UNLABELLED: Trypanosomatids are among the most extensively studied protists due to their parasitic interactions with insects, vertebrates, and plants. Recently, Blastocrithidia nonstop was found to depart from the canonical genetic code, with all three stop codons reassigned to encode amino acids (UAR for glutamate and UGA for tryptophan), and UAA having dual meaning also as a termination signal (glutamate and stop). To explore features linked to this phenomenon, we analyzed the genomes of four Blastocrithidia and four Obscuromonas species, the latter representing a sister group employing the canonical genetic code. We found that all Blastocrithidia species encode cognate tRNAs for UAR codons, possess a distinct 4 bp anticodon stem tRNATrpCCA decoding UGA, and utilize UAA as the only stop codon. The distribution of in-frame reassigned codons is consistently non-random, suggesting a translational burden avoided in highly expressed genes. Frame-specific enrichment of UAA codons immediately following the genuine UAA stop codon, not observed in Obscuromonas, points to a specific mode of termination. All Blastocrithidia species possess specific mutations in eukaryotic release factor 1 and a unique acidic region following the prion-like N-terminus of eukaryotic release factor 3 that may be associated with stop codon readthrough. We infer that the common ancestor of the genus Blastocrithidia already exhibited a GC-poor genome with the non-canonical genetic code. Our comparative analysis highlights features associated with this extensive stop codon reassignment. This cascade of mutually dependent adaptations, driven by increasing AU-richness in transcripts and frequent emergence of in-frame stops, underscores the dynamic interplay between genome composition and genetic code plasticity to maintain vital functionality. IMPORTANCE: The genetic code, assigning amino acids to codons, is almost universal, yet an increasing number of its alterations keep emerging, mostly in organelles and unicellular eukaryotes. One such case is the trypanosomatid genus Blastocrithidia, where all three stop codons were reassigned to amino acids, with UAA also serving as a sole termination signal. We conducted a comparative analysis of four Blastocrithidia species, all with the same non-canonical genetic code, and their close relatives of the genus Obscuromonas, which retain the canonical code. This across-genome comparison allowed the identification of key traits associated with genetic code reassignment in Blastocrithidia. This work provides insight into the evolutionary steps, facilitating an extensive departure from the canonical genetic code that occurred independently in several eukaryotic lineages.

Department of Parasitology Faculty of Science Charles University BIOCEV Vestec Czechia

Division of Infectious Diseases Department of Medicine Faculty of Medicine and Dentistry University of Alberta Edmonton Alberta Canada

Faculty of Science University of South Bohemia České Budějovice Czechia

Institute of Microbiology Czech Academy of Sciences Prague Czechia

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czechia

Life Science Research Centre Faculty of Science University of Ostrava Ostrava Czechia

Scripps Institution of Oceanography University of California San Diego La Jolla California USA

Zoological Institute Russian Academy of Sciences St Petersburg Russia

See more in PubMed

Ivanova NN, Schwientek P, Tripp HJ, Rinke C, Pati A, Huntemann M, Visel A, Woyke T, Kyrpides NC, Rubin EM. 2014. Stop codon reassignments in the wild. Science 344:909–913. doi: 10.1126/science.1250691 PubMed DOI

Bezerra AR, Guimarães AR, Santos MAS. 2015. Non-standard genetic codes define new concepts for protein engineering. Life (Basel) 5:1610–1628. doi: 10.3390/life5041610 PubMed DOI PMC

Shulgina Y, Eddy SR. 2021. A computational screen for alternative genetic codes in over 250,000 genomes. Elife 10:e71402. doi: 10.7554/eLife.71402 PubMed DOI PMC

Kollmar M, Mühlhausen S. 2017. Nuclear codon reassignments in the genomics era and mechanisms behind their evolution. Bioessays 39. doi: 10.1002/bies.201600221 PubMed DOI

Lozupone CA, Knight RD, Landweber LF. 2001. The molecular basis of nuclear genetic code change in ciliates. Curr Biol 11:65–74. doi: 10.1016/s0960-9822(01)00028-8 PubMed DOI

McGowan J, Kilias ES, Alacid E, Lipscombe J, Jenkins BH, Gharbi K, Kaithakottil GG, Macaulay IC, McTaggart S, Warring SD, Richards TA, Hall N, Swarbreck D. 2023. Identification of a non-canonical ciliate nuclear genetic code where UAA and UAG code for different amino acids. PLoS Genet 19:e1010913. doi: 10.1371/journal.pgen.1010913 PubMed DOI PMC

Zürcher JF, Robertson WE, Kappes T, Petris G, Elliott TS, Salmond GPC, Chin JW. 2022. Refactored genetic codes enable bidirectional genetic isolation. Science 378:516–523. doi: 10.1126/science.add8943 PubMed DOI PMC

Nyerges A, Vinke S, Flynn R, Owen SV, Rand EA, Budnik B, Keen E, Narasimhan K, Marchand JA, Baas-Thomas M, Liu M, Chen K, Chiappino-Pepe A, Hu F, Baym M, Church GM. 2023. A swapped genetic code prevents viral infections and gene transfer. Nature 615:720–727. doi: 10.1038/s41586-023-05824-z PubMed DOI PMC

de la Torre D, Chin JW. 2021. Reprogramming the genetic code. Nat Rev Genet 22:169–184. doi: 10.1038/s41576-020-00307-7 PubMed DOI

Záhonová K, Kostygov AY, Ševčíková T, Yurchenko V, Eliáš M. 2016. An unprecedented non-canonical nuclear genetic code with all three termination codons reassigned as sense codons. Curr Biol 26:2364–2369. doi: 10.1016/j.cub.2016.06.064 PubMed DOI

Kachale A, Pavlíková Z, Nenarokova A, Roithová A, Durante IM, Miletínová P, Záhonová K, Nenarokov S, Votýpka J, Horáková E, Ross RL, Yurchenko V, Beznosková P, Paris Z, Valášek LS, Lukeš J. 2023. Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment. Nature 613:751–758. doi: 10.1038/s41586-022-05584-2 PubMed DOI

Čapková Pavlíková Z, Miletínová P, Roithová A, Pospíšilová K, Záhonová K, Kachale A, Becker T, Durante IM, Lukeš J, Paris Z, Beznosková P, Valášek LS. 2025. Ribosomal A-site interactions with near-cognate tRNAs drive stop codon readthrough. Nat Struct Mol Biol 32:662–674. doi: 10.1038/s41594-024-01450-z PubMed DOI

Afonin DA, Gerasimov ES, Škodová-Sveráková I, Záhonová K, Gahura O, Albanaz ATS, Myšková E, Bykova A, Paris Z, Lukeš J, Opperdoes FR, Horváth A, Zimmer SL, Yurchenko V. 2024. Blastocrithidia nonstop mitochondrial genome and its expression are remarkably insulated from nuclear codon reassignment. Nucleic Acids Res 52:3870–3885. doi: 10.1093/nar/gkae168 PubMed DOI PMC

Kostygov AY, Albanaz ATS, Butenko A, Gerasimov ES, Lukeš J, Yurchenko V. 2024. Phylogenetic framework to explore trait evolution in Trypanosomatidae. Trends Parasitol 40:96–99. doi: 10.1016/j.pt.2023.11.009 PubMed DOI

Albanaz ATS, Carrington M, Frolov AO, Ganyukova AI, Gerasimov ES, Kostygov AY, Lukeš J, Malysheva MN, Votýpka J, Zakharova A, Záhonová K, Zimmer SL, Yurchenko V, Butenko A. 2023. Shining the spotlight on the neglected: new high-quality genome assemblies as a gateway to understanding the evolution of Trypanosomatidae. BMC Genomics 24:471. doi: 10.1186/s12864-023-09591-z PubMed DOI PMC

Grybchuk D, Galan A, Klocek D, Macedo DH, Wolf YI, Votýpka J, Butenko A, Lukeš J, Neri U, Záhonová K, Kostygov AY, Koonin EV, Yurchenko V. 2024. Identification of diverse RNA viruses in Obscuromonas flagellates (Euglenozoa: Trypanosomatidae: Blastocrithidiinae). Virus Evol 10:veae037. doi: 10.1093/ve/veae037 PubMed DOI PMC

Frolov AO, Malysheva MN, Ganyukova AI, Spodareva VV, Králová J, Yurchenko V, Kostygov AY. 2020. If host is refractory, insistent parasite goes berserk: Trypanosomatid Blastocrithidia raabei in the dock bug Coreus marginatus. PLoS One 15:e0227832. doi: 10.1371/journal.pone.0227832 PubMed DOI PMC

Malysheva MN, Ganyukova AI, Frolov AO. 2020. Blastocrithidia frustrata sp. n. (Kinetoplastea, Trypanosomatidae) from the brown marmorated stink bug Halyomorpha halys (Stål) (Hemiptera, Pentatomidae). Protistology 14:130–146. doi: 10.21685/1680-0826-2020-14-3-3 DOI

Lukeš J, Tesařová M, Yurchenko V, Votýpka J. 2021. Characterization of a new cosmopolitan genus of trypanosomatid parasites, Obscuromonas gen. nov. (Blastocrithidiinae subfam. nov.). Eur J Protistol 79:125778. doi: 10.1016/j.ejop.2021.125778 PubMed DOI

Tice AK, Žihala D, Pánek T, Jones RE, Salomaki ED, Nenarokov S, Burki F, Eliáš M, Eme L, Roger AJ, Rokas A, Shen X-X, Strassert JFH, Kolísko M, Brown MW. 2021. PhyloFisher: a phylogenomic package for resolving eukaryotic relationships. PLoS Biol 19:e3001365. doi: 10.1371/journal.pbio.3001365 PubMed DOI PMC

Tamura K, Battistuzzi FU, Billing-Ross P, Murillo O, Filipski A, Kumar S. 2012. Estimating divergence times in large molecular phylogenies. Proc Natl Acad Sci USA 109:19333–19338. doi: 10.1073/pnas.1213199109 PubMed DOI PMC

Reduth D, Schaub GA, Pudney M. 1989. Cultivation of Blastocrithidia triatomae (Trypanosomatidae) on a cell line of its host Triatoma infestans (Reduviidae). Parasitology 98 Pt 3:387–393. doi: 10.1017/s0031182000061461 PubMed DOI

Strassert JFH, Irisarri I, Williams TA, Burki F. 2021. A molecular timescale for eukaryote evolution with implications for the origin of red algal-derived plastids. Nat Commun 12:1879. doi: 10.1038/s41467-021-22044-z PubMed DOI PMC

Negreira GH, de Groote R, Van Giel D, Monsieurs P, Maes I, de Muylder G, Van den Broeck F, Dujardin J-C, Domagalska MA. 2023. The adaptive roles of aneuploidy and polyclonality in Leishmania in response to environmental stress. EMBO Rep 24:e57413. doi: 10.15252/embr.202357413 PubMed DOI PMC

Reis-Cunha JL, Pimenta-Carvalho SA, Almeida LV, Coqueiro-Dos-Santos A, Marques CA, Black JA, Damasceno J, McCulloch R, Bartholomeu DC, Jeffares DC. 2024. Ancestral aneuploidy and stable chromosomal duplication resulting in differential genome structure and gene expression control in trypanosomatid parasites. Genome Res 34:441–453. doi: 10.1101/gr.278550.123 PubMed DOI PMC

Porcel BM, Denoeud F, Opperdoes F, Noel B, Madoui M-A, Hammarton TC, Field MC, Da Silva C, Couloux A, Poulain J, et al. 2014. The streamlined genome of Phytomonas spp. relative to human pathogenic kinetoplastids reveals a parasite tailored for plants. PLoS Genet 10:e1004007. doi: 10.1371/journal.pgen.1004007 PubMed DOI PMC

Charrière F, Helgadóttir S, Horn EK, Söll D, Schneider A. 2006. Dual targeting of a single tRNA PubMed DOI PMC

Shen N, Guo L, Yang B, Jin Y, Ding J. 2006. Structure of human tryptophanyl-tRNA synthetase in complex with tRNA PubMed DOI PMC

Cheng Z, Saito K, Pisarev AV, Wada M, Pisareva VP, Pestova TV, Gajda M, Round A, Kong C, Lim M, Nakamura Y, Svergun DI, Ito K, Song H. 2009. Structural insights into eRF3 and stop codon recognition by eRF1. Genes Dev 23:1106–1118. doi: 10.1101/gad.1770109 PubMed DOI PMC

Kryukov GV, Castellano S, Novoselov SV, Lobanov AV, Zehtab O, Guigó R, Gladyshev VN. 2003. Characterization of mammalian selenoproteomes. Science 300:1439–1443. doi: 10.1126/science.1083516 PubMed DOI

