Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II

Karen W Hughes,Samuel D Morris,Ana Reboredo Segovia

doi:10.3897/mycokeys.10.5136

Abstract

Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II

Highlights

There has been speculation that chimeras may be the result of incomplete extension of PCR products which subsequently act as primers for the amplification cycle and, there seems to be a reduction in chimeric PCR products when extension times are increased (Meyerhans et al 1990; Qiu et al 2001a; Smyth et al 2010; Thompson et al 2002) or by optomizing the PCR protocol (Qiu et al 2001b; Wang and Wang 1997)
The possibility of identical chimeras occurring in GenBank and being interpreted as valid taxa was noted by Nilsson et al (2012)
We investigated the possibility that secondary structure formation during the PCR process might lead to non-random chimera formation, perhaps by briefly stalling taq polymerase transcription at the point of secondary folding and allowing template switching

Summary

Introduction

Sequence chimeras are common when pooled DNA s are co-amplified by a PCR process (Edgar et al 2011; Judo et al 1998; Jumpponen 2007; Meyerhans et al 1990; Odelberg et al 1995; Qiu et al 2001a; Smyth et al 2010; Tedersoo et al 2014; Wang and Wang 1997). Odelberg et al (1995) demonstrated that chimeras can be generated in a single round of PCR amplification in the absence of heat denaturation and re-annealing which suggests that some polymerase template switching may occur. Fonseca et al (2012) demonstrated that nuclear SSU chimeras were produced at high levels during the PCR-process when mixed templates were present. Their results with a nematode population demonstrated that chimera formation is higher in species-diverse PCR pools than in pools that are genetically less diverse but that the breakpoints were in regions of sequence similarity

Methods

Results

Conclusion