A 16S rRNA gene sequencing and analysis protocol for the Illumina MiniSeq platform

Abstract

High-throughput sequencing of the 16S rRNA gene on the Illumina platform is commonly used to assess microbial diversity in environmental samples. The MiniSeq, Illumina's latest benchtop sequencer, enables more cost-efficient DNA sequencing relative to larger Illumina sequencing platforms (e.g., MiSeq). Here we used a modified custom primer sequencing approach to test the fidelity of the MiniSeq for high-throughput sequencing of the V4 hypervariable region of 16S rRNA genes from complex communities in environmental samples. To this end, we designed additional sequencing primers that enabled application of a dual-index barcoding method on the MiniSeq. A mock community was sequenced alongside the environmental samples in four different sequencing runs as a quality control benchmark. We were able to recapture a realistic richness of the mock community in all sequencing runs, and identify meaningful differences in alpha and beta diversity in the environmental samples. Furthermore, rarefaction analysis indicated diversity in many environmental samples was close to saturation. These results show that the MiniSeq can produce similar quantities of high-quality V4 reads compared to the MiSeq, yet is a cost-effective option for any laboratory interested in performing high-throughput 16S rRNA gene sequencing.

Keywords: 16S rRNA gene; Illumina; high-throughput sequencing; microbial diversity.

Figure 1

Schematic description of the dual‐index sequencing strategy on the MiniSeq. Reading the figure from top to bottom shows the sequential order of paired‐end sequencing steps (four total). “Turn around” indicates the step of paired‐end turn around on the flow cell surface. The sequencing proceeds in the direction of the flow cell surface, which in this figure is located on the right side (arrows point in direction of sequencing reaction). Sequencing starts by using Read 1 primer to sequence Read 1, followed by Index 1 primer to generate Index 1. The MiniSeq only uses the oligonucleotides on the flow cell for bridging and both the second index and the paired read are sequenced after the clusters are turned around. Hence an Index 2 primer is needed to sequence Index 2. Read 2 is then sequenced by using the Read 2 primer (after Kozich et al., 2013). Sequencing primers for only the forward primer 515F‐Y (Parada et al., 2016) are shown, for sequencing primers needed for the 515F primer (Caporaso et al., 2012) please see Table 1 for all sequencing primers

Figure 2

OTU assessment for the mock community composed of 18 defined species. UPARSE generated an accurate estimate of the microbial community in all performed 16S rRNA sequencing runs, given the low number of spurious OTUs

Figure 3

Nonmetric multidimensional scaling analysis showing microbial beta diversity of the 16S data sets. (1) mock community replicates, (2) pond sediments (Niederlibbach, Germany), (3) saltmarsh sediments (Cape Cod, MA), (4) salt water aquaria, (5) marine sponge, (6) sandy beach sediments (Obidos lagoon), (7) microbial mats (Obidos lagoon, Portugal), (8) salt marsh sediments (Pt Judith, RI), (9) pond sediments (Niederlibbach, Germany), and (10) carbonate biofilms (Liguria Springs, Italy)