In Moving Pictures, a frozen Sunkern fell from the sky near Ash and his friends. It was later thawed out after being put in a basin of hot water. Nested PCR experiments [ 127] were performed during the clean surface decontamination procedures, using two pairs of primers designed to detect lambda phage DNA in the artificial ice section samples. The external primer set LamouterF (5′-CAACTACACGGCTCACCTGT-3′) and LamouterR (5′-ACGGAACGAGATTTCCGCTT-3′) amplifies a 674 bp fragment, and the nested primer set LaminnerF (5′-GAAGCTGCATGTGCTGGAAG-3′) and LaminnerR (5′-CACACTCTGGAGAGCACCAC-3′) amplifies a 189 bp fragment within the previous fragment. In the first PCR with the external primer sets, the 25 μl reaction mixture consisted of 12.5 μl 2× commercial mix (Cat No. M712B, GoTaq® Green Master Mix, Promega), 1.25 μl of each external primer (LamouterF/LamouterR, 10 uM), 5.0 μl template DNA, and 5 μl of ddH 2O. The amplification included a 5-min denaturation step at 95°C, followed by 40 cycles of 30 s at 95°C, 30 s at 56°C, and 50 s at 72°C, with a final extension of 5 min at 72°C. For the nested PCR, the reaction mixture was identical to the first PCR, except that 5.0 μl of the first PCR product and 1.25 μl of each nested primer (LaminnerF/LaminnerR, 10 μΜ) were included. The amplification conditions were also identical to the first PCR except for the extension time of 20 s at 72°C for 40 cycles of amplifications. In Full Moon and Many Arms!, a wild Necrozma was briefly frozen by an Ice Beam from Lana's Ride Dragonair before the former broke out of the ice with brute force.

Rohde RA, Price PB. Diffusion-controlled metabolism for long-term survival of single isolated microorganisms trapped within ice crystals. Proc Natl Acad Sci U S A. 2007;104(42):16592–7. https://doi.org/10.1073/pnas.0708183104. Roux S, Enault F, Hurwitz BL, Sullivan MB. VirSorter: mining viral signal from microbial genomic data. PeerJ. 2015;3:e985. https://doi.org/10.7717/peerj.985. In An Old Family Blend!, Ash's Glalie froze its Trainer with an Ice Beam upon seeing him again for the Lily of the Valley Conference. Ash was later thawed out by a Flamethrower from his Torkoal.In Ash, Iris, and Trip: Then There Were Three!, during Ash's match against Iris in the Junior Cup semifinals, Ash's Krokorok was briefly frozen by an Ice Beam from Iris's Dragonite before the latter sent him flying with a Flamethrower. Solonenko SA, Ignacio-Espinoza JC, Alberti A, Cruaud C, Hallam S, Konstantinidis K, et al. Sequencing platform and library preparation choices impact viral metagenomes. BMC Genomics. 2013;14(1):320. https://doi.org/10.1186/1471-2164-14-320. Touchon M, Bernheim A, Rocha EP. Genetic and life-history traits associated with the distribution of prophages in bacteria. ISME J. 2016;10(11):2744–54. https://doi.org/10.1038/ismej.2016.47. Emerson JB, Roux S, Brum JR, Bolduc B, Woodcroft BJ, Jang HB, et al. Host-linked soil viral ecology along a permafrost thaw gradient. Nat Microbiol. 2018;3(8):870–80. https://doi.org/10.1038/s41564-018-0190-y. Yao TD, Liu YQ, Kang SC, Jiao NZ, Zeng YH, Liu XB, et al. Bacteria variabilities in a Tibetan ice core and their relations with climate change. Global Biogeochem Cy. 2008;22:GB4017.

Miteva V, Sowers T, Schupbach S, Fischer H, Brenchley J. Geochemical and microbiological studies of nitrous oxide variations within the new NEEM Greenland ice core during the Last Glacial period. Geomicrobiol J. 2016;33(8):647–60. https://doi.org/10.1080/01490451.2015.1074321. In Rescuing the Unwilling!, Lusamine's Absol used an Ice Beam to freeze the feet of Gladion's Silvally to the ground. In the next episode, however, Gladion places a Fire Memory in Silvally's RKS System, changing its type to Fire and allowing to melt its feet out of the ice. The viral genomic DNA from three samples (D25, D49, and RiverV) was subjected to low-input library preparation pipeline using the Nextera® XT Library Prep Kit (Cat No. 15032354, Illumina) in the clean room, according to our methods described previously [ 46, 47, 63]. The metagenomes were sequenced by Illumina HiSeq 2000 platform (1×100 bp) at JP Sulzberger Genome Center at Columbia University. Viromic analysis and characterization of viral communities

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Miteva V, Teacher C, Sowers T, Brenchley J. Comparison of the microbial diversity at different depths of the GISP2 Greenland ice core in relationship to deposition climates. Environ Microbiol. 2009;11(3):640–56. https://doi.org/10.1111/j.1462-2920.2008.01835.x.

In A Flood of Torrential Gains!, Leon's Mr. Rime froze Ash's Gengar with Freeze-Dry during their Masters Eight Tournament battle, giving it a chance to finish off Gengar soon afterward. Ng TF, Chen LF, Zhou Y, Shapiro B, Stiller M, Heintzman PD, et al. Preservation of viral genomes in 700-y-old caribou feces from a subarctic ice patch. Proc Natl Acad Sci USA. 2014;111(47):16842–7. https://doi.org/10.1073/pnas.1410429111.

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Yu DT, He JZ, Zhang LM, Han LL. Viral metagenomics analysis and eight novel viral genomes identified from the Dushanzi mud volcanic soil in Xinjiang, China. J Soils Sediments. 2019;19(1):81–90. https://doi.org/10.1007/s11368-018-2045-9.

In Arrival of a Rival!, during the Jubilife City Pokémon Contest, Dawn's Buneary froze a Coordinator's Bidoof with an Ice Beam. A form of water also known as superionic water or superionic ice in which oxygen ions develop a crystalline structure while hydrogen ions move freely.

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Vikram S, Guerrero LD, Makhalanyane TP, Le PT, Seely M, Cowan DA. Metagenomic analysis provides insights into functional capacity in a hyperarid desert soil niche community. Environ Microbiol. 2016;18(6):1875–88. https://doi.org/10.1111/1462-2920.13088. In Shorting out Electivire, Volkner's Raichu was frozen by a Blizzard from Platinum's Empoleon. It managed to break out of the ice after eating an Aspear Berry. Miteva VI, Brenchley JE. Detection and isolation of ultrasmall microorganisms from a 120,000-year-old Greenland glacier ice core. Appl Environ Microbiol. 2005;71(12):7806–18. https://doi.org/10.1128/AEM.71.12.7806-7818.2005. Department of Plant Pathology and Nebraska Center for Virology, University of Nebraska–Lincoln, Lincoln, NE, USA Lima-Mendez G, Van Helden J, Toussaint A, Leplae R. Reticulate representation of evolutionary and functional relationships between phage genomes. Mol Biol Evol. 2008;25(4):762–77. https://doi.org/10.1093/molbev/msn023.