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Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

World Journal of Gastroenterology 2010年第33期16卷 4135-4144页

作者： Carlos W Nossa William E Oberdorf Jφrn A Aas Bruce J Paster Todd Z DeSantis Eoin L Brodie daniel malamud Michael A Poles Zhiheng Pei Department of Medicine New York University School of Medicine Department of Molecular Genetics The Forsyth Institute Faculty of Dentistry University of Oslo Harvard School of Dental Medicine Lawrence Berkeley National Laboratory Center for Environmental Biotechnology New York University College of Dentistry Depar tment of Pa thology and Medicine New York University School of MedicineNew YorkNY 10016United States Department of Veterans Affairs New York Harbor Health SystemNewYorkNY 10010United States

AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut ***:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial *** primers evaluated were from the European rRNA *** assess the effect of sequence length on accuracy of classification,16S rRNA genes of various lengths were created by trimming the full length *** spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer *** sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP).The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes,represented by 36 ***:Truncation to 100 nucleotides(nt)downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87(39.7%)of the 219 sequences,compared with misclassification of only 29(13.2%)sequences with truncation to 350 *** 350-nt sequence reads within various regions of the 16S rRNA gene,the reverse read of an amplicon generated using the 343F/798R primers had the least(8.2%)effect on *** comparison,truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0%of the 219 *** 343F/798R amplicon accurately assigned 91.8%of the 219 sequences at the species *** by abundance of the species in the esophageal dataset,the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage(92%).Modification of the 343F/798R primers to 347F/803R increased their universality among foregut ***

关键词： Foregut Microbiome 16S 454 sequencing Primer

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