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 indicate that amplifying sequences from environmental samples in a two-step PCR process, with the addition of the multiplex identifiers and 454 adapters in a second round of PCR, further improved primer specificity. Primers that included 3' phosphorothioate bonds, which were designed to block primer degradation, did not amplify consistently across samples. The different forward primers do not appear to bias the bacterial communities detected. We provide a methodological framework for reducing chloroplast reads in high-throughput sequencing data sets that can be applied to a number of environmental samples and sequencing techniques.

Keywords: 16S rRNA Gene; 454 Pyrosequencing; Chloroplast; Insect; Symbiosis.

Fig 1

Relative abundances of sequence reads at the bacterial order taxonomic classification sequenced with the tested forward primers.

Fig 2

MDS plot illustrating the differences between the bacterial communities in two arthropod samples sequenced with 8 primer pairs. Pairwise community distances determined using the unweighted Unifrac algorithm (A) and the weighted Unifrac algorithm (B). These two panels show that while there may be more bacterial taxa detected with different primers (A), when abundance is taken into account, the communities in each respective insect are nearly indistinguishable (B).