Throughput and accuracy of microbial community analysis using full-length 16S rDNA amplicon sequence

来源 :第七届全国微生物资源学术暨国际微生物系统与分类学研讨会 | 被引量 : 0次 | 上传用户:caoerduo
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  Universally conserved 16S rRNA gene sequences generated using high-throughput sequencing technique has become powerful tool to analysis the robust diversity and characterizing microbial communities.Recently,even full-length 16S rRNA sequence can be obtained from PacBio(R)SMRT sequencer with high yield and accuracy.While partial region sequences have been used as sequence tags in microbial community analysis with sequencing bias and absence of taxonomic classification below genus level,the analysis using full-length 16S rRNA is expected to improve the result significantly.In this study,soil metagenome,fecal metagenome and a synthetic mock community DNA were profiled for bacterial 16S with SMRT sequencing using P6/C4chemistry.Triplicate 16S rDNA PCR and its representing SMRT sequencing were performed five times repeatedly.Each SMRT cell of full-length 16S rDNA reads analyzed using three different CCS filtering condition,CCS with minimum 6 full passes,minimum 90%,and 99%predicted accuracy.Bareode sorting and 12~18kbp length filtering was followed by primer trimming process.Homopolymer checked as error correction and UCHIME from USEARCH program used for detect chimeras.Non-chimeric CCS reads analyzed for community profiling through in house pipeline.CCS reads accuracy evaluated by number of mismatches and insertion/deletion errors.Mock community profile evaluated in classification rate at taxonomic levels and its accuracy,taxon composition.From soil and fecal data,we were able to sort out non-chimeric sequences based on the reproduction of highly similar sequences from multiple PCR reactions.Overall,we demonstrate the usefulness of full-length 16S rRNA gene amplicon sequencing in microbial ecology,and suggest the optimal method for generation and analysis of barcoded full-length 16S rDNA sequence data.
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