Abstract
Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519–539, E969–983, E1063–1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies.
Highlights
IntroductionThe 16S ribosomal RNA (rRNA) genes are essential and occur in at least one copy in a genome [1]
In prokaryotes, the 16S ribosomal RNA genes are essential and occur in at least one copy in a genome [1]
Most of the conservative archaeal and eubacterial fragments were numbered according to approximate positions on the E. coli 16S ribosomal RNA (rRNA) gene, and only four fragments lacked any counterparts: eubacterial fragments 104–120, 683–707, and 1177–1197, and archaeal fragment 1225–1242 (Table 1)
Summary
The 16S ribosomal RNA (rRNA) genes are essential and occur in at least one copy in a genome [1]. They are present in all mitochondrial genomes, which have lost most of their ancestral gene content in the long evolutionary history of symbiosis [2]. The products of the rRNA genes can fold into a complex, stable secondary structure, consisting of stems and loops [4]. Since the invention of the polymerase chain reaction (PCR) technique [7], the variant regions, V1–V9, of the 16S rRNA genes (rDNAs) have been used for species identification [8]
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