Recombination shapes the 2022 monkeypox outbreak

Supplemental File 1. The Newick tree of the MPXV sequence dataset for Supplemental File 2. Supplemental File 2. Rooted phylogenetic tree of MPXV genomes during 2022 pandemics. Alignments of viral sequences were generated using MAFFT, and the phylogenetic tree was visualized using FigTree. Rooting was done by introducing the camelpox virus sequence (MZ300860) as an outgroup virus. Group U, M, and I of MPXV genomes is labeled in green, blue, and red, respectively. Recombinant MPXV isolates based on TR analysis are labeled in orange. Supplemental File 3. Genomic sequences of MPXV (B.1 clade) from January 1 to July 20, 2022 (N=415) in this study. The camelpox virus sequence (MZ300860) is included for phylogenetic analysis in Supplemental File 2. Supplemental File 4. TRAL analysis of the annotated TRs in MPXV genomic sequences of Supplemental File 3 and Figure S6. Supplemental Table 1. Summary of MPXV genome sequence information in this study. Figure S1. Sequences that contain SNP pairs with strong linkage disequilibrium, which the upper 95% confidence bound of the D’ is above 0.98 and the lower bound is above 0.7. Figure S2. TR C in the intergenic region of monkeypox A47R and A49R. (A) The map of TR C, A47R, and A49R gene. Arrows indicate the gene direction. (B) The location of TR C at the 3’-untranslated region of A47R transcript. The 3’ termination UUUUUNU sequence for adding poly(A) tail is boxed in red. Reference MPXV sequence is OP19276. Figure S3. TR B in the intergenic region of monkeypox A41L and A42R. (A) The map of TR B, A41L, and A42R gene. Arrows indicate the gene direction. Sequence b and c are shown in (B) and (C). (B) Sequence of TR B insertion in monkeypox genome as in (A) compared to vaccinia sequence. (C) Alignment of monkeypox and vaccinia in A42R promoter. TAAAT and AAAAAA element are boxed in red. Reference MPXV sequence is OP19276. Figure S4. TR D in the intergenic region of monkeypox B18R and B19R. (A) (A) The map of TR D, B18R, and B19R gene. Arrows indicate the gene direction. Sequence b, c and d are shown in (B), (C) and (D). (B) Alignment of monkeypox sequence B with vaccinia B19R promoter, including TAAAT and AAAAAAA element only found in vaccinia. (C and D) Sequences of B19R promoter of monkeypox. Sequence of TR D insertion in monkeypox genome as in (A). Reference MPXV sequence is OP19276. Figure S5. Comparison of vaccinia K3L and monkeypox C3L sequence. The stop codon was introduced at bp 130-132, causing C3L a truncated protein compared to K3L. Figure S6. (A) Table of divergence value ranges of TR A to F. The value of TR B is not available because its sequence is AT only. (B) Each box (TR A, C, D, E, F) contains individual TRNs with different colors associated with the parental viral sequences: IRBA22-14 (I, ON755040), MUW1527495 (M, ON019276) or USA_2022_CA002 (U, ON954773) as Figure 1F. TRs and TRNs were validated by the TRAL algorithm with p-value < 10-4.

补充文件1：用于补充文件2的猴痘病毒（Monkeypox Virus, MPXV）序列数据集的Newick格式系统发育树文件。
补充文件2：2022年全球大流行期间猴痘病毒基因组的有根系统发育树。病毒序列的多序列比对通过MAFFT生成，系统发育树使用FigTree进行可视化。以骆驼痘病毒序列（登录号MZ300860）作为外群完成系统发育树的根化处理。猴痘病毒基因组的U、M、I三组分别以绿色、蓝色和红色标注；基于串联重复序列（Tandem Repeat, TR）分析得到的重组猴痘病毒分离株以橙色标注。
补充文件3：本研究中2022年1月1日至7月20日的猴痘病毒B.1进化分支基因组序列（共415条，N=415）。补充文件2的系统发育分析中纳入了该骆驼痘病毒序列（登录号MZ300860）。
补充文件4：针对补充文件3与图S6中猴痘病毒基因组序列内已注释串联重复序列（TR）的TRAL分析结果。
补充表1：本研究中猴痘病毒基因组序列信息汇总。
图S1：包含强连锁不平衡单核苷酸多态性（Single Nucleotide Polymorphism, SNP）对的序列，其D’的95%置信区间上限高于0.98，下限高于0.7。
图S2：猴痘病毒A47R与A49R基因间区的串联重复序列C（TR C）。（A）TR C、A47R与A49R基因的示意图，箭头指示基因转录方向；（B）TR C位于A47R转录本的3’非翻译区（3’-UTR）。用于添加poly(A)尾的3’末端UUUUUNU序列以红色方框标注。本研究参考猴痘病毒序列为OP19276。
图S3：猴痘病毒A41L与A42R基因间区的串联重复序列B（TR B）。（A）TR B、A41L与A42R基因的示意图，箭头指示基因转录方向，序列b与序列c分别见于（B）与（C）；（B）与牛痘病毒序列相比，猴痘病毒基因组中TR B的插入序列（对应图A）；（C）猴痘病毒与牛痘病毒A42R启动子区的序列比对，TAAAT与AAAAAA元件以红色方框标注。本研究参考猴痘病毒序列为OP19276。
图S4：猴痘病毒B18R与B19R基因间区的串联重复序列D（TR D）。（A）TR D、B18R与B19R基因的示意图，箭头指示基因转录方向，序列b、c与d分别见于（B）、（C）与（D）；（B）猴痘病毒B序列与牛痘病毒B19R启动子区的比对结果，其中仅牛痘病毒含有TAAAT与AAAAAAA元件；（C、D）猴痘病毒B19R启动子区序列，以及对应图A的猴痘病毒基因组中TR D的插入序列。本研究参考猴痘病毒序列为OP19276。
图S5：牛痘病毒K3L与猴痘病毒C3L的序列比对。在第130~132位碱基处引入终止密码子，使得C3L相较于K3L形成截短蛋白。
图S6：（A）TR A至TR F的分歧值范围统计表。由于TR B的序列仅由AT碱基组成，故无对应数值；（B）每个方框（对应TR A、C、D、E、F）包含与亲本病毒序列关联的不同颜色的独立串联重复核苷酸单元（Tandem Repeat Nucleotide, TRN）：IRBA22-14（群I，登录号ON755040）、MUW1527495（群M，登录号ON019276）或USA_2022_CA002（群U，登录号ON954773），与图1F一致。所有TR与TRN均通过TRAL算法验证，显著性p值小于10^-4。