Abstract
High-throughput amplicon sequencing (HTAS) of conserved DNA regions is a powerful technique to characterize microbial communities. Recently, spike-in mock communities have been used to measure accuracy of sequencing platforms and data analysis pipelines. To assess the ability of sequencing platforms and data processing pipelines using fungal internal transcribed spacer (ITS) amplicons, we created two ITS spike-in control mock communities composed of cloned DNA in plasmids: a biological mock community, consisting of ITS sequences from fungal taxa, and a synthetic mock community (SynMock), consisting of non-biological ITS-like sequences. Using these spike-in controls we show that: (1) a non-biological synthetic control (e.g., SynMock) is the best solution for parameterizing bioinformatics pipelines, (2) pre-clustering steps for variable length amplicons are critically important, (3) a major source of bias is attributed to the initial polymerase chain reaction (PCR) and thus HTAS read abundances are typically not representative of starting values. We developed AMPtk, a versatile software solution equipped to deal with variable length amplicons and quality filter HTAS data based on spike-in controls. While we describe herein a non-biological SynMock community for ITS sequences, the concept and AMPtk software can be widely applied to any HTAS dataset to improve data quality.
Highlights
High-throughput amplicon sequencing (HTAS) is a powerful tool that is frequently used for examining community composition of environmental samples
Given the small percentage of ITS1 and ITS2 regions that are greater than 450 bp, the number of taxa in the reference database that are unlikely to sequence on the Ion Torrent due to amplicon length is relatively small (Table 1)
We propose that HTAS studies of fungal internal transcribed spacer (ITS) communities can be improved by employing synthetic mock community (SynMock) or a similar non-biological community as a technical control
Summary
High-throughput amplicon sequencing (HTAS) is a powerful tool that is frequently used for examining community composition of environmental samples. HTAS has proven to be a robust and cost-effective solution due to the ability to multiplex hundreds of samples on a single next-generation sequencing (NGS) run. HTAS output from environmental samples requires careful interpretation and appropriate and consistent use of positive and negative controls (Nguyen et al, 2015). One of the major challenges in HTAS is to differentiate sequencing error versus real biological sequence variation. How to cite this article Palmer et al (2018), Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data.
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