Minimization of chloroplast contamination in 16S rRNA gene pyrosequencing of insect herbivore bacterial communities.

ABSTRACT: Chloroplast sequence contamination in 16S ribosomal RNA gene (16S) analyses can be particularly problematic when sampling microbial communities in plants and folivorous arthropods. We previously encountered high levels of plastid contamination in herbivorous insect samples when we used the predominant 454 pyrosequencing 16S methodologies described in the literature. 799F, a primer previously found to exclude chloroplast sequences, was modified to enhance its efficacy, and we describe, in detail, our methodology throughout amplicon pyrosequencing. Thirteen versions of 799F were assessed for the exclusion of chloroplast sequences from our samples. We found that a shift in the mismatch between 799F and chloroplast 16S resulted in significant reduction of chloroplast reads. Our results also in

SUBMITTER: Hanshew AS 

PROVIDER: S-EPMC4133986 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature