Getting started with microbiome analysis: sample acquisition to bioinformatics

Abstract

Historically, in order to study microbes, it was necessary to grow them in the laboratory. It was clear though that many microbe communities were refractory to study because none of the members could be grown outside of their native habitat. The development of culture-independent methods to study microbiota using high-throughput sequencing of the 16S ribosomal RNA gene variable regions present in all prokaryotic organisms has provided new opportunities to investigate complex microbial communities. In this unit, the process for a microbiome analysis is described. Many of the components required for this process may already exist. A pipeline is described for acquisition of samples from different sites on the human body, isolation of microbial DNA, and DNA sequencing using the Illumina MiSeq sequencing platform. Finally, a new analytical workflow for basic bioinformatics data analysis, QWRAP, is described, which can be used by clinical and basic science investigators.

Keywords: 16S rRNA genes; QWRAP bioinformatics analysis; microbe communities; microbiome analysis pipeline.

Figure 1

The 16 S, 23S and 5S rRNA gene. The expanded 16S gene depicts the variable regions. The target V4 region for microbiome analysis is depicted in black.

Figure 2

V4 region and 5′ and 3′ PCR primers. The target nucleotides for the PCR primers are depicted in grey (5′region) and black (3′region). The 5′ and 3′ primers include adaptor Illumina sequences for NexGen sequences and “pad “sequences to facilitate PCR amplification. The 3′ primer also contains 6 base index primers (see Figure 3).

Figure 3

Listing of 96 6 base pair index sequences. For location of the 6 base pair sequences see Figure 2.

Figure 4

Sequencing primers for 16S PCR product. The 5′ and 3′ sequencing primers used for MiSeq sequencing are depicted. The “index” sequencing primer is also depicted.

Figure 5

The QWRAP workflow for data analysis.

Figure 6

Sample Microbiome Analysis Report from QWRAP.

Figure 7

Sample Information Form. A template form given to patients for donation of sample for microbiome analysis.

Figure 8

Bar graph depicting bacterial taxa at the phylum level. The eight samples (A1, A2, B1, B2, C1, C2 and D1, D2) are shown. The figure shows a taxa summary chart showing distribution of top ten bacterial taxa (at phylum level) for 8 human fecal samples.

Figure 9

Heat map. The four samples (A1, B1, C1 and D1) are shown. The darker color in the map represents greater abundance of the microbe. On the right the microbe at the phylum level is identified.

Figure 10

Rarefaction curve for 8 fecal samples using Shannon’s diversity. The samples with the highest Shannon values corresponding to samples with most diversity.

Figure 11

Principal Coordinate Analysis plot. The eight samples (A1, A2, B1, B2 etc.) are presented in the PCoA analysis; note that the sample designated as A consists of A1 and A2.