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Fuyi Xu, Tianzhu Chao, Yiyin Zhang, Shixian Hu, Yuxun Zhou, Hongyan Xu, Junhua Xiao, Kai Li, "Chromosome 1 Sequence Analysis of C57BL/6J-Chr1KM Mouse Strain", International Journal of Genomics, vol. 2017, Article ID 1712530, 9 pages, 2017. https://doi.org/10.1155/2017/1712530
Chromosome 1 Sequence Analysis of C57BL/6J-Chr1KM Mouse Strain
The Chinese Kunming (KM) mouse is a widely used outbred mouse stock in China. However, its genetic structure remains unclear. In this study, we sequenced the genome of the C57BL/6J-Chr1KM (B6-Chr1KM) strain, the chromosome 1 (Chr 1) of which was derived from one KM mouse. With 36.6× average coverage of the entire genome, 0.48 million single nucleotide polymorphisms (SNPs) and 96,679 indels were detected on Chr 1 through comparison with reference strain C57BL/6J. Moreover, 46,590 of them were classified as novel mutations. Further functional annotation identified 155 genes harboring potentially functional variants, among which 27 genes have been associated with human diseases. We then performed sequence similarity and Bayesian concordance analysis using the SNPs identified on Chr 1 and their counterparts in three subspecies, Mus musculus domesticus, M. m. musculus, and M. m. castaneus. Both analyses suggested that the Chr 1 sequence of B6-Chr1KM was predominantly derived from M. m. domesticus while 9.7% of the sequence was found to be from M. m. musculus. In conclusion, our analysis provided a detailed description of the genetic variations on Chr 1 of B6-Chr1KM and a new perspective on the subspecies origin of KM mouse which can be used to guide further genetic studies with this mouse strain.
The Chinese Kunming (KM) mouse colony, the largest outbred mouse stock maintained by commercial dealers nationwide in China, has been widely used in pharmaceutical and genetic studies . Unlike other outbred mice, KM mouse has a complex evolutionary history. In 1944 during the World War II, Swiss mice were initially introduced into Kunming, Yunnan Province, China, from the Indian Haffkine Institute by Professor Feifan Tang via the Hump route with the help of the American Volunteer Group . These mice were named KM mice after their initial location in China. Because most other mouse strains were lost and mouse facilities were damaged during the World War II, KM mouse became the only laboratory mouse available afterwards. They were gradually distributed throughout most of the country for medical studies. However, despite the importance of this outbred mouse, its underlying genetic structure remains unclear.
According to the Mouse Genome Informatics (http://www.informatics.jax.org/), over one thousand quantitative trait loci (QTLs) have been mapped on mouse chromosome 1 (hereafter referred to as Chr 1) including large amounts of QTLs related to metabolism disorder. However, very few candidate genes have been identified partly because of the large QTL intervals. In order to fine map the metabolism disorder QTLs on Chr 1 and identify the candidate genes, we established a population of Chr 1 substitution mouse strains, in which C57BL6/J (B6) was the host strain, and one KM mouse, five inbred strains, and twenty-four wild mice captured from various locations in China were selected as the Chr 1 donors . In order to dissect the genetic structure and variations of this population and better severe further genetic studies, we have resequenced 18 strains of this population including C57BL/6J-Chr1KM (B6-Chr1KM) with next-generation sequencing technology .
In this study, we analyzed the genome sequence data from B6-Chr1KM strain and identified 0.48 million single nucleotide polymorphisms (SNPs) and 96,679 indels on Chr 1, of which 6.4% SNPs and 16.3% indels were considered to be novel. Functional annotation suggested that 474 variants had deleterious effect on gene functions. In addition, we explored the KM mouse genetic structure by performing sequence similarity and Bayesian concordance analysis (BCA) on Chr 1. Results suggested that KM mouse was predominately originated from Mus musculus domesticus and part of the sequence was from M. m. musculus.
2. Materials and Methods
B6 and KM mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., China. One male KM mouse was mated with female B6 to produce hybrid F1, followed by 8 generations of backcrossing with B6 using marker-assisted selection, then brother × sister mating to create a B6-Chr1KM Chr 1 substitution strain . All mice were maintained under specific pathogen-free (SPF) conditions according to the People’s Republic of China Laboratory Animal Regulations, and the study was conducted in accordance with the recommendations of and was approved by the Laboratory Animal Committee of Donghua University.
2.2. DNA Sequencing
B6-Chr1KM genomic DNA was extracted from tail tissue of a male mouse using an AxyPrep™ Multisource Genomic DNA Miniprep Kit (Axygen, Hangzhou, China) according to the manufacturer’s protocol.
Purified genomic DNA was sheared and size selected (300–500 bp). Paired-end sequencing (2 × 125 bp) was carried out with an Illumina HiSeq 2500 instrument (Illumina Inc., San Diego, CA, USA) on two lanes by WuXi AppTec (Shanghai, China) according to the manufacturer’s protocol.
2.3. Read Alignment
Raw reads were filtered using NGS QC toolkit v2.3  to remove reads containing more than 30% low-quality (Q20) bases. Filtered reads were aligned to the C57BL/6J reference genome (December 2011 release of the mouse reference genome (mm10) from Ensembl) using BWA (version 0.7.10-r789) with 12 threads . The resulting SAM file was converted to a binary format and sorted with SAMtools v1.1 , followed by the marking of duplicate reads using picard-tools v1.119 (http://picard.sourceforge.net). To improve SNP and indel calling, indel realignment was conducted with Genome Analysis Toolkit (GATK v3.3) .
2.4. SNP/Indel Identification and Annotation
SNPs and indels were called using SAMtools mpileup and BCFtools call functions , with the '-uf' and '-cv' parameters, respectively. To identify a high-quality variant data set, variants were filtered using the BCFtools filter and VCFtools varFilter function . The following parameters were used: for BCFtools filter, '-g 10 -G 3 -i 'QUAL>10 && MIN(MQ)>25 && MIN(DP)>6 && MAX(DP)<199 && (DP4+DP4)> 2', and for VCFtools varFilter, '-2 0'.
Ensembl Variant Effect Predictor tool (VEP, v78)  was used to characterize the SNPs and indels, and the algorithm SIFT was used to predict whether a missense variant would have a deleterious effect on a protein-coding gene.
2.5. Sequence Similarity Analysis
SNP information for WSB/EiJ (WSB), PWK/PhJ (PWK), and CAST/EiJ (CAST) was downloaded from the Mouse Genome Project (MGP) database of the Sanger Institute. The Chr 1 consensus sequence for each strain was constructed using the SAMtools consensus parameters. The repeat-masked B6-Chr1KM Chr 1 sequence was divided into 1955 100 kb segments. The similarities of each segment with the corresponding segments in the WSB, CAST, and PWK were evaluated. Sliding window similarity analysis was also performed using 500 kb windows and 100 kb sliding intervals.
2.6. Phylogenetic Analysis
Phylogenetic analysis was conducted with the previously reported BCA method , with the Rattus norvegicus Chr 1 sequence (version rn5) downloaded from Ensembl used as the out-group. Briefly, consensus sequences from the WSB, PWK, and CAST strains were mapped to the alignment and gaps filled with Ns. Collinear segments were partitioned into 830 loci using a minimum description length algorithm with a default maximum cost.
2.7. Phylogenetic Tree Evaluation
Nexus files corresponding to the WSB-derived or PWK-derived regions were converted to FASTA files, and then a neighbor-joining phylogenetic tree was constructed using MEGA6 program . Subsequently, 1000 bootstrap replicates were performed to generate branch support values.
3.1. B6-Chr1KM Genome Background
Chromosome substitution strains, also named as consomic strains, are designed to simplify the genome background and increase the power and speed of QTL mapping. The characteristic of consomic strain is that it only contains a single chromosome from the donor strain substituting the corresponding chromosome in the host strain. For B6-Chr1KM consomic strain, Chr 1 sequence was derived from one KM mouse, while the genome background was from the B6 strain (Figure 1). In addition, sequences in the primary mouse reference assembly come from the same B6 strain. Therefore, our analysis of B6-Chr1KM whole genome resequencing data only focused on Chr 1.
3.2. SNP and Indel Discovery
In this study, approximately one billion reads from the B6-Chr1KM mouse strain were generated on two lanes of Illumina HiSeq 2500. A total of 78.65% of the reads were considered to be clean reads after quality control evaluation. Of them, more than 99% were aligned to the B6 mouse reference genome (mm10) using BWA with a mean genome-wide coverage of 36.6×.
A total of 479,956 SNPs and 96,679 indels were detected using SAMtools/BCFtools on Chr 1, in which 462,755 (96.42%) of the sites were homozygous. These variants were compared with variant calls from 36 key mouse strains from the Sanger Institute  as well as NCBI dbSNP142 variant data sets. This led to the identification of 449,089 SNPs (93.6%) as known, and the remaining 30,867 SNPs (6.4%) were classified as novel. For indels, 15,723 (16.3%) were classified as novel. In addition, we evaluated the variant calls using Sanger sequencing in our previous study which achieved high accuracy with 0.57% false positive and 0% false negative rate .
Next, we detected the distribution and density of SNPs over 100 kb window sizes. The observed average SNP density across the entire Chr 1 was 250 per 100 kb. However, different regions showed varying densities. For example, 29.5% of the Chr 1 sequence had an extremely low (0–5 SNPs per 100 kb) SNP density, while 9.1% had a high density (800 or more SNPs per 100 kb). The proximal region of Chr 1 was the longest region with a low SNP density encompassing nearly 25 Mb (Figure 2).
3.3. Functional Consequences of the SNPs and Indels
The putative consequences of SNPs and indels were cataloged using VEP from Ensembl (Table 1). The majority of the SNPs were located in intergenic (224,557, 18.7%) and intronic regions (575,013, 47.8%), and nearly 12% were classified as noncoding transcript variants. With regard to splice sites, 40 splice variants (including splice donor and splice acceptor variants) were found. The numbers of SNPs causing a premature stop codon or stop loss were 19 and 5, respectively. In addition, 2,378 (0.2%) missense variants were detected in 358 genes (one or more variants per gene). Among them, 380 variants (31.6%) from 113 genes were considered to have deleterious effects (). Similar to the SNPs, the majority of indels were intronic (49.3%) and intergenic (17.1%) or within 5 kb upstream or downstream of a gene (16.9%). Only a small number of indels caused frameshift (22) and stop gain or loss (2). Among the novel variants, 7 caused a disruption of the translational reading frame; 10 were predicted as premature truncation of the protein due to gain or loss of stop codons; and 9 were located in splice donor regions. In addition, 104 novel missense variants from 20 genes had deleterious effects.
|Consequences were predicted using Ensembl VEP and gene models from Ensembl version 76. Novel SNPs or indels are defined as variants that were not in MGP and dbSNP142 data sets.|
Next, we annotated these genes containing amino acid altering variants () and those with stop gain or loss, frameshift, and splice region variant genes with the Human-Mouse: Disease Connection database from Mouse Genome Informatics . This analysis, which contained 155 genes, resulted in 27 genes associated with 49 different human disease-related phenotypes (Table 2), including macular degeneration, breast cancer, and immunodeficiency. Among these 27 disease genes, 9 have been investigated with mouse models, which had an in-depth phenotype information in different mouse genome background.
|OMIM: online Mendelian inheritance in man. Numbers in italic in OMIM ID column indicate that these diseases have mouse models. Human disease-related phenotypes come from “Human-Mouse: Disease Connection” database (http://www.informatics.jax.org/humanDisease.shtml) in Mouse Genome Informatics website.|
3.4. Sequence Similarity Analysis
The house mouse, Mus musculus, consists of three principal subspecies, with M. m. domesticus in Western Europe and the Middle East, M. m. musculus in Eastern Europe and Asia, and M. m. castaneus in Southeast Asia and India. Three genome sequences of the wild-derived inbred mouse strains, WSB, PWK, and CAST, which are broadly used to represent each of the subspecies, were selected for phylogenetic analysis. A Chr 1 consensus sequence was constructed for each strain using the SNP information from MGP. Because the simplest way to analyze phylogenetic divergence is by assessing sequence similarity, the Chr 1 sequence was separated into 1955 100 kb blocks and the similarities between each fragment and the corresponding sequences from WSB, PWK, and CAST were determined. The Chr 1 sequence was found to contain a large number of fragments with high sequence similarity to the corresponding sequence in WSB (Figure 3(a)), which is consistent with previous reports showing that KM mouse is derived from Swiss mice originated from the M. m. domesticus subspecies . In addition, a bimodal distribution of blocks with two peaks of similarity was observed in a comparison of B6-Chr1KM Chr 1 with PWK counterpart (Figure 3(a)). The first peak had only 99.05–99.1% sequence similarity to PWK, indicating the intersubspecies genome divergence of the Chr 1 sequence from M. m. musculus. The second peak had >99.7% sequence similarity to PWK (Figure 3(a)), indicating that the sequence of M. m. musculus introgressed into the KM mouse Chr 1. For the comparison of B6-Chr1KM and CAST, we just observed one peak which suggested no signs of introgression of M. m. castaneus into the KM mouse Chr 1.
We next performed sliding window similarity analysis using 500 kb windows and 100 kb sliding intervals (Figure 3(b)). We found that 13.5% and 6.4% of the Chr 1 sequences had high similarity (>99.7%) with the corresponding sequences of PWK and CAST, respectively. The distal portion of the B6-Chr1KM Chr 1 was found to have several regions that were highly similar to the corresponding regions of PWK with sharp boundaries between the regions of high and low similarity. However, we did not find any distinct boundaries between B6-Chr1KM and CAST Chr 1 sequence.
3.5. Bayesian Concordance Analysis
To determine the extent of phylogenetic discordance in B6-Chr1KM Chr 1, we assessed the discordance along Chr 1 by BCA. A total of 886 partitioned individual locus trees were used to estimate Bayesian concordance factors. In BCA, 87.7% of the loci supported a single KM/WSB topology with higher posterior probability, and 9.7% supported a single KM/PWK topology. None of the loci supported a KM/CAST topology, and the remaining 2.6% had a complicated topology (Figure 4(a)). Highly conserved genomic regions (Figure 3(b)) between the KM and PWK were almost found to have a relatively close topological relationship (Figure 4(a)). Furthermore, five loci with KM/WSB or KM/PWK topology were randomly selected, and the phylogenetic trees were confirmed by Mega software (Figure 4(b)).
Because the KM mouse is used regularly in pharmaceutical and genetic studies, its detailed genetic structure is of great value to the research community. In this study, we sequenced the genome of a male B6-Chr1KM mouse, in which Chr 1 was derived from one KM mouse. The detailed sequence analysis would provide new insights into the application of B6-Chr1KM in biomedical research.
In this study, we identified 479,956 SNPs and 96,679 indels on Chr 1, of which 8.1% did not exist in the MGP and dbSNP142 data sets, indicating that these variants were unique to the B6-Chr1KM mice. Therefore, these variants can be used as unique genetic markers for the genetic quality control of KM mouse. As the most common types of genetic variants, SNPs and indels have been increasingly recognized as having a wide range of effects on gene functions. Among the variants identified on Chr 1, most were located within intergenic or intronic regions. However, we also identified 474 functional variants (missense variant with , stop gain or loss variant, frameshift variant, and splice donor or acceptor variant) which influenced 155 genes. Additionally, several genes have been identified to be associated with human diseases, making them interesting candidates for further functional studies using KM mouse or our newly build B6-Chr1KM strain. For example, Rd3, which is associated with retinal degeneration, was identified as a missense substitution (A->T) with significant deleterious effects (). Previous studies have shown that mice with a homozygous mutation in Rd3 exhibit retinal degeneration at three weeks after birth . We also identified a splice acceptor variant in Lamb3 gene, which is associated with blistering of the skin. The mouse models with homozygous Lamb3 628 G->A showed blistering and erosions after birth .
Since KM mouse is originated from Swiss mice, it has been speculated to be contaminated with M. m. castaneus. In 1991, the morphological characteristics and isozyme polymorphisms of KM and Swiss mice were evaluated, revealing the presence of distinct genetic differences between them . Comparison of KM mouse with wild mice of M. m. castaneus captured in Kunming has revealed that the former is more closely related to M. m. domesticus than to M. m. castaneus. Conversely, contamination of KM mouse by M. m. castaneus has been previously demonstrated using the isozyme test . In 2003, the results of a study involving the detection of isozyme polymorphisms also supported the grouping of KM and Swiss mice with M. m. domesticus and not with M. m. musculus or M. m. castaneus . However, it has not yet been confirmed whether KM mouse contains part of the genome of M. m. musculus or M. m. castaneus. Therefore, high resolution studies of Chr 1 of KM mouse by next-generation sequencing may clarify whether these mice were originated from Swiss mice and/or other mice. Our sequence similarity analysis provided substantial evidence that KM mouse was derived from M. m. domesticus, which means that Swiss mice were their ancestor. Both 100 kb blocks and sliding window similarity analysis demonstrated that the Chr 1 of KM mouse was largely composed of M. m. domesticus sequences with the rest may derive from M. m. musculus or M. m. castaneus. Therefore, further analysis is needed to determine the proportion of each subspecies contribution to the Chr 1 of KM mouse.
With the increasing number of whole genome data sets, the reconstruction of phylogenetic trees at a genomic scale has become feasible. Exploration of these large data sets has revealed that there may be discordance among the topologies in different genomic regions [19, 20]. Although these differences may be caused by incorrect estimations of gene genealogies, incongruent gene trees can also be attributed to the differing evolutionary histories of different genomic regions, especially for close species or subspecies. Traditionally, there are two types of phylogenetic analysis methods, the consensus method and the total evidence method. Both methods barely quantify the topological discordance across the entire genome. Recently, BCA, which is an improvement upon the consensus method, has been used to statistically quantify the discordance, as well as to generate phylogenetic trees . A few studies using BCA have demonstrated its great potential for the reconstruction of phylogenic trees of mouse subspecies [11, 13, 22]. These studies indicate that BCA is a suitable method to quantify the proportions of Chr 1 sequence in B6-Chr1KM derived from the different subspecies. Through BCA, we found approximately that 90% and 10% of the sequences of Chr 1 were derived from M. m. domesticus and M. m. musculus, respectively. Although the sequence similarity analysis revealed that there were some regions which had higher sequence similarity with CAST, we did not observed the same results in the BCA. Therefore, we cannot make the conclusion that some of Chr 1 sequence of B6-Chr1KM came from CAST which represent M. m. castaneus. While for PWK, highly conserved genomic regions (Figure 3(b)) with KM aligned well with the BCA results (Figure 4(a)). Thus, from both analyses, we can make the conclusion that Chinese KM mouse has a mosaic genome structure with sequences predominately derived from M. m. domesticus and with at least some of the remaining sequences derived from M. m. musculus.
In summary, we presented the analysis of a high-quality genome sequence of the B6-Chr1KM. These data allow better understanding of the structure and origin of the genetic variations in the B6-Chr1KM mouse strain, which provides insights into the utility of this mouse strain and the KM outbred stock for further biomedical research and the study of complex diseases.
All raw reads were submitted to NCBI Sequence Read Archive under the Accession no. SRR2954707 associated with BioProject Accession no. PRJNA298468 and BioSample Accession no. SAMN04159475.
Conflicts of Interest
The authors declare that they have no competing interests.
Fuyi Xu and Tianzhu Chao contributed equally to this work.
This work was supported by the Key Project of Science & Technology Commission of Shanghai Municipality (no. 13140900300), the National Science Foundation of China (no. 31171199), the Fundamental Research Funds for the Central Universities (no. 2232013A3-06), and the DHU Distinguished Young Professor Program (B201308).
- X. Zhang, Z. Zhu, Z. Huang, P. Tan, and R. Z. Ma, “Microsatellite genotyping for four expected inbred mouse strains from KM mice,” Journal of Genetics and Genomics, vol. 34, no. 3, pp. 214–222, 2007.
- B. Yue, S. Liu, D. Liu et al., “Comparative studies on the genetic biological markers of five closed colonies of Kunming mice,” Laboratory Animal Science and Management, vol. 20, no. S1, pp. 58–62, 2003.
- J. Xiao, Y. Liang, K. Li et al., “A novel strategy for genetic dissection of complex traits: the population of specific chromosome substitution strains from laboratory and wild mice,” Mammalian Genome, vol. 21, no. 7-8, pp. 370–376, 2010.
- F. Xu, T. Chao, Y. Liang et al., “Genome sequencing of chromosome 1 substitution lines derived from Chinese wild mice revealed a unique resource for genetic studies of complex traits,” G3 (Bethesda), vol. 6, no. 11, pp. 3571–3580, 2016.
- R. K. Patel and M. Jain, “NGS QC Toolkit: a toolkit for quality control of next generation sequencing data,” PloS One, vol. 7, no. 2, article e30619, 2012.
- H. Li and R. Durbin, “Fast and accurate short read alignment with Burrows-Wheeler transform,” Bioinformatics, vol. 25, no. 14, pp. 1754–1760, 2009.
- H. Li, B. Handsaker, A. Wysoker et al., “The sequence alignment/map format and SAMtools,” Bioinformatics, vol. 25, no. 16, pp. 2078–2079, 2009.
- A. McKenna, M. Hanna, E. Banks et al., “The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data,” Genome Research, vol. 20, no. 9, pp. 1297–1303, 2010.
- P. Danecek, A. Auton, G. Abecasis et al., “The variant call format and VCFtools,” Bioinformatics, vol. 27, no. 15, pp. 2156–2158, 2011.
- W. McLaren, B. Pritchard, D. Rios, Y. Chen, P. Flicek, and F. Cunningham, “Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor,” Bioinformatics, vol. 26, no. 16, pp. 2069–2070, 2010.
- M. A. White, C. Ané, C. N. Dewey, B. R. Larget, and B. A. Payseur, “Fine-scale phylogenetic discordance across the house mouse genome,” PLoS Genetics, vol. 5, no. 11, article e1000729, 2009.
- K. Tamura, G. Stecher, D. Peterson, A. Filipski, and S. Kumar, “MEGA6: molecular evolutionary genetics analysis version 6.0,” Molecular Biology and Evolution, vol. 30, no. 12, pp. 2725–2729, 2013.
- T. M. Keane, L. Goodstadt, P. Danecek et al., “Mouse genomic variation and its effect on phenotypes and gene regulation,” Nature, vol. 477, no. 7364, pp. 289–294, 2011.
- J. A. Blake, J. T. Eppig, J. A. Kadin et al., “Mouse Genome Database (MGD)-2017: community knowledge resource for the laboratory mouse,” Nucleic Acids Research, vol. 45, no. D1, pp. D723–D729, 2017.
- J. S. Friedman, B. Chang, C. Kannabiran et al., “Premature truncation of a novel protein, RD3, exhibiting subnuclear localization is associated with retinal degeneration,” American Journal of Human Genetics, vol. 79, no. 6, pp. 1059–1070, 2006.
- J. Hammersen, J. Hou, S. Wunsche, S. Brenner, T. Winkler, and H. Schneider, “A new mouse model of junctional epidermolysis bullosa: the LAMB3 628G>A knockin mouse,” The Journal of Investigative Dermatology, vol. 135, no. 3, pp. 921–924, 2015.
- S. Shi, H. Wang, B. Cui et al., “Study on genetic variants of Chinese KM mouse subcolonies,” Chinese Journal of Laboratory Animal Science, vol. 1, no. 1, pp. 29–36, 1991.
- G. Zhao, S. Bao, D. Zhang, R. Zhang, and M. Jin, “Probing into the course of formation of genetic character of KM mouse,” Shanghai Laboratory Animal Science, vol. 14, no. 1, pp. 1–4, 1994.
- D. A. Pollard, V. N. Iyer, A. M. Moses, and M. B. Eisen, “Widespread discordance of gene trees with species tree in Drosophila: evidence for incomplete lineage sorting,” PLoS Genetics, vol. 2, no. 10, article e173, 2006.
- G. Giribet, G. D. Edgecombe, and W. C. Wheeler, “Arthropod phylogeny based on eight molecular loci and morphology,” Nature, vol. 413, no. 6852, pp. 157–161, 2001.
- C. Ane, B. Larget, D. A. Baum, S. D. Smith, and A. Rokas, “Bayesian estimation of concordance among gene trees,” Molecular Biology and Evolution, vol. 24, no. 2, pp. 412–426, 2007.
- T. Takada, T. Ebata, H. Noguchi et al., “The ancestor of extant Japanese fancy mice contributed to the mosaic genomes of classical inbred strains,” Genome Research, vol. 23, no. 8, pp. 1329–1338, 2013.
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