Methodology challenges in studying human gut microbiota – comparison of illumina and Ion Torrent NGS platforms

Hana Čipčić Paljetak1, Anja Barešić 2, Mihaela Perić 1, Marina Panek 1, Mario Matijašić 1, Darija Vranešić Bender 3, Ana Kunović 3, Željko Krznarić 3, Donatella Verbanac1

1 University of Zagreb School of Medicine, Zagreb, Croatia; 2 Imperial College London, London, UK; 3 University Hospital Centre Zagreb, Croatia hana.paljetak@mef.hr

INTRODUCTION

The human gut contains dense and diverse microbial communities which have profound impact on human health. To understand microbiota complexity, reliable technological solutions for determination of bacterial community structure are needed. The quality of final output is influenced by the methodologies along the pipeline – sample collection, storage, DNA extraction and sequencing approach [1]. As number of microbiota studies increases continually, it is important to ensure comparability and meaningful data interpretation both within and between studies. Therefore, efforts aiming at comparing different workflows are useful for much needed methodology standardisation.

REFERENCES

[1] Salipante et al. Appl. Environ. Microbiol. 2014, 80 (24), 7583-7591. [2] Caporaso et al. Nat. Methods 2010, 7 (5), 335-336. [3] DeSantis et al. Applied and Environmental Microbiology 2006, 72 (7), 5069-5072. [4] Perić et al. Evaluation of human faecal microbiota content by 16S rRNA analysis using different collection, storage and DNA extraction methods – OMNIgene.GUT case study. 3rd World Congress on Targeting Microbiota, Paris, France, Oct 21-23, 2015.

This work has been fully supported by the Croatian Science Foundation (project number 5467)

AIM

The objective of this study was to compare results obtained by 16S rRNA gene sequencing of human faeces samples on two NGS platforms – illumina MiSeq and Ion Torrent PGM.

MATERIALS & METHODS

Samples from 4 healthy donors were collected fresh and in OMNIgene.GUT device, then stored for 14 days at -20°C and at room temperature, respectively. Three commercially available DNA extraction kits (MO BIO Power faecal DNA isolation kit, QIAamp Fast DNA Stool Mini Kit and MP Biomedicals Fast DNA spin kit for feces) were employed. In total, six samples per donor were prepared. After extraction DNA samples integrity was tested on 1% agarose gel electrophoresis while purity and concentration were verified using Qubit fluorometer.

Faecal bacterial communities were profiled by 16S rRNA amplicon sequencing using two NGS platforms MiSeq (illumina MiSeq, regions V3-V4) and IT (Ion Torrent PGM, regions V2-V9). Manufacturer recommended reagents and protocols were applied: for MiSeq Nextera XT Index Kit, MiSeq Reagent Kit v3 and pair end sequencing protocol and for IT Ion Hi-Q Sequencing kit and 4 316 chip.

Sequence analyses were performed using QIIME protocol [2]: OTU assignment procedure for demultiplexed and trimmed reads was performed against the Greengenes reference database version 13_8 (May 2013) [3] by the uclust algorithm.

RESULTS

In our previous work, we have shown that DNA yield and quality varied between DNA extraction kits (MP Biomedicals>QIAGEN>MO BIO) [4].

Here we compare MiSeq and Ion Torrent NGS platforms at the level of overall discrimination at different taxonomic levels, as well as in terms of correlation with other experimental parameters.

Donor-specific patterns of bacterial diversity were maintained irrespective of the collection, storage or DNA extraction method used (principal component analysis (PCA), Figure 1). Platform-based differentiation could be observed (PC2 vs PC3). Kit-based trends are indicated as well and the main differentiation seems to occur between MP Biomedicals and MO BIO kits. Variability due to collection and storage methodology was not observed in PCA (not shown).

The number of families detected in the entire sample pool, as presented on Venn diagram, was 118 and 130 for MiSeq and Ion Torrent, respectively, while the number of overlapping families was 86 (Figure 2A). When families detected only once per platform were removed, the number of overlapping families was 74 (MiSeq detected 109 and IT 92 families) (Figure 2B). The total number of families per sample detected on both platforms is shown in Figure 2C.

To check if distinct relative abundance patterns due to sequencing on different platforms can be observed, relative abundance of the overlapping families within the entire sample pool detected by each NGS platform was compared, in order to exclude variances due to other experimental parameters. Different levels of abundance were observed depending on the platform, especially among the less abundant families (Figure 3). However, comparison at the phylum level reveals the ten most abundant phyla, covering 99,9% of the assigned OTUs, occurred in similar ratios on both platforms, with the exception of Fusobacteria and Verrucomicrobia (Figure 4). These phyla were represented in higher abundance in the samples sequenced on MiSeq.

CONCLUSION

The results reveal differentiation between NGS platforms as indicated by PCA analysis. However, donor specific microbiota composition is maintained irrespective of the platform, DNA extraction kit, collection and storage methodology. Although comparable numbers of families are identified across platforms, the families identified are not fully overlapping. The relative abundance ratio for the most abundant phyla was comparable across platforms, with the exception of Fusobacteria and Verrucomicrobia.

Figure 2. A) Venn diagram of number of families detected using MiSeq and Ion Torrent NGS platforms. B) Number of families detected when those detected only once per platform are removed. C) Number of families detected on each platform per sample, coloured by donor.

C

Figure 3: Relative abundance ratio of overlapping families detected on both NGS platforms.

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Figure 4: Relative abundance ratio of overlapping phyla detected on both NGS platforms.

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Figure 1: Principle component analysis (PCA) of families detected. MiSeq samples are designated by dots and Ion Torrent by triangles. A) Coloured by donor B) Coloured by DNA extraction kit. First four components cover 99,88 % of total variance.

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