Cassago A, Rodrigues EM, Prieto EL, Gaston KW, Alfonzo JD, Iribar MP, Berry MJ, Cruz AK, Thiemann OH. 2006. Identification of Leishmania selenoproteins and SECIS element. Mol Biochem Parasitol 149:128–134. doi: 10.1016/j.molbiopara.2006.05.002 PubMed DOI

Lobanov AV, Gromer S, Salinas G, Gladyshev VN. 2006. Selenium metabolism in Trypanosoma: characterization of selenoproteomes and identification of a Kinetoplastida-specific selenoprotein. Nucleic Acids Res 34:4012–4024. doi: 10.1093/nar/gkl541 PubMed DOI PMC

Brown A, Shao S, Murray J, Hegde RS, Ramakrishnan V. 2015. Structural basis for stop codon recognition in eukaryotes. Nature 524:493–496. doi: 10.1038/nature14896 PubMed DOI PMC

Inagaki Y, Blouin C, Doolittle WF, Roger AJ. 2002. Convergence and constraint in eukaryotic release factor 1 (eRF1) domain 1: the evolution of stop codon specificity. Nucleic Acids Res 30:532–544. doi: 10.1093/nar/30.2.532 PubMed DOI PMC

Seit-Nebi A, Frolova L, Kisselev L. 2002. Conversion of omnipotent translation termination factor eRF1 into ciliate-like UGA-only unipotent eRF1. EMBO Rep 3:881–886. doi: 10.1093/embo-reports/kvf178 PubMed DOI PMC

Conard SE, Buckley J, Dang M, Bedwell GJ, Carter RL, Khass M, Bedwell DM. 2012. Identification of eRF1 residues that play critical and complementary roles in stop codon recognition. RNA 18:1210–1221. doi: 10.1261/rna.031997.111 PubMed DOI PMC

Trubitsina N, Zemlyanko O, Moskalenko S, Zhouravleva G. 2019. From past to future: suppressor mutations in yeast genes encoding translation termination factors. BioComm 64:89–109. doi: 10.21638/spbu03.2019.202 DOI

Edskes HK, Khamar HJ, Winchester C-L, Greenler AJ, Zhou A, McGlinchey RP, Gorkovskiy A, Wickner RB. 2014. Sporadic distribution of prion-forming ability of Sup35p from yeasts and fungi. Genetics 198:605–616. doi: 10.1534/genetics.114.166538 PubMed DOI PMC

Mangkalaphiban K, Fu L, Du M, Thrasher K, Keeling KM, Bedwell DM, Jacobson A. 2024. Extended stop codon context predicts nonsense codon readthrough efficiency in human cells. Nat Commun 15:2486. doi: 10.1038/s41467-024-46703-z PubMed DOI PMC

Lukeš J, Wheeler R, Jirsová D, David V, Archibald JM. 2018. Massive mitochondrial DNA content in diplonemid and kinetoplastid protists. IUBMB Life 70:1267–1274. doi: 10.1002/iub.1894 PubMed DOI PMC

Kostygov AY, Karnkowska A, Votýpka J, Tashyreva D, Maciszewski K, Yurchenko V, Lukeš J. 2021. Euglenozoa: taxonomy, diversity and ecology, symbioses and viruses. Open Biol 11:200407. doi: 10.1098/rsob.200407 PubMed DOI PMC

Gerasimov ES, Afonin DA, Škodová-Sveráková I, Saura A, Trusina N, Gahura O, Zakharova A, Butenko A, Baráth P, Horváth A, Opperdoes FR, Pérez-Morga D, Zimmer SL, Lukeš J, Yurchenko V. 2025. Evolutionary divergent kinetoplast genome structure and RNA editing patterns in the trypanosomatid Vickermania. Proc Natl Acad Sci USA 122:e2426887122. doi: 10.1073/pnas.2426887122 PubMed DOI PMC

Heaphy SM, Mariotti M, Gladyshev VN, Atkins JF, Baranov PV. 2016. Novel ciliate genetic code variants including the reassignment of all three stop codons to sense codons in Condylostoma magnum. Mol Biol Evol 33:2885–2889. doi: 10.1093/molbev/msw166 PubMed DOI PMC

Swart EC, Serra V, Petroni G, Nowacki M. 2016. Genetic codes with no dedicated stop codon: Context-dependent translation termination. Cell 166:691–702. doi: 10.1016/j.cell.2016.06.020 PubMed DOI PMC

Chen W, Geng Y, Zhang B, Yan Y, Zhao F, Miao M. 2023. Stop or not: Genome-wide profiling of reassigned stop codons in ciliates. Mol Biol Evol 40. doi: 10.1093/molbev/msad064 PubMed DOI PMC

Osawa S, Jukes TH. 1989. Codon reassignment (codon capture) in evolution. J Mol Evol 28:271–278. doi: 10.1007/BF02103422 PubMed DOI

Saks ME, Sampson JR, Abelson J. 1998. Evolution of a transfer RNA gene through a point mutation in the anticodon. Science 279:1665–1670. doi: 10.1126/science.279.5357.1665 PubMed DOI

Schultz DW, Yarus M. 1994. Transfer RNA mutation and the malleability of the genetic code. J Mol Biol 235:1377–1380. doi: 10.1006/jmbi.1994.1094 PubMed DOI

Santos MAS, Cheesman C, Costa V, Moradas‐Ferreira P, Tuite MF. 1999. Selective advantages created by codon ambiguity allowed for the evolution of an alternative genetic code in Candida spp. Mol Microbiol 31:937–947. doi: 10.1046/j.1365-2958.1999.01233.x PubMed DOI

Mühlhausen S, Schmitt HD, Plessmann U, Mienkus P, Sternisek P, Perl T, Weig M, Urlaub H, Bader O, Kollmar M. 2021. Proteogenomics analysis of CUG codon translation in the human pathogen Candida albicans. BMC Biol 19:258. doi: 10.1186/s12915-021-01197-9 PubMed DOI PMC

Heneghan PG, Salzberg LI, Ó Cinnéide E, Dewald JA, Weinberg CE, Wolfe KH. 2025. Ancient origin and high diversity of zymocin-like killer toxins in the budding yeast subphylum. Proc Natl Acad Sci USA 122:e2419860122. doi: 10.1073/pnas.2419860122 PubMed DOI PMC

Sueoka N. 1988. Directional mutation pressure and neutral molecular evolution. Proc Natl Acad Sci USA 85:2653–2657. doi: 10.1073/pnas.85.8.2653 PubMed DOI PMC

Stadtman TC. 1996. Selenocysteine. Annu Rev Biochem 65:83–100. doi: 10.1146/annurev.bi.65.070196.000503 PubMed DOI

Turanov AA, Lobanov AV, Fomenko DE, Morrison HG, Sogin ML, Klobutcher LA, Hatfield DL, Gladyshev VN. 2009. Genetic code supports targeted insertion of two amino acids by one codon. Science 323:259–261. doi: 10.1126/science.1164748 PubMed DOI PMC

Bachvaroff TR. 2019. A precedented nuclear genetic code with all three termination codons reassigned as sense codons in the syndinean Amoebophrya sp. ex Karlodinium veneficum. PLoS One 14:e0212912. doi: 10.1371/journal.pone.0212912 PubMed DOI PMC

Baejen C, Torkler P, Gressel S, Essig K, Söding J, Cramer P. 2014. Transcriptome maps of mRNP biogenesis factors define pre-mRNA recognition. Mol Cell 55:745–757. doi: 10.1016/j.molcel.2014.08.005 PubMed DOI

Hosoda N, Kobayashi T, Uchida N, Funakoshi Y, Kikuchi Y, Hoshino S, Katada T. 2003. Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation. J Biol Chem 278:38287–38291. doi: 10.1074/jbc.C300300200 PubMed DOI

Seah BKB, Singh A, Swart EC. 2022. Karyorelict ciliates use an ambiguous genetic code with context-dependent stop/sense codons. Peer Community Journal 2:e42. doi: 10.24072/pcjournal.141 DOI

Merritt EA, Arakaki TL, Gillespie R, Napuli AJ, Kim JE, Buckner FS, Van Voorhis WC, Verlinde CLMJ, Fan E, Zucker F, Hol WGJ. 2011. Crystal structures of three protozoan homologs of tryptophanyl-tRNA synthetase. Mol Biochem Parasitol 177:20–28. doi: 10.1016/j.molbiopara.2011.01.003 PubMed DOI PMC

Flegontov P, Butenko A, Firsov S, Kraeva N, Eliáš M, Field MC, Filatov D, Flegontova O, Gerasimov ES, Hlaváčová J, et al. 2016. Genome of Leptomonas pyrrhocoris: a high-quality reference for monoxenous trypanosomatids and new insights into evolution of Leishmania. Sci Rep 6:23704. doi: 10.1038/srep23704 PubMed DOI PMC

Albanaz ATS, Gerasimov ES, Shaw JJ, Sádlová J, Lukeš J, Volf P, Opperdoes FR, Kostygov AY, Butenko A, Yurchenko V. 2021. Genome analysis of Endotrypanum and Porcisia spp., closest phylogenetic relatives of Leishmania, highlights the role of amastins in shaping pathogenicity. Genes (Basel) 12:444. doi: 10.3390/genes12030444 PubMed DOI PMC

Beznosková P, Cuchalová L, Wagner S, Shoemaker CJ, Gunišová S, von der Haar T, Valášek LS. 2013. Translation initiation factors eIF3 and HCR1 control translation termination and stop codon read-through in yeast cells. PLoS Genet 9:e1003962. doi: 10.1371/journal.pgen.1003962 PubMed DOI PMC

Delhi P, Queiroz R, Inchaustegui D, Carrington M, Clayton C. 2011. Is there a classical nonsense-mediated decay pathway in trypanosomes? PLoS One 6:e25112. doi: 10.1371/journal.pone.0025112 PubMed DOI PMC

Wang P, Siao W, Zhao X, Arora D, Wang R, Eeckhout D, Van Leene J, Kumar R, Houbaert A, De Winne N, Mylle E, Vandorpe M, Korver RA, Testerink C, Gevaert K, Vanneste S, De Jaeger G, Van Damme D, Russinova E. 2023. Adaptor protein complex interaction map in Arabidopsis identifies P34 as a common stability regulator. Nat Plants 9:355–371. doi: 10.1038/s41477-022-01328-2 PubMed DOI PMC

Nenarokova A, Záhonová K, Krasilnikova M, Gahura O, McCulloch R, Zíková A, Yurchenko V, Lukeš J. 2019. Causes and effects of loss of classical nonhomologous end joining pathway in parasitic eukaryotes. mBio 10:e01541-19. doi: 10.1128/mBio.01541-19 PubMed DOI PMC

Lukeš J, Butenko A, Hashimi H, Maslov DA, Votýpka J, Yurchenko V. 2018. Trypanosomatids are much more than just trypanosomes: clues from the expanded family tree. Trends Parasitol 34:466–480. doi: 10.1016/j.pt.2018.03.002 PubMed DOI

Cayla M, Nievas YR, Matthews KR, Mottram JC. 2022. Distinguishing functions of trypanosomatid protein kinases. Trends Parasitol 38:950–961. doi: 10.1016/j.pt.2022.08.009 PubMed DOI

Králová J, Grybchuk-Ieremenko A, Votýpka J, Novotný V, Kment P, Lukeš J, Yurchenko V, Kostygov AY. 2019. Insect trypanosomatids in Papua New Guinea: high endemism and diversity. Int J Parasitol 49:1075–1086. doi: 10.1016/j.ijpara.2019.09.004 PubMed DOI

Votýpka J, Kment P, Kriegová E, Vermeij MJA, Keeling PJ, Yurchenko V, Lukeš J. 2019. High prevalence and endemism of trypanosomatids on a small Caribbean island. J Eukaryot Microbiol 66:600–607. doi: 10.1111/jeu.12704 PubMed DOI

Yurchenko VY, Lukes J, Tesarová M, Jirků M, Maslov DA. 2008. Morphological discordance of the new trypanosomatid species phylogenetically associated with the genus crithidia. Protist 159:99–114. doi: 10.1016/j.protis.2007.07.003 PubMed DOI

Green MR, Sambrook JF. 2012. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Bushnell B, Rood J, Singer E. 2017. BBMerge - accurate paired shotgun read merging via overlap. PLoS One 12:e0185056. doi: 10.1371/journal.pone.0185056 PubMed DOI PMC

Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi: 10.1089/cmb.2012.0021 PubMed DOI PMC

Allam A, Kalnis P, Solovyev V. 2015. Karect: accurate correction of substitution, insertion and deletion errors for next-generation sequencing data. Bioinformatics 31:3421–3428. doi: 10.1093/bioinformatics/btv415 PubMed DOI

Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. doi: 10.1093/bioinformatics/btt086 PubMed DOI PMC

Kajitani R, Toshimoto K, Noguchi H, Toyoda A, Ogura Y, Okuno M, Yabana M, Harada M, Nagayasu E, Maruyama H, Kohara Y, Fujiyama A, Hayashi T, Itoh T. 2014. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res 24:1384–1395. doi: 10.1101/gr.170720.113 PubMed DOI PMC

Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, He G, Chen Y, Pan Q, Liu Y, et al. 2012. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience 1:18. doi: 10.1186/2047-217X-1-18 PubMed DOI PMC

Laetsch DR, Blaxter ML. 2017. BlobTools: Interrogation of genome assemblies. F1000Res 6:1287. doi: 10.12688/f1000research.12232.1 DOI

Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009. BLAST+: architecture and applications. BMC Bioinformatics 10:421. doi: 10.1186/1471-2105-10-421 PubMed DOI PMC

Buchfink B, Xie C, Huson DH. 2015. Fast and sensitive protein alignment using DIAMOND. Nat Methods 12:59–60. doi: 10.1038/nmeth.3176 PubMed DOI

Flynn JM, Hubley R, Goubert C, Rosen J, Clark AG, Feschotte C, Smit AF. 2020. RepeatModeler2 for automated genomic discovery of transposable element families. Proc Natl Acad Sci USA 117:9451–9457. doi: 10.1073/pnas.1921046117 PubMed DOI PMC

Tempel S. 2012. Using and understanding RepeatMasker. Methods Mol Biol 859:29–51. doi: 10.1007/978-1-61779-603-6_2 PubMed DOI

Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM. 2021. BUSCO Update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol 38:4647–4654. doi: 10.1093/molbev/msab199 PubMed DOI PMC

Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. 2010. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515. doi: 10.1038/nbt.1621 PubMed DOI PMC

Shulgina Y, Eddy SR. 2023. Codetta: predicting the genetic code from nucleotide sequence. Bioinformatics 39:btac802. doi: 10.1093/bioinformatics/btac802 PubMed DOI PMC

Opperdoes FR, Záhonová K, Škodová-Sveráková I, Bučková B, Chmelová Ľ, Lukeš J, Yurchenko V. 2024. In silico prediction of the metabolism of Blastocrithidia nonstop, a trypanosomatid with non-canonical genetic code. BMC Genomics 25:184. doi: 10.1186/s12864-024-10094-8 PubMed DOI PMC

Shanmugasundram A, Starns D, Böhme U, Amos B, Wilkinson PA, Harb OS, Warrenfeltz S, Kissinger JC, McDowell MA, Roos DS, Crouch K, Jones AR. 2023. TriTrypDB: an integrated functional genomics resource for kinetoplastida. PLoS Negl Trop Dis 17:e0011058. doi: 10.1371/journal.pntd.0011058 PubMed DOI PMC

Fiebig M, Gluenz E, Carrington M, Kelly S. 2014. SLaP mapper: a webserver for identifying and quantifying spliced-leader addition and polyadenylation site usage in kinetoplastid genomes. Mol Biochem Parasitol 196:71–74. doi: 10.1016/j.molbiopara.2014.07.012 PubMed DOI PMC

Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. 2019. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol 37:907–915. doi: 10.1038/s41587-019-0201-4 PubMed DOI PMC

Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842. doi: 10.1093/bioinformatics/btq033 PubMed DOI PMC

Steinbiss S, Silva-Franco F, Brunk B, Foth B, Hertz-Fowler C, Berriman M, Otto TD. 2016. Companion: a web server for annotation and analysis of parasite genomes. Nucleic Acids Res 44:W29–34. doi: 10.1093/nar/gkw292 PubMed DOI PMC

Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, et al. 2021. Highly accurate protein structure prediction with AlphaFold. Nature 596:583–589. doi: 10.1038/s41586-021-03819-2 PubMed DOI PMC

Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. 2022. ColabFold: making protein folding accessible to all. Nat Methods 19:679–682. doi: 10.1038/s41592-022-01488-1 PubMed DOI PMC

Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J, et al. 2024. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630:493–500. doi: 10.1038/s41586-024-07487-w PubMed DOI PMC

Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, Morris JH, Ferrin TE. 2021. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci 30:70–82. doi: 10.1002/pro.3943 PubMed DOI PMC

Blum M, Chang HY, Chuguransky S, Grego T, Kandasaamy S, Mitchell A, Nuka G, Paysan-Lafosse T, Qureshi M, Raj S, et al. 2021. The InterPro protein families and domains database: 20 years on. Nucleic Acids Res 49:D344–D354. doi: 10.1093/nar/gkaa977 PubMed DOI PMC

Mistry J, Chuguransky S, Williams L, Qureshi M, Salazar GA, Sonnhammer ELL, Tosatto SCE, Paladin L, Raj S, Richardson LJ, Finn RD, Bateman A. 2021. Pfam: The protein families database in 2021. Nucleic Acids Res 49:D412–D419. doi: 10.1093/nar/gkaa913 PubMed DOI PMC

Cantalapiedra CP, Hernández-Plaza A, Letunic I, Bork P, Huerta-Cepas J. 2021. eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Mol Biol Evol 38:5825–5829. doi: 10.1093/molbev/msab293 PubMed DOI PMC

Chan PP, Lowe TM. 2019. tRNAscan-SE: searching for tRNA genes in genomic sequences. Methods Mol Biol 1962:1–14. doi: 10.1007/978-1-4939-9173-0_1 PubMed DOI PMC

Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16. doi: 10.1093/nar/gkh152 PubMed DOI PMC

Pedersen BS, Quinlan AR. 2018. Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics 34:867–868. doi: 10.1093/bioinformatics/btx699 PubMed DOI PMC

Sharp PM, Li WH. 1987. The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res 15:1281–1295. doi: 10.1093/nar/15.3.1281 PubMed DOI PMC

da Silva MTA, Silva IRE, Faim LM, Bellini NK, Pereira ML, Lima AL, de Jesus TCL, Costa FC, Watanabe TF, Pereira HD, Valentini SR, Zanelli CF, Borges JC, Dias MVB, da Cunha JPC, Mittra B, Andrews NW, Thiemann OH. 2020. Trypanosomatid selenophosphate synthetase structure, function and interaction with selenocysteine lyase. PLoS Negl Trop Dis 14:e0008091. doi: 10.1371/journal.pntd.0008091 PubMed DOI PMC

Mariotti M, Guigó R. 2010. Selenoprofiles: profile-based scanning of eukaryotic genome sequences for selenoprotein genes. Bioinformatics 26:2656–2663. doi: 10.1093/bioinformatics/btq516 PubMed DOI PMC

Lancaster AK, Nutter-Upham A, Lindquist S, King OD. 2014. PLAAC: a web and command-line application to identify proteins with prion-like amino acid composition. Bioinformatics 30:2501–2502. doi: 10.1093/bioinformatics/btu310 PubMed DOI PMC

Jones RE, Tice AK, Eliáš M, Eme L, Kolísko M, Nenarokov S, Pánek T, Rokas A, Salomaki E, Strassert JFH, Shen X-X, Žihala D, Brown MW. 2024. Create, analyze, and visualize phylogenomic datasets using PhyloFisher. Curr Protoc 4:e969. doi: 10.1002/cpz1.969 PubMed DOI PMC

Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. 2018. UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol 35:518–522. doi: 10.1093/molbev/msx281 PubMed DOI PMC

Sanderson MJ. 2003. R8s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock. Bioinformatics 19:301–302. doi: 10.1093/bioinformatics/19.2.301 PubMed DOI

Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. 2009. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25:1972–1973. doi: 10.1093/bioinformatics/btp348 PubMed DOI PMC

Gray J, Boucot AJ. 1989. Is Moyeria a euglenoid. Lethaia 22:447–456. doi: 10.1111/j.1502-3931.1989.tb01449.x DOI

Poinar G Jr, Poinar R. 2004. Paleoleishmania proterus n. gen., n. sp., (Trypanosomatidae: Kinetoplastida) from Cretaceous Burmese amber. Protist 155:305–310. doi: 10.1078/1434461041844259 PubMed DOI

Edgar RC. 2022. Muscle5: high-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny. Nat Commun 13:6968. doi: 10.1038/s41467-022-34630-w PubMed DOI PMC

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. doi: 10.1093/molbev/mst197 PubMed DOI PMC

Emms DM, Kelly S. 2015. OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol 16:157. doi: 10.1186/s13059-015-0721-2 PubMed DOI PMC

Csurös M. 2010. Count: evolutionary analysis of phylogenetic profiles with parsimony and likelihood. Bioinformatics 26:1910–1912. doi: 10.1093/bioinformatics/btq315 PubMed DOI

Kanehisa M, Sato Y, Morishima K. 2016. BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J Mol Biol 428:726–731. doi: 10.1016/j.jmb.2015.11.006 PubMed DOI