Toward the robust resolution of taxonomic ambiguity within Lepocinclis (Euglenida) based on DNA sequencing and morphology.
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ABSTRACT:
SUBMITTER: Chaber K
PROVIDER: S-EPMC9300208 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The amount of host DNA poses a major challenge to metagenome analysis. However, there is no guidance on the levels of host DNA, nor on the depth of sequencing needed to acquire meaningful information from whole metagenome sequencing (WMS). Here, we evaluated the impact of a wide range of amounts of host DNA and sequencing depths on microbiome taxonomic profiling using WMS. Synthetic samples with increasing levels of host DNA were created by spiking DNA of a mock bacterial community, with DNA from a mouse-derived cell line. Taxonomic analysis revealed that increasing proportions of host DNA led to decreased sensitivity in detecting very low and low abundant species. Reduction of sequencing depth had major impact on the sensitivity of WMS for profiling samples with 90% host DNA, increasing the number of undetected species. Finally, analysis of simulated datasets with fixed depth of 10 million reads confirmed that microbiome profiling becomes more inaccurate as the level of host DNA increases in a sample. In conclusion, samples with high amounts of host DNA coupled with reduced sequencing depths, decrease WMS coverage for characterization of the microbiome. This study highlights the importance of carefully considering these aspects in the design of WMS experiments to maximize microbiome analyses.
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Project description:BACKGROUND: Subterranean mammals have been of great interest for evolutionary biologists because of their highly specialized traits for the life underground. Owing to the convergence of morphological traits and the incongruence of molecular evidence, the phylogenetic relationships among three subfamilies Myospalacinae (zokors), Spalacinae (blind mole rats) and Rhizomyinae (bamboo rats) within the family Spalacidae remain unresolved. Here, we performed de novo transcriptome sequencing of four RNA-seq libraries prepared from brain and liver tissues of a plateau zokor (Eospalax baileyi) and a hoary bamboo rat (Rhizomys pruinosus), and analyzed the transcriptome sequences alongside a published transcriptome of the Middle East blind mole rat (Spalax galili). We characterize the transcriptome assemblies of the two spalacids, and recover the phylogeny of the three subfamilies using a phylogenomic approach. RESULTS: Approximately 50.3 million clean reads from the zokor and 140.8 million clean reads from the bamboo ratwere generated by Illumina paired-end RNA-seq technology. All clean reads were assembled into 138,872 (the zokor) and 157,167 (the bamboo rat) unigenes, which were annotated by the public databases: the Swiss-prot, Trembl, NCBI non-redundant protein (NR), NCBI nucleotide sequence (NT), Gene Ontology (GO), Cluster of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG). A total of 5,116 nuclear orthologous genes were identified in the three spalacids and mouse, which was used as an outgroup. Phylogenetic analysis revealed a sister group relationship between the zokor and the bamboo rat, which is supported by the majority of gene trees inferred from individual orthologous genes, suggesting subfamily Myospalacinae is more closely related to subfamily Rhizomyinae. The same topology was recovered from concatenated sequences of 5,116 nuclear genes, fourfold degenerate sites of the 5,116 nuclear genes and concatenated sequences of 13 protein coding mitochondrial genes. CONCLUSIONS: This is the first report of transcriptome sequencing in zokors and bamboo rats, representing a valuable resource for future studies of comparative genomics in subterranean mammals. Phylogenomic analysis provides a conclusive resolution of interrelationships of the three subfamilies within the family Spalacidae, and highlights the power of phylogenomic approach to dissect the evolutionary history of rapid radiations in the tree of life.
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