Abstract
BackgroundAdvancements in Next Generation Sequencing (NGS) technologies regarding throughput, read length and accuracy had a major impact on microbiome research by significantly improving 16S rRNA amplicon sequencing. As rapid improvements in sequencing platforms and new data analysis pipelines are introduced, it is essential to evaluate their capabilities in specific applications. The aim of this study was to assess whether the same project-specific biological conclusions regarding microbiome composition could be reached using different sequencing platforms and bioinformatics pipelines.ResultsChicken cecum microbiome was analyzed by 16S rRNA amplicon sequencing using Illumina MiSeq, Ion Torrent PGM, and Roche 454 GS FLX Titanium platforms, with standard and modified protocols for library preparation. We labeled the bioinformatics pipelines included in our analysis QIIME1 and QIIME2 (de novo OTU picking [not to be confused with QIIME version 2 commonly referred to as QIIME2]), QIIME3 and QIIME4 (open reference OTU picking), UPARSE1 and UPARSE2 (each pair differs only in the use of chimera depletion methods), and DADA2 (for Illumina data only). GS FLX+ yielded the longest reads and highest quality scores, while MiSeq generated the largest number of reads after quality filtering. Declines in quality scores were observed starting at bases 150–199 for GS FLX+ and bases 90–99 for MiSeq. Scores were stable for PGM-generated data. Overall microbiome compositional profiles were comparable between platforms; however, average relative abundance of specific taxa varied depending on sequencing platform, library preparation method, and bioinformatics analysis. Specifically, QIIME with de novo OTU picking yielded the highest number of unique species and alpha diversity was reduced with UPARSE and DADA2 compared to QIIME.ConclusionsThe three platforms compared in this study were capable of discriminating samples by treatment, despite differences in diversity and abundance, leading to similar biological conclusions. Our results demonstrate that while there were differences in depth of coverage and phylogenetic diversity, all workflows revealed comparable treatment effects on microbial diversity. To increase reproducibility and reliability and to retain consistency between similar studies, it is important to consider the impact on data quality and relative abundance of taxa when selecting NGS platforms and analysis tools for microbiome studies.
Highlights
Advancements in Generation Sequencing (NGS) technologies regarding throughput, read length and accuracy had a major impact on microbiome research by significantly improving 16S rRNA amplicon sequencing
We have previously shown that prebiotics have a modulatory effect on the gut microbiome [13, 39], not by dramatically altering its composition but by impacting very specific bacterial groups
The different bioinformatics pipelines were named as follows; QIIME1 refers to the pipeline based on the de novo operational taxonomic units (OTU) picking without chimera checking, QIIME2, the de novo OTU picking pipeline with chimera detection, QIIME3 refers to the open reference OTU picking pipeline without chimera checking, and QIIME4 the open reference OTU picking pipeline with chimera detection
Summary
Advancements in Generation Sequencing (NGS) technologies regarding throughput, read length and accuracy had a major impact on microbiome research by significantly improving 16S rRNA amplicon sequencing. While bioinformatics tools like removal of chimeric sequences can reduce some of the intrinsic errors of sequencing data, it is challenging to eliminate the bias introduced by primer design [22], library preparation [23], DNA isolation methods [24], and PCR amplification artifacts, each of which introduce unique biases that can result in over or underrepresentation of individual microbes within complex communities [25]. These biases are unavoidable and rarely impact the overall merit of a study
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