16S rRNA gene sequence anomalies

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Anomalous 16S rDNA

In this context, an anomalous, or artifactual 16S rDNA sequence, is one which differs from the original DNA template from which it was generated, as a result of some methodological error. Anomalous sequences can by classified as follows:

PCR amplification errors

Sequencing errors
Poor sequence assembly

Construction of sequence from non-contiguous fragments
Incorrect ordering of fragments
Incorrect orientation of fragments

Programs such as Pintail and Mallard attempt to recognise and characterise anomalous sequences by comparative analysis with error free reliable sequences, by means of the the Pintail algorithm.

The Pintail algorithm works on the assumption that if two error-free sequences are aligned together, the evolutionary distance between the two, as measured by the number of base mismatches at homologous positions, will be roughly the same along the length of the alignment, having allowed for the pattern of hypervariability inherent in the 16S rRNA gene.

Normally this is reasonable assumption to make. However there are a small, and as yet unquantified, number of 16S rRNA genes in nature that fail to show this uniformity, and have all the appearance of being chimeras. They are, in fact, the consequence of natural recombination events, possibly as a result of horizontal gene transfer, and are normally recognised when comparisons are made between 16S rDNA sequences derived from the same genome.