RESUMO
The 16S rRNA gene has been used as master key for studying prokaryotic diversity in almost every environment. Despite the claim of several researchers to have the best universal primers, the reality is that no primer has been demonstrated to be truly universal. This suggests that conserved regions of the gene may not be as conserved as expected. The aim of this study was to evaluate the conservation degree of the so-called conserved regions flanking the hypervariable regions of the 16S rRNA gene. Data contained in SILVA database (release 123) were used for the study. Primers reported as matches of each conserved region were assembled to form contigs; sequences sizing 12 nucleotides (12-mers) were extracted from these contigs and searched into the entire set of SILVA sequences. Frequency analysis shown that extreme regions, 1 and 10, registered the lowest frequencies. 12-mer frequencies revealed segments of contigs that were not as conserved as expected (≤90%). Fragments corresponding to the primer contigs 3, 4, 5b and 6a were recovered from all sequences in SILVA database. Nucleotide frequency analysis in each consensus demonstrated that only a small fraction of these so-called conserved regions is truly conserved in non-redundant sequences. It could be concluded that conserved regions of the 16S rRNA gene exhibit considerable variation that has to be considered when using this gene as biomarker.
RESUMO
The classification performance of Kraken was evaluated in terms of sensitivity and specificity when using short and long 16S rRNA sequences. A total of 440,738 sequences from bacteria with complete taxonomic classifications were downloaded from the high quality ribosomal RNA database SILVA. Amplicons produced (86,371 sequences; 1450 bp) by virtual PCR with primers covering the V1-V9 region of the 16S-rRNA gene were used as reference. Virtual PCRs of internal fragments V3-V4, V4-V5 and V3-V5 were performed. A total of 81,523, 82,334 and 82,998 amplicons were obtained for regions V3-V4, V4-V5 and V3-V5 respectively. Differences in depth of taxonomic classification were detected among the internal fragments. For instance, sensitivity and specificity of sequences classified up to subspecies level were higher when the largest internal fraction (V3-V5) was used (54.0 and 74.6% respectively), compared to V3-V4 (45.1 and 66.7%) and V4-V5 (41.8 and 64.6%) fragments. Similar pattern was detected for sequences classified up to more superficial taxonomic categories (i.e. family, order, class ). Results also demonstrate that internal fragments lost specificity and some could be misclassified at the deepest taxonomic levels (i.e. species or subspecies). It is concluded that the larger V3-V5 fragment could be considered for massive high throughput sequencing reducing the loss of sensitivity and sensibility.
RESUMO
Ultrafast-metagenomic sequence classification using exact alignments (Kraken) is a novel approach to classify 16S rDNA sequences. The classifier is based on mapping short sequences to the lowest ancestor and performing alignments to form subtrees with specific weights in each taxon node. This study aimed to evaluate the classification performance of Kraken with long 16S rDNA random environmental sequences produced by cloning and then Sanger sequenced. A total of 480 clones were isolated and expanded, and 264 of these clones formed contigs (1352 ± 153 bp). The same sequences were analyzed using the Ribosomal Database Project (RDP) classifier. Deeper classification performance was achieved by Kraken than by the RDP: 73% of the contigs were classified up to the species or variety levels, whereas 67% of these contigs were classified no further than the genus level by the RDP. The results also demonstrated that unassembled sequences analyzed by Kraken provide similar or inclusively deeper information. Moreover, sequences that did not form contigs, which are usually discarded by other programs, provided meaningful information when analyzed by Kraken. Finally, it appears that the assembly step for Sanger sequences can be eliminated when using Kraken. Kraken cumulates the information of both sequence senses, providing additional elements for the classification. In conclusion, the results demonstrate that Kraken is an excellent choice for use in the taxonomic assignment of sequences obtained by Sanger sequencing or based on third generation sequencing, of which the main goal is to generate larger sequences.
Assuntos
Bactérias/genética , DNA Ribossômico/classificação , Metagenômica/métodos , RNA Ribossômico 16S/classificação , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Bactérias/classificação , Clonagem de Organismos/métodos , Biologia Computacional/métodos , Código de Barras de DNA Taxonômico/métodos , DNA Ribossômico/análise , DNA Ribossômico/genética , Bases de Dados Genéticas , Escherichia coli/genética , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , SoftwareRESUMO
La acuicultura, como actividad económica en crecimiento, requiere el empleo de técnicas y estrategias que le permitan solucionar problemas para ser eficiente. Dentro del contexto de mejoramiento genético de los organismos cultivables, se requiere de la selección de organismos reproductores que posean las mejores características en tasa de crecimiento, de conversión alimenticia y de resistencia a enfermedades, entre otras. Hoy en día se dispone de la metodología que permite identificar a nivel genómico las características requeridas para seleccionar a los mejores pies de cría, además de determinar la diversidad genética entre familias y poblaciones, establecer líneas de pedigrí y determinar paternidad. La técnica denominada AFLP (amplified fragment length polymorphism) ha sido exitosa para generar marcadores moleculares que son aplicados en estudios genéticos en una amplia gama de disciplinas, incluyendo genómica de organismos cultivables, donde ha aportado importantes avances para la domesticación de especies acuícolas. Este trabajo revisa los hallazgos y usos más relevantes de la aplicación de AFLP en acuicultura