AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities

Abstract

High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches (“AmpliconDuo filter”). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.

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“…Briefly, in order to cover a broad taxonomic spectrum, two primers with different wobble positions were combined in a ratio of 10% : 90%: 5'-GCTGCGCCCTTCATCGKTG-3' (ITS2_Dino; 10%) and 5'-GCTGCGTTCTTCATC-GWTR-3' (ITS2_broad; 90%). We used the Amplicon-Duo pipeline (Lange et al 2015) to separate true biological variation from sequence artefacts. For each sample, two technical replicates of the extracted DNA were independently amplified using primers with different sample identifiers [see Lange et al 2015 for details].…”

“…We used the Amplicon-Duo pipeline (Lange et al 2015) to separate true biological variation from sequence artefacts. For each sample, two technical replicates of the extracted DNA were independently amplified using primers with different sample identifiers [see Lange et al 2015 for details]. The concentrations of the PCR reaction were as follows: 1 μl of DNA template (depending on the concentration, different dilutions of 1:1, 1:10, and 1:100 were used) in 25 μl PCR reaction with 0.4 units of Phusion Taq (Biozym), 0.25 μM primers, 0.4 mM dNTPs and 1 x Phusion buffer (Biozym).…”

“…Base quality of the sequence reads was checked using the FastQC software (Andrews 2015). A split-sample filtering protocol for Illumina amplicon sequencing was used as described in Lange et al (Lange et al 2015). In brief, the raw sequences were quality filtered to remove reads with an average Phred quality score below 25 using PRINSEQ-lite [v0.20.4, (Schmieder and Edwards 2011)].…”

“…, (Edgar et al 2011] with default parameters. Finally, sequences that were not found in both sample branches using AmpliconDuo (Lange et al 2015) were discarded.…”

“…We applied the AmpliconDuo filter (Lange et al 2015) for separating true biological diversity from PCR and sequencing artefacts. This novel cleaning step based on technical replication effectively discarded sequence artefacts, resulting in a total number of 100,516,809 reads.…”