Barcoded primers used in multiplex amplicon pyrosequencing bias amplification

Abstract

"Barcode-tagged" PCR primers used for multiplex amplicon sequencing generate a thus-far-overlooked amplification bias that produces variable terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing data from the same environmental DNA template. We propose a simple two-step PCR approach that increases reproducibility and consistently recovers higher genetic diversity in pyrosequencing libraries.

Fig. 1.

Barcoded pyrosequencing primers affect reproducibility of community profiles obtained via T-RFLP (A and B) or 454 sequencing (C and D). All T-RFLP experiments were performed in triplicate. (A and B) Average pairwise Euclidean distances of T-RFLP profiles are shown, as measured by T-RF relative abundances (A) and T-RF presence/absence (B). From left to right, the bars show comparisons made using T-RFLP replicates obtained from application of a single barcoded primer for bcPCR using DNA from a single extraction, T-RFLP replicates obtained from application of a single barcoded primer for bcPCR using DNA extracted separately for each replicate from the same homogenized gut sample, and T-RFLP profiles obtained after amplification of DNA from a single extraction using a mixture of 11 randomly chosen barcoded PCR primers with either 1-step or 2-step bcPCR. (C and D) Average pairwise community similarities from 454 sequencing libraries prepared from the same DNA extraction (also DNA from the mouse gut lumen, but different extraction than that used for panels A and B) using 16 barcoded primers with either 1-step or 2-step bcPCR are compared. Bray-Curtis (C) and unweighted UniFrac (D) distances are shown. Error bars indicate standard deviations, and asterisks indicate statistical significance at P values of <0.05 (*) and <0.001 (***).

Fig. 2.

The bcPCR method affects alpha diversity and reproducibility of taxonomic classification. (A) Comparison of 11 randomly selected barcoded primers (6 used for both bcPCR methods). Relative abundance for each taxon (family level) present on average at ≥1% is plotted on a heatmap for each barcode used. Average relative abundance and relative standard deviation are listed for each method and taxon. (B) A box plot of the paired difference for several alpha diversity metrics for operational taxonomic units (OTUs) is shown for 1-step and 2-step bcPCR using an identical set of 6 randomly selected barcoded primers (for details about metrics, see the supplemental material). The dashed line indicates a difference of zero.