, 1354–1366 (2022). Article  PubMed  PubMed Central  Google

Scholar  * Kikuta, H. et al. Genomic regulatory blocks encompass multiple neighboring genes and maintain conserved synteny in vertebrates. _Genome Res._ 17, 545–555 (2007). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Lazar, N. H. et al. Epigenetic maintenance of topological domains in the highly rearranged gibbon genome. _Genome Res._ 28, 983–997 (2021). Article

  Google Scholar  * Zhao, T. et al. Whole-genome microsynteny-based phylogeny of angiosperms. _Nat. Commun._ 12, 3498 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhou, Z.

W. et al. GenomeSyn: a bioinformatics tool for visualizing genome synteny and structural variations. _J. Genet. Genomics._ 49, 1174–1176 (2022). Article  PubMed  Google Scholar  * Shtolz,

N. & Mishmar, D. The metazoan landscape of mitochondrial DNA gene order and content is shaped by selection and affects mitochondrial transcription. _Commun. Biol._ 6, 93 (2023). Article

  CAS  PubMed  PubMed Central  Google Scholar  * Stein, J. C. et al. Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the

genus Oryza. _Nat. Genet._ 50, 285–296 (2018). Article  CAS  PubMed  Google Scholar  * Grueber, C. E. Comparative genomics for biodiversity conservation. _Comput. Struct. Biotechnol. J._ 13,

370–375 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wallace, H. A. et al. Manipulating the mouse genome to engineer precise functional syntenic replacements with human

sequence. _Cell_ 128, 197–209 (2007). Article  CAS  PubMed  Google Scholar  * Nutzmann, H. W. et al. Plant metabolic clusters—from genetics to genomics. _N. Phytol._ 211, 771–789 (2016).

Article  Google Scholar  * Graham, L. A. et al. Horizontal gene transfer in vertebrates: a fishy tale. _Trends Genet._ 37, 501–503 (2020). Article  Google Scholar  * Waterhouse, R. M. et al.

Evolutionary superscaffolding and chromosome anchoring to improve Anopheles genome assemblies. _BMC Biol._ 18, 1 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Meyer, A. et

al. Giant lungfish genome elucidates the conquest of land by vertebrates. _Nature_ 590, 284–289 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Albertin, C. B. et al. Genome

and transcriptome mechanisms driving cephalopod evolution. _Nat. Commun._ 13, 2427 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Rhie, A. et al. Towards complete and

error-free genome assemblies of all vertebrate species. _Nature_ 592, 737–746 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Nurk, S. et al. The complete sequence of a human

genome. _Science_ 376, 44–53 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Nakatani, Y. et al. Reconstruction of proto-vertebrate, proto-cyclostome and proto-gnathostome

genomes provides new insights into early vertebrate evolution. _Nat. Commun._ 12, 4489 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ip, J. C. et al. Host–endosymbiont

genome integration in a deep-sea chemosymbiotic clam. _Mol. Biol. Evol._ 38, 502–518 (2021). Article  CAS  PubMed  Google Scholar  * Kim, J. et al. Reconstruction and evolutionary history of

eutherian chromosomes. _Proc. Natl Acad. Sci. USA_ 114, E5379–E5388 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Simakov, O. et al. Insights into bilaterian evolution

from three spiralian genomes. _Nature_ 493, 526–531 (2013). Article  CAS  PubMed  Google Scholar  * Li, Y. et al. Contrasting modes of macro and microsynteny evolution in a eukaryotic

subphylum. _Curr. Biol._ 32, 1–9 (2022). Article  Google Scholar  * Fernández, R. & Gabaldón, T. Gene gain and loss across the metazoan tree of life. _Nat. Ecol. Evol._ 4, 524–533

(2020). Article  PubMed  PubMed Central  Google Scholar  * Ocaña–Pallarès, E. et al. Divergent genomic trajectories predate the origin of animals and fungi. _Nature_ 609, 747–753 (2022).

Article  PubMed  PubMed Central  Google Scholar  * Irimia, M. et al. Extensive conservation of ancient microsynteny across metazoans due to _cis_-regulatory constraints. _Genome Res._ 22,

2356–2367 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. _J. Mol.

Biol._ 215, 403–410 (1990). Article  CAS  PubMed  Google Scholar  * Buchfink, B., Reuter, K. & Drost, H. G. Sensitive protein alignments at tree-of-life scale using DIAMOND. _Nat.

Methods_ 18, 366–368 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Bryson, A. E. et al. Uncovering a miltiradiene biosynthetic gene cluster in the Lamiaceae reveals a

dynamic evolutionary trajectory. _Nat. Commun._ 14, 343 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * Krzywinski, M. et al. Circos: an information aesthetic for comparative

genomics. _Genome Res._ 19, 1639–1645 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Emms, D. M. & Kelly, S. OrthoFinder: phylogenetic orthology inference for

comparative genomics. _Genome Biol._ 20, 238 (2019). Article  PubMed  PubMed Central  Google Scholar  * Lopez-Delisle, L. et al. pyGenomeTracks: reproducible plots for multivariate genomic

datasets. _Bioinformatics_ 37, 422–423 (2021). Article  CAS  PubMed  Google Scholar  * Ayad, L. A. K., Pissis, S. P. & Polychronopoulos, D. CNEFinder: finding conserved non-coding

elements in genomes. _Bioinformatics_ 34, i743–i747 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Tan, G., Polychronopoulos, D. & Lenhard, B. CNEr: a toolkit for

exploring extreme noncoding conservation. _PLoS Comput. Biol._ 15, e1006940 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kumar, S., Tamura, K. & Nei, M. MEGA:

molecular evolutionary genetics analysis software for microcomputers. _Comput. Appl. Biosci._ 10, 189–191 (1994). CAS  PubMed  Google Scholar  * de Hoon, M. J., Imoto, S., Nolan, J. &

Miyano, S. Open source clustering software. _Bioinformatics_ 20, 1453–1454 (2004). Article  PubMed  Google Scholar  * Anand, L. & Rodriguez Lopez, C. M. ChromoMap: an R package for

interactive visualization of multi-omics data and annotation of chromosomes. _BMC Bioinforma._ 23, 33 (2022). Article  CAS  Google Scholar  * Quigley, S., Damas, J., Larkin, D. M. &

Farré, M. syntenyPlotteR: a user-friendly R package to visualize genome synteny, ideal for both experienced and novice bioinformaticians. _Bioinforma. Adv._ 3, vbad161 (2023). Article 

Google Scholar  * Servant, N. et al. HiC-Pro: an optimized and flexible pipeline for Hi-C data processing. _Genome Biol._ 16, 259 (2015). Article  PubMed  PubMed Central  Google Scholar  *

Adhikari, B., Trieu, T. & Cheng, J. Chromosome3D: reconstructing three-dimensional chromosomal structures from Hi-C interaction frequency data using distance geometry simulated

annealing. _BMC Genomics_ 17, 886 (2016). Article  PubMed  PubMed Central  Google Scholar  * DeLano, W. L. PyMOL: an open-source molecular graphics tool. _CCP4 Newsl. Protein Crystallogr._

40, 82–92 (2002). Google Scholar  * Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. _Protein Sci._ 30, 70–82 (2021). Article  CAS 

PubMed  Google Scholar  * Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. _J. Mol. Graph._ 14, 33–38 (1996). Article  CAS  PubMed  Google Scholar  * Csurös, M.

Count: evolutionary analysis of phylogenetic profiles with parsimony and likelihood. _Bioinformatics_ 26, 1910–1912 (2010). Article  PubMed  Google Scholar  * Cantalapiedra, C. P.,

Hernández-Plaza, A., Letunic, I., Bork, P. & Huerta-Cepas, J. eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. _Mol. Biol.

Evol._ 38, 5825–5829 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Stevens, T. J. et al. 3D structures of individual mammalian genomes studied by single-cell Hi-C. _Nature_

544, 59–64 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhou, Y. et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. _Nat.

Commun._ 10, 1523 (2019). Article  PubMed  PubMed Central  Google Scholar  * Engström, P. G., Ho Sui, S. J., Drivenes, O., Becker, T. S. & Lenhard, B. Genomic regulatory blocks underlie

extensive microsynteny conservation in insects. _Genome Res._ 17, 1898–1908 (2007). Article  PubMed  PubMed Central  Google Scholar  * Dong, X., Fredman, D. & Lenhard, B. Synorth:

exploring the evolution of synteny and long-range regulatory interactions in vertebrate genomes. _Genome Biol._ 10, R86 (2009). Article  PubMed  PubMed Central  Google Scholar  * Muffato, M.

et al. Reconstruction of hundreds of reference ancestral genomes across the eukaryotic kingdom. _Nat. Ecol. Evol._ 7, 355–366 (2023). Article  PubMed  PubMed Central  Google Scholar  *

Damas, J. et al. Evolution of the ancestral mammalian karyotype and syntenic regions. _Proc. Natl Acad. Sci. USA_ 119, e2209139119 (2022). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Van de Peer, Y., Maere, S. & Meyer, A. The evolutionary significance of ancient genome duplications. _Nat. Rev. Genet._ 10, 725–732 (2009). Article  PubMed  Google Scholar  *

Van de Peer, Y., Mizrachi, E. & Marchal, K. The evolutionary significance of polyploidy. _Nat. Rev. Genet._ 18, 411–424 (2017). Article  PubMed  Google Scholar  * Lee, T. H., Tang, H.,

Wang, X. & Paterson, A. H. PGDD: a database of gene and genome duplication in plants. _Nucleic Acids Res._ 41, D1152–D1158 (2013). Article  CAS  PubMed  Google Scholar  * Zhao, T. &

Schranz, M. E. Network approaches for plant phylogenomic synteny analysis. _Curr. Opin. Plant Biol._ 36, 129–134 (2017). Article  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS

We thank all developers of useful genome comparison algorithms and tools that have been integrated in the PanSyn pipeline. We also wish to thank J. Zhang (Novogene Bioinformatics Institute)

and X. Dai (University of Michigan) for assisting in the early development of macrosynteny pipeline and PanSyn protocol testing, respectively. This research is part of the ongoing M10K+

genome project that is proposed by M10K+ Consortium and targets sequencing of 10,000 molluscan genomes. We acknowledge the grant support from the Science & Technology Innovation Project

of Laoshan Laboratory (LSKJ202203001, LSKJ202202804), National Natural Science Foundation of China (32130107, 32222085), National Key Research and Development Program of China

(2022YFD2400301), Key R&D Project of Shandong Province (2021ZLGX03, 2022ZLGX01), the Fundamental Research Funds for the Central Universities (842341005) and Taishan Scholar Project Fund

of Shandong Province of China. AUTHOR INFORMATION Author notes * These authors contributed equally: Hongwei Yu, Yuli Li. AUTHORS AND AFFILIATIONS * Fang Zongxi Center for Marine Evo-Devo

& MOE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China Hongwei Yu, Yuli Li, Wentao Han, Fuyun Liu, Yuanting Ma, 

Zhongqi Pu, Qifan Zeng, Lingling Zhang, Zhenmin Bao & Shi Wang * Laboratory for Marine Biology and Biotechnology, Laoshan Laboratory, Qingdao, China Yuli Li, Lingling Zhang & Shi

Wang * Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao, China Lisui Bao * Southern Marine Science and Engineering Guangdong Laboratory, Guangzhou, China

Zhenmin Bao & Shi Wang * Key Laboratory of Tropical Aquatic Germplasm of Hainan Province, Sanya Oceanographic Institution, Ocean University of China, Sanya, China Zhenmin Bao & Shi

Wang * Laboratory for Marine Fisheries and Aquaculture, Laoshan Laboratory, Qingdao, China Zhenmin Bao Authors * Hongwei Yu View author publications You can also search for this author

inPubMed Google Scholar * Yuli Li View author publications You can also search for this author inPubMed Google Scholar * Wentao Han View author publications You can also search for this

author inPubMed Google Scholar * Lisui Bao View author publications You can also search for this author inPubMed Google Scholar * Fuyun Liu View author publications You can also search for

this author inPubMed Google Scholar * Yuanting Ma View author publications You can also search for this author inPubMed Google Scholar * Zhongqi Pu View author publications You can also

search for this author inPubMed Google Scholar * Qifan Zeng View author publications You can also search for this author inPubMed Google Scholar * Lingling Zhang View author publications You

can also search for this author inPubMed Google Scholar * Zhenmin Bao View author publications You can also search for this author inPubMed Google Scholar * Shi Wang View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS S.W. and Y.L. conceived and designed the protocol. H.Y., Y.L., W.H., L.B., F.L., Y.M. and Z.P.

developed, optimized and tested the protocol. Q.Z., L.Z. and Z.B. participated in discussions and provided suggestions for protocol improvement. S.W., Y.L. and H.Y. wrote the protocol with

the input from other authors. CORRESPONDING AUTHORS Correspondence to Yuli Li or Shi Wang. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW

PEER REVIEW INFORMATION _Nature Protocols_ thanks Steven Cannon, Xiyin Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL

INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. RELATED LINKS KEY REFERENCES USING THIS

PROTOCOL Wang, S. et al. _Nat. Ecol. Evol_. 1, 120 (2017): https://doi.org/10.1038/s41559-017-0120 Han, W. et al. _Nat. Ecol. Evol_. 6, 1891–1906 (2022):

https://doi.org/10.1038/s41559-022-01898-6 Wei, J. et al. _Nucleic Acids Res_. 51, D913–D923 (2023): https://doi.org/10.1093/nar/gkac944 EXTENDED DATA EXTENDED DATA FIG. 1 EXTENDED

DEMONSTRATIONS OF MICROSYNTENY ANALYSES. (A) Dot plot or Circos plot visualization of polyploid plant genomes (left) and microbial genomes (right). In the dot plot, homologous gene pairs are

shown as dots, and syntenic gene pairs are aligned together. In the Circos plot, lines linking two chromosomes indicate the location of microsynteny genes. (B) Microsynteny analysis of

heteromorphic and homomorphic sex chromosomes in X/Y and Z/W sexual systems, respectively. Lines linking two sex chromosomes indicate the location of microsynteny genes. The location of

sex-determining gene is indicated by a green line. (C) Genomic organization of plant gene clusters, which are tandemly connected in metabolic pathways. Homologous genes are represented with

rectangles of the same color. Microsynteny between two species is shown with grey curves. (D) Integrative analysis of microsynteny with genomic structural variations. _Oryza sativa_ L.

indica rice varieties Minghui63 (MH63) and Zhenshan97 (ZS97) genomes are used for displaying the association of genome synteny and different structural variations (insertions/deletions or

inversions). Source data EXTENDED DATA FIG. 2 COMPUTATIONAL PROCEDURE AND VISUALIZATION OF NETWORK-BASED MICROSYNTENY ANALYSIS. (A, B) Schematic overview of network-based approaches

developed for microsynteny network detection and macroevolutionary history inference (see Zhao et al.108 and Robert et al.27 for detailed algorithm descriptions). (C) Network-based

microsynteny analysis in 18 animal genomes. The heatmap in the top panel shows the pairwise comparisons for microsynteny conservation between any two species. The adjacent network shows

several example clusters after microsynteny network clustering. The middle panel shows a binary matrix constructed by the phylogenomic profiling of all clusters, where rows represent

clusters and columns represent species. The bottom panel shows the network representation of one conserved (left) and one Eutheria-specific (right) microsynteny genes. Source data EXTENDED

DATA FIG. 3 FUNCTIONAL CHARACTERIZATION AND REGULATORY ANALYSIS OF MICROSYNTENY GENE CLUSTERS. (A) Association of microsynteny cluster with single-cell transcriptome data of _Amphimedon

queenslandica_ (left) and _Trichoplax adhaerens_ (right), with microsynteny genes associated with cell type (top), cell lineage (middle) and co-expression pattern (bottom). (B)

Identification of the conserved regulatory CNEs for the well-known pharyngeal gene cluster across four placental mammals. Blue and orange rectangles represent the positions of CNEs on

chromosomes that are presented in each species (blue) or conserved across all species (orange). Pink rectangles represent the position of conserved gene cluster on the chromosome of the

reference species (human Chr14). (C) Distribution and comparison of TADs around the conserved pharyngeal gene cluster in human and mouse. The chromatin interaction heatmap was generated

using the 3D Genome Browser (http://3dgenome.fsm.northwestern.edu/). Source data EXTENDED DATA FIG. 4 ANCESTRAL GENOME RECONSTRUCTION AND MACROSYNTENY ANALYSIS. (A) Schematic overview of

ancestral genome reconstruction approaches for macrosynteny analysis, which are suitable for a wide range of evolutionary distance (see Kim et al.74 and Simakov et al.75 for detailed

algorithm descriptions). (B) Various visualizations of genome macrosynteny for human, chimpanzee and mouse in comparison with the deduced karyotype of the eutherian ancestor, including

profiling of karyotype evolution and conservation (CI values), identification of chromosome breakage and fusion events, and genome-wide profiling of macrosynteny landscapes for both genomic

DNA-based and protein-based analyses. Source data EXTENDED DATA FIG. 5 MACROSYNTENY ANALYSIS OF 34 REPRESENTATIVE SPECIES ACROSS THE ANIMAL KINGDOM. Macrosynteny analysis using the ancestral

linkage groups represented by the ancestral genome of _Nematostella vectensis_ is presented, with orange and blue dots representing chromosome-level and scaffold-level genomes,

respectively. In the dot plots, dots represent homologous genes distributed in the chromosomes of compared species (x-axis: extant species, y-axis: bilaterian ancestor). Conserved

macrosynteny blocks (with statistical significance) are indicated by red dots. Source data EXTENDED DATA FIG. 6 VISUALIZATION OF KARYOTYPE EVOLUTION AND INTEGRATION WITH FUNCTIONAL GENOMICS

DATA. (A) Visualization of karyotype comparison of the bilaterian ancestor with human (top) or mouse (bottom). Each color represents one of the 17 chromosomes of bilaterian ancestor. (B)

Integrative analysis of karyotype and regulatory evolution in humans (top) and mice (bottom). The color density in the heatmap represents the relative number of contacts observed within

chromosomes. Various epigenetic and regulatory data are collectively shown for the chromosomes under investigation. The chromatin interaction heatmap was generated using the 3D Genome

Browser (http://3dgenome.fsm.northwestern.edu/). Source data EXTENDED DATA FIG. 7 SCHEMATIC OVERVIEW OF PAN-EVOLUTIONARY ANALYSIS OF MICROSYNTENY AND MACROSYNTENY. (A) Recovering

macrosyntenic ancient blocks from microsyntenic gene clusters in extant species. Coloured circles correspond to different OGs. The lengths of the edges in the networks are proportional to

the intergenic distance. The schematic overview is adapted from the SYNPHONI pipeline27. (B) Tracing evolutionary trajectories and dynamics of gene contents and gene orders from ancestors to

extant species in the aspects of gene gain and loss events and conservative transitions from macrosynteny to microsynteny. EXTENDED DATA FIG. 8 WHOLE-GENOME DISTRIBUTION AND

THREE-DIMENSIONAL EXHIBITION OF ANCIENT/NOVEL GENE CLUSTERS. (A) Distribution of Eutheria-conserved (green), Boreoeutheria-conserved (pink) and Simian-conserved (blue) microsynteny genes

along the human chromosomes. (B) Three-dimensional chromosome model of human (top) and mouse (bottom), with color labeling Eutheria-conserved (left), Euarchontoglires-conserved (middle) and

Simian/Glires-specific (right) microsynteny locations. Gray thick threads represent the 3D structure of the entire chromosome. 3D genome structures are visualized using the Nucleome Browser

(http://www.nucleome.org). Source data EXTENDED DATA FIG. 9 INTEGRATED PAN-EVOLUTIONARY AND REGULATORY ANALYSIS OF GENOME MICROSYNTENY AND MACROSYNTENY. (A) Chromosomal distribution of

macrosynteny, microsynteny and various regulatory information derived from the ENCODE project in human (left) and mouse (right). Rectangles represent conserved synteny genes (blue:

macrosynteny, green: microsynteny, red: microsynteny & macrosynteny). Visualization of various associated regulatory data types is shown below. (B) Functional enrichment analysis of

macrosynteny/microsynteny genes based on KEGG (top) and GO (bottom) annotation. In the bubble diagrams, the color and size of the bubbles are utilized to convey statistical information, such

as the _P_-value and the number of overlapping genes with the pathway. In the bar charts, the enriched annotated GO terms are shown, with statistical significance indicated by the height of

the bars. (C) Detailed presentation of CNEs and other regulatory information from ENCODE for the _HOXA_ gene cluster in human and mouse. Blue and red rectangles represent the position of

the _HOXA_ gene cluster and identified CNEs on chromosomes, respectively. Various ENCODE data types are collectively shown for the chromosomal regions under investigation. Source data

SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLE 1 The sources of all the input datasets used in demonstration. SUPPLEMENTARY CODE 1 All PanSyn source codes and user guidance. SOURCE DATA

SOURCE DATA FIG. 2 Statistical source data. SOURCE DATA FIG. 3 Statistical source data. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 1 Statistical source data.

SOURCE DATA EXTENDED DATA FIG. 2 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data. SOURCE DATA

EXTENDED DATA FIG. 5 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 6 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 8 Statistical source data. SOURCE DATA EXTENDED DATA

FIG. 9 Statistical source data. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement

with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and

applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Yu, H., Li, Y., Han, W. _et al._ Pan-evolutionary and regulatory genome architecture delineated by an integrated

macro- and microsynteny approach. _Nat Protoc_ 19, 1623–1678 (2024). https://doi.org/10.1038/s41596-024-00966-4 Download citation * Received: 07 April 2022 * Accepted: 20 December 2023 *

Published: 21 March 2024 * Issue Date: June 2024 * DOI: https://doi.org/10.1038/s41596-024-00966-4 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this

content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative