N^(6)-methyladenine(6mA)is a prevalent DNA modification and is involved in a wide range of human diseases.Previous studies have indicated that 6mA is enriched in mitochondrial DNA(mtDNA)of mammals.By employing an evol...N^(6)-methyladenine(6mA)is a prevalent DNA modification and is involved in a wide range of human diseases.Previous studies have indicated that 6mA is enriched in mitochondrial DNA(mtDNA)of mammals.By employing an evolved adenine deaminase,we developed a deaminase-mediated sequencing(DM-seq)method that could achieve genome-wide mapping of 6mA in mammalian mtDNA at single-base resolution.In this study,we used an engineered adenine deaminase,known as TadA8e protein,to map 6mA in mtDNA of hepatocellular carcinoma(HCC)by DM-seq.Through high-throughput sequencing,we identified sixteen 6mA sites in both HCC and adjacent normal tissue mtDNA.The results revealed an increased overall 6mA level in mtDNA associated with HCC.Furthermore,an elevation in 6mA level was observed alongside a decrease in the m RNA levels of the corresponding genes,indicating that increased6mA level hindered transcription processes related to these genes.These findings demonstrate that 6mA in mtDNA is correlated with HCC and provide evidence supporting the inhibitory effect of elevated 6mA level on subsequent transcriptional activity.This research illuminates the intricate relationship between 6mA modification and transcriptional regulation in the context of HCC,offering valuable insights into the role of 6mA modification in HCC pathogenesis.展开更多
To understand the gene-based biological processes in-depth,the single-molecule real-time sequencing has drawn increasing attention with promoted by the Human Genome Project.Herein,a set of newly designed canonical flu...To understand the gene-based biological processes in-depth,the single-molecule real-time sequencing has drawn increasing attention with promoted by the Human Genome Project.Herein,a set of newly designed canonical fluorescent bases(A_(y),tC,G_(b),T_(p))are proposed for four-color DNA sequencing.These quasi-intrinsic probes are derived from the fluorophore replacement and ring expansion on natural bases,which still keep the pyrimidine or purine underlying skeleton and Watson–Crick hydrogen bonding face to allow minimal perturbation to the native DNA duplex.More importantly,these nucleobase analogues possess red-shifted absorption and efficient photoluminescence due to the enhancedπ-conjugation in character.Meanwhile,the four analogues could generate distinct emission wavelength(Δλ~50 nm)for real-time sequencing.To assess the biological employment of the proposed biosensors,the effects of base pairing and linking deoxyribose are also considered.展开更多
The efficacy of DNA sequencing,particularly long reads nanopore sequencing,is critically dependent on the amount and quality of the input DNA.However,extracting high concentrations of DNA from low biomass samples,espe...The efficacy of DNA sequencing,particularly long reads nanopore sequencing,is critically dependent on the amount and quality of the input DNA.However,extracting high concentrations of DNA from low biomass samples,especially from solid matrices,presents significant challenges,this limitation not only substantially hampers the scope of environmental microbiology studies but also makes enhancing DNA yield indispensable in many instances.Therefore,in this study,we systematically evaluated the impact of four different DNA enrichment methods on both amplicon and metagenomic community analyses of solid-phase,low-biomass samples:permafrost soil and biofilm of sand filter.These methods include multiple displacement amplification(MDA),centrifugal filtration(CF),freeze vacuum drying at(FVD)as well as vacuum centrifugal at 35,45,and 60°C(namely VC35,VC45,VC60).Our results indicate that FVD was the most effective for increasing DNA concentration,while VC methods best preserved DNA fragment length.In contrast,the widely used MDA and CF methods exhibited biases,preferentially enriching low-GC content sequences,which affected both assembly and annotation outcomes.Metagenomic assembly from MDA and CF samples was suboptimal,with fewer contigs and no middle quality MAGs recovered compared to other methods.Community composition analysis revealed significant shifts across all enrichment methods,with Sphingomonas and Sphingorhabdus genera could be obviously enriched.These findings highlight the necessity and importance of carefully selecting DNA enrichment methods to ensure reliable metagenomic investigation of low-biomass environmental samples.展开更多
基金supported by the National Natural Science Foundation of China(No.22277093)the Key Research and Development Project of Hubei Province(No.2023BCB094)。
文摘N^(6)-methyladenine(6mA)is a prevalent DNA modification and is involved in a wide range of human diseases.Previous studies have indicated that 6mA is enriched in mitochondrial DNA(mtDNA)of mammals.By employing an evolved adenine deaminase,we developed a deaminase-mediated sequencing(DM-seq)method that could achieve genome-wide mapping of 6mA in mammalian mtDNA at single-base resolution.In this study,we used an engineered adenine deaminase,known as TadA8e protein,to map 6mA in mtDNA of hepatocellular carcinoma(HCC)by DM-seq.Through high-throughput sequencing,we identified sixteen 6mA sites in both HCC and adjacent normal tissue mtDNA.The results revealed an increased overall 6mA level in mtDNA associated with HCC.Furthermore,an elevation in 6mA level was observed alongside a decrease in the m RNA levels of the corresponding genes,indicating that increased6mA level hindered transcription processes related to these genes.These findings demonstrate that 6mA in mtDNA is correlated with HCC and provide evidence supporting the inhibitory effect of elevated 6mA level on subsequent transcriptional activity.This research illuminates the intricate relationship between 6mA modification and transcriptional regulation in the context of HCC,offering valuable insights into the role of 6mA modification in HCC pathogenesis.
基金Project supported by the National Natural Science Foundation of China(Grant Nos.11804195,11847224,11674198,and 12274265)the Natural Science Foundation of Shandong Province,China(Grant Nos.ZR2018BA034 and ZR2022MA006)。
文摘To understand the gene-based biological processes in-depth,the single-molecule real-time sequencing has drawn increasing attention with promoted by the Human Genome Project.Herein,a set of newly designed canonical fluorescent bases(A_(y),tC,G_(b),T_(p))are proposed for four-color DNA sequencing.These quasi-intrinsic probes are derived from the fluorophore replacement and ring expansion on natural bases,which still keep the pyrimidine or purine underlying skeleton and Watson–Crick hydrogen bonding face to allow minimal perturbation to the native DNA duplex.More importantly,these nucleobase analogues possess red-shifted absorption and efficient photoluminescence due to the enhancedπ-conjugation in character.Meanwhile,the four analogues could generate distinct emission wavelength(Δλ~50 nm)for real-time sequencing.To assess the biological employment of the proposed biosensors,the effects of base pairing and linking deoxyribose are also considered.
基金supported by the National Natural Science Foundation of China (No. 42277103)the Shenzhen Science and Technology Innovation Committee, China (Nos. JCYJ20240813095659001 and KCXFZ20240903094205008)+1 种基金the Guangdong Provincial Key Laboratory of Soil and Groundwater Pollution Control, China (No. 2023B1212060002)the Guangdong Provincial Key Laboratory of Environmental Health and Land Resource, China (No. 2020B121201014) and the high level special funds, China (Nos. G03050K001 and G030290001)。
文摘The efficacy of DNA sequencing,particularly long reads nanopore sequencing,is critically dependent on the amount and quality of the input DNA.However,extracting high concentrations of DNA from low biomass samples,especially from solid matrices,presents significant challenges,this limitation not only substantially hampers the scope of environmental microbiology studies but also makes enhancing DNA yield indispensable in many instances.Therefore,in this study,we systematically evaluated the impact of four different DNA enrichment methods on both amplicon and metagenomic community analyses of solid-phase,low-biomass samples:permafrost soil and biofilm of sand filter.These methods include multiple displacement amplification(MDA),centrifugal filtration(CF),freeze vacuum drying at(FVD)as well as vacuum centrifugal at 35,45,and 60°C(namely VC35,VC45,VC60).Our results indicate that FVD was the most effective for increasing DNA concentration,while VC methods best preserved DNA fragment length.In contrast,the widely used MDA and CF methods exhibited biases,preferentially enriching low-GC content sequences,which affected both assembly and annotation outcomes.Metagenomic assembly from MDA and CF samples was suboptimal,with fewer contigs and no middle quality MAGs recovered compared to other methods.Community composition analysis revealed significant shifts across all enrichment methods,with Sphingomonas and Sphingorhabdus genera could be obviously enriched.These findings highlight the necessity and importance of carefully selecting DNA enrichment methods to ensure reliable metagenomic investigation of low-biomass environmental samples.
