Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences

Speksnijder, A.G.C.L.; Kowalchuk, George A.; Jong, Steven de; Kline, Elizabeth; Stephen, J.; Laanbroek, Hendrikus J.

doi:10.1128/aem.67.1.469-472.2001

Cited by 209 publications

(144 citation statements)

References 28 publications

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“…Surplus alleles occurred in seven individuals (Table 1), but the number of sqs clades (Table 1, Figure 3) did not exceed the ploidy level. Given the high sequence similarity within the clades, these additional 'alleles' may be attributed to polymerase errors that were not identifiable as such (Speksnijder et al, 2001) or to a failure to decide which of several very similar sequences were recombinant within a sample, especially if the putative recombination point was close to one end of the sequence. The inclusion of sequences into the final alignment was done in a conservative way in order not to dismiss small, but real differences, which may be easily overlooked if alleles are inferred from consensus sequences of clones.…”

Section: Discussionmentioning

confidence: 99%

Reconstruction of phylogenetic relationships in a highly reticulate group with deep coalescence and recent speciation (Hieracium, Asteraceae)

et al. 2012

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Phylogeny reconstruction based on multiple unlinked markers is often hampered by incongruent gene trees, especially in closely related species complexes with high degrees of hybridization and polyploidy. To investigate the particular strengths and limitations of chloroplast DNA (cpDNA), low-copy nuclear and multicopy nuclear markers for elucidating the evolutionary history of such groups, we focus on Hieracium s.str., a predominantly apomictic genus combining the above-mentioned features. Sequences of the trnV-ndhC and trnT-trnL intergenic spacers were combined for phylogenetic analyses of cpDNA. Part of the highly variable gene for squalene synthase (sqs) was applied as a low-copy nuclear marker. Both gene trees were compared with previous results based on the multicopy external transcribed spacer (ETS) of the nuclear ribosomal DNA. The power of the different markers to detect hybridization varied, but they largely agreed on particular hybrid and allopolyploid origins. The same crown groups of species were recognizable in each dataset, but basal relationships were strongly incongruent among cpDNA, sqs and ETS trees. The ETS tree was considered as the best approximation of the species tree. Both cpDNA and sqs trees showed basal polytomies as well as merging or splitting of species groups of non-hybrid taxa. These patterns can be best explained by a rapid diversification of the genus with ancestral polymorphism and incomplete lineage sorting. A hypothetical scenario of Hieracium speciation based on all available (including non-molecular) evidence is depicted. Incorporation of seemingly contradictory information helped to better understand species origins and evolutionary patterns in this notoriously difficult agamic complex.

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Section: Discussionmentioning

confidence: 99%

Reconstruction of phylogenetic relationships in a highly reticulate group with deep coalescence and recent speciation (Hieracium, Asteraceae)

et al. 2012

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“…If these repetitive procedures confirmed the double signals in the reads, PCR products were cloned following the same protocol a s u s e d f o r t h e s e p a r a t i o n o f t h e n a t i v e a n d contaminating DNA fragments. The resulting clones were checked for errors, e.g., as those reported by Speksnijder et al (2001) and Acinas et al (2005), against the alignment of the approved direct sequences. Clones with unique nucleotides or gaps in the conservative sites were disregarded.…”

Section: Taxonomic Scopementioning

confidence: 99%

Molecular phylogenies challenge the classification of Polymastiidae (Porifera, Demospongiae) based on morphology

et al. 2016

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Polymastiidae Gray, 1867 is a worldwide distributed sponge family, which has a great significance for understanding of the demosponge deep phylogeny since the former order Hadromerida Topsent, 1894 has been recently split based on the molecular evidence and a new separate order has been established for the polymastiids. However, molecular data obtained from Polymastiidae so far are scarce, while the phylogenetic reconstruction based on morphology has faced a deficit of characters along with the vagueness of their states. The present study is a phylogenetic reconstruction of Polymastiidae based on novel data on two molecular markers, cytochrome oxidase subunit I and large subunit ribosomal DNA, obtained from a broad set of species. Monophyly of the family and nonmonophyly of four polymastiid genera are revealed, suggesting a high level of homoplasy of morphological characters, which are therefore not an appropriate base for the natural classification of Polymastiidae. Although the presented phylogenies cannot yet provide an alternative classification scheme, several strongly supported clades, which may be used as reference points in future classification, are recovered and three taxonomic actions are proposed: transfer of one species from Radiella to Polymastia Bowerbank, 1862; transfer of three species from Radiella Schmidt, 1870 to Spinularia Gray, 1867; and the consequent abandonment of Radiella.

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“…Several biases which may cause pitfalls in the interpretation of the DGGE patterns have been reported, like heteroduplex formation, amplification errors, the presence of multiple copies of 16S rRNA operons (Speksnijder et al, 2001;Wintzingerode et al, 1997), co-migration of different DNA fragments (Sekiguchi et al, 2001) and the presence of several melting domains in a DNA fragment (Kisand and Wikner, 2003). Nevertheless, some of these problems can be solved by the excision of the bands followed by reamplification and sequencing, as the identity of the organisms can be ascertained if the DNA fragment analysed is polymorphic.…”

Section: Introductionmentioning

confidence: 99%

Testing of primers for the study of cyanobacterial molecular diversity by DGGE

Boutte

Grubisic

Balthasart

et al. 2006

Journal of Microbiological Methods

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Denaturing Gradient Gel electrophoresis (DGGE) is a PCR-based technique which is widely used in the study of microbial communities. Here, the use of the three specific 16S rRNA cyanobacterial specific primers CYA359F, CYA781R(a) and CYA781R(b) on the assessment of the molecular diversity of cyanobacterial communities is examined. Assignments of the reverse primers CYA781R(a) and CYA781R(b) with cyanobacterial strain sequences showed that the former preferentially targets filamentous cyanobacteria whereas the latter targets unicellular cyanobacteria. The influence of the GC clamp position on the forward or on reverse primer and the use of the two reverse primers separately or in equimolar mixture were investigated. Three environmental samples were subjected to amplification with 6 combinations of primers. The 6 banding patterns as well as the sequences of the bands extracted were analysed and compared. In addition, to assess the effect of the position of the GC clamp, the melting profiles of the sequences of Aphanizomenon flos-aquae PMC9707 and Synechococcus sp. MH305 were determined, with the GC clamp in the 3V or 5V position. Results showed that the use of two separate amplifications allowed a more complete study of the molecular diversity of the cyanobacterial community investigated. Furthermore, similar richness and identical phylogenetic assignments of extracted bands were obtained irrespective of the positioning of the GC clamp. D

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Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences

Cited by 209 publications

References 28 publications

Reconstruction of phylogenetic relationships in a highly reticulate group with deep coalescence and recent speciation (Hieracium, Asteraceae)

Reconstruction of phylogenetic relationships in a highly reticulate group with deep coalescence and recent speciation (Hieracium, Asteraceae)

Molecular phylogenies challenge the classification of Polymastiidae (Porifera, Demospongiae) based on morphology

Testing of primers for the study of cyanobacterial molecular diversity by DGGE

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