Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

Summary

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

Abstract

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.

Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.

Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

Introduction

Concentrated efforts have been made to better understand microbiome diversity and abundance, as another aspect of capturing difference and similarities between individuals in healthy and pathological conditions. Age^2,3, geography⁴, lifestyle^5,6, and illness⁵ were shown to be associated with the composition of the gut microbiome, but many conditions and populations have not yet been fully characterized. Recently it has been reported that the microbiome can be modified for therapeutic applications^7,8,9. Therefore, additional insight into the relationship between various physiological conditions and the microbial composition is the first step toward optimization of potential future modifications.

The traditional microbial culture methods are limited by low yields^10,11, and are conceptualized as a binary state where a bacteria is either present in the gut or not. High-throughput DNA-based sequencing has revolutionized microbial ecology, enabling the capture of all members of the microbial community. However, sequence read length and quality remain significant barriers to accurate taxonomy assignment¹². Furthermore, high-throughput based experiments may suffer from batch effects, where measurements are affected by non-biological or non-scientific variables¹³. In recent years, several programs have been established to study the human microbiome, including the American Gut project, the United States (US) Human Microbiome Project, and the United Kingdom (UK) MetaHIT project. These initiatives have generated vast amounts of data that are not easily comparable due to a lack of consistency in their approaches. A variety of international projects such as the International Human Microbiome Consortium, the International Human Microbiome Standards project, and the National Institute of Standards and Technology (NIST) attempted to address some of these issues¹⁴, and developed standards for microbiome measurements which should enable the achievement of reliable reproductive results. Described here is an integrated protocol of several broadly used methods^15,16 for 16S rRNA high-throughput sequencing (16S-seq) starting from fecal sample collection thru data analyses. The protocol describes a column-free PCR approach, originally designed for direct extraction of plant DNA¹⁶, to enable the simultaneous handling of large numbers of fecal samples in a relatively short time with high quality amplified DNA for targeted sequencing of the microbial variable V4 region on a common sequencing platform. This protocol aims to guide scientists interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way, using important controls. Having a guided and detailed step-by step protocol may minimize batch effect and thus will allow more comparable sequencing results between labs.

Protocol

Ethical approval for the study was granted by the Sheba Local Research Ethics Committee and all methods were performed in accordance with the relevant guidelines and regulations. The protocol received a patient consent exception from the local Ethical Review Board, since the fecal material that were used were already submitted to the microbiology core as part of clinical workup and without identifiable patient information other than age, gender, and microbial results. Written, informed consent was obtained from healthy volunteers and the Institutional Review Board approved the study. Some of those samples have already been included in a previous analysis¹.

1. Sample Handling

1. Collect an approximately 5 mm² smear (roughly the size of a pencil eraser) from a fresh fecal sample with a sterile swab in a test tube (see Table of Materials). Store all swabs containing fecal samples at -80 °C within 24 h. The fecal swabs can remain there until further processing.

2. DNA Extraction

1. Thaw the Extraction and Dilution solutions at room temperature (see Table of Materials).
2. Transfer the fecal swab into an empty 2 mL collection tube (see Table of Materials). Adjust the swab stick size by cutting it using a clean scissors to enable tube closure with minimum cross-contamination. Add 250 μL of Extraction solution to each collection tube containing the fecal swab and vortex to mix.
3. Heat the samples for 10 min in a boiling water bath (95–100 °C). Add 250 μL of Dilution solution to each sample and vortex to mix.
4. Store the 2 mL tubes containing the extracted DNA and the swab at 4 °C.

3. PCR and Library Preparation

For steps 3.1 and 3.2, work in a PCR workstation that provides clean, template and amplicon free environment.

1. Label the primers (Table 1) according to their barcode. Dilute each primer in double distilled water (DDW) to a 50 μM concentration and store at -20 °C.
2. Use a 96-well plate for PCR reactions. Each plate can contain 32 different samples, which are tagged by 32 different index primers. Thaw the forward primer and the 32 reverse primers at room temperature and dilute them to 5 μM.
3. Prepare PCR reaction mixes for 100 reactions (final volume in each well will be 20 μl) by mixing 100 μL of 5 μM Forward primer, 1 mL 2X PCR Master mix, and 400 μL DDW. Put 15 μL of this PCR mix in each well (total of 96 wells). Add 1 μL of each 5 μM Reverse indexed primer to 3 different wells (32 different primers in triplicates gives a total of 96 wells).
4. In a pre-PCR dedicated zone, meaning a clean bench that is template and amplicon free, add 4 μL of each extracted DNA sample to reaction mixtures (each extracted DNA sample is amplified in triplicate — 32 samples per 96-well plate).
5. Run PCR with the following settings: initial denaturation of 94 °C for 3 min; followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min; and a final extension at 72 °C for 10 min.
6. On a different bench, which will be defined as a post-PCR dedicated zone, combine each triplicate PCR reaction into a single volume tube (60 μL per sample).
7. To assess the quality of PCR amplicons, run 4 μL of each combined-PCR reaction on an ethidium bromide-stained 1% agarose gel. Under UV wavelength of 260 nm, the positive amplicons will appear in an expected band size of 375–425 bp. Only these amplicons will be included in the subsequent steps.
   NOTE: Each sample is amplified in triplicate, meaning that each sample is amplified in 3 different PCR reactions. Do not scale up.

4. Library Quantification and Cleaning

1. In order to get an equimolar concentration pool of all PCR samples, quantify each amplicon by a double stranded DNA (dsDNA quantify reagent) florescent nucleic acid stain (see Table of Materials), suitable for the simultaneous quantification of a large amount of samples.
   NOTE: Since the size range of the amplicon is equivocal, the actual amount in nanograms is used to load an equimolar concentration.
2. Combine 500 ng of each sample into a single, sterile tube. Vortex to mix.
3. Run 200 μL of the pooled library on an Ethidium Bromide-stained 1% agarose gel. Extract the 375–425 bp bands from the gel using a gel extraction kit according to the manufacturer's instructions (see Table of Materials) and elute the pooled library in 80 μL DDW.
   NOTE: The pooled library should be size selected to reduce non-specific amplification products from host DNA.
4. Measure the final library concentration using a highly sensitive dsDNA detecting kit according to the manufacturer's instructions (see Table of Materials). Measure the accurate library size using a highly sensitive separation and analysis kit for DNA libraries according to the manufacturer's instructions (see Table of Materials).

5. Sequencing

1. Dilute the pooled library to 7 pM with addition of 20% control library (see Table of Materials), according to the sequencing machine protocol.
2. For sequencing, use custom designed read primers that are complimentary to the V4 amplification primers (see Table 1).
3. Use a third custom designed sequencing primer that reads the barcode in an additional cycle (see Table 1) and generates paired-end reads of 175 bases in length in each direction, according to the manufacturer's specifications.
4. Run the sequencing machine and obtain FASTQ files according to the manufacturer's protocol.

6. Data Processing

1. Stitch together and process the overlapping paired-end FASTQ files in a data curation pipeline implemented in QIIME 2 version 2017.7.0¹⁷. Demultiplex the reads according to sample specific barcodes.
2. Use DADA2¹⁸ for quality control and sequence variant (SVs) detection. Truncate reads at 13 bases from the 3' end and 15 bases from the 5' end, discard reads with more than 2 expected errors. Identify and remove the chimeras using the consensus method — chimeras are detected in samples individually, and sequences found chimeric in a sufficient fraction of samples are removed.
3. Perform SVs taxonomic classification using a Naive Bayes fitted classifier, trained on the August 2013 99% identity Greengenes database⁶, for 175 long reads and the Forward/Reverse primer set.
4. Rarefy all samples to depth of 2,146 sequences.
5. Use Unweighted UniFrac for measurement of β-diversity (between sample diversity^19,20) on the rarefied samples, to avoid a sample size effect.
6. Use the resulting distance matrix to perform a principal coordinates analysis (PCoA).
   NOTE: The data processing script and mapping files are provided as supplementary material (Supplementary Material 1 and 2).

Results

A schematic illustration of the protