Here, we compared four commonly used pipelines (QIIME2, Bioconductor, UPARSE and mothur) run on two operating systems (OS) (Linux and Mac), to evaluate the impact of bioinformatic pipeline and OS on the taxonomic classification of 40 human stool samples. Microbial identification might be influenced by several factors, including the choice of bioinformatic pipelines, making comparisons across studies difficult. 6Memory Clinic and LANVIE – Laboratory of Neuroimaging of Aging, University Hospitals and University of Geneva, Geneva, SwitzerlandĪmplicon high-throughput sequencing of 16S ribosomal RNA (rRNA) gene is currently the most widely used technique to investigate complex gut microbial communities.5Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.4Institut de Microbiologie de l’Université de Lausanne, Lausanne, Switzerland.3Laboratory of Biological Psychiatry, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy.2Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland.1Laboratory of Neuroimaging and Alzheimer’s Epidemiology, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy.Chichester: Wiley.Moira Marizzoni 1*†, Thomas Gurry 2†, Stefania Provasi 3, Gilbert Greub 4, Nicola Lopizzo 3, Federica Ribaldi 1,5,6, Cristina Festari 1, Monica Mazzelli 3, Elisa Mombelli 3, Marco Salvatore 7, Peppino Mirabelli 7, Monica Franzese 7, Andrea Soricelli 7, Giovanni B. In Nucleic Acid Techniques in Bacterial Systematics. ExampleĬonsider a partial 16S rRNA gene sequence from the strain Nocardia carnea that’s 606 bp in length (Accession AY756546.1, 606 bp):Ĭompleteness is 42.1% because the query 16S rRNA sequence (indicated in blue) only spans from 19~625 bp of the complete 16S rRNA sequence (indicated in red), which is 1439 bp long. The suggested minimum threshold for using a 16S rRNA gene sequence for taxonomic purposes is 95% completeness, as incomplete or partial sequences with low completeness scores will have insufficient resolving power, resulting in erroneous identification results. The most similar sequence in the database of complete sequences is identified by using an algorithm called USEARCH. Where L is the length of a query sequence and C is the length of the most similar sequence that is regarded as complete (using the definition above). Mathematically, completeness is defined as ( Kim et al., 2012): Then how do we determine whether a 16S rRNA gene segment that was sequenced from a sample is complete or nearly complete? We use a measure called completeness.Ĭompleteness is an objective measure of the degree of coverage of a query 16S rRNA gene sequence with respect to the full-length, complete 16S rRNA gene sequence. The complete 16S rRNA gene sequence serves as a reference against which partial 16S rRNA gene sequences (obtained from high throughput sequencing) can be compared. Complete 16S rRNA gene lengths vary depending on species, and a complete or nearly complete sequence is generally required for taxonomic analyses. 2014).Ī complete 16S rRNA gene sequence is the DNA between PCR primers 27F and 1492R for Bacteria, and between PCR primers A25F and U1492R for Archaea. (2012) in the latter study, the determination of 16S cutoff, i.e., 98.7% similarity, for species delineation was proposed on the basis of this region as full-length 16S ( Kim et al. The use of these particular regions in EzBioCloud database was given by Kim et al.
This allows the fair calculation of sequence similarity between PCR-derived and genome-derived reference sequences. For the purposes of all necessary bioinformatics calculations, a complete 16S rRNA gene sequence is defined as the DNA sequence region between universal PCR primers 27F and 1492R for Bacteria (Lane, 1991), and between PCR primers A25F and U1492R for Archaea ( Dojka et al.
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