Simultaneous pyrosequencing of the 16S rRNA, IncP-1 trfA, and merA genes

• Amplicons pyrosequencing of three genes of different length
• Proposal for new linker sequences for tailed PCR library construction
• Determination clustering level of IncP-1 trfA sequences
• Amplicon pyrosequencing of IncP-1 trfA gene and cluster analysis of these
• Amplicon pyrosequencing of the merA gene

The use of amplicon pyrosequencing makes it possible to produce thousands of sequences of the same gene at relatively low costs. Here we show that it is possible to simultaneously sequence the 16S rRNA gene, IncP-1 trfA gene and mercury reductase gene (merA) as a way for screening the diversity of several genes in the same samples. As a proof-of-concept two different soil samples and a wastewater sample were screened. Multiplexing identifiers (MIDs) and sequencing adapters were added to amplicons using a tailed PCR approach and the universal overhangs U1 and U2 for this approach were redesigned. Furthermore, this is the first time the IncP-1 plasmid diversity was studied by amplicon pyrosequencing and for this purpose a clustering threshold of 89% nucleotide sequence similarity was determined to differentiate the IncP-1 subgroups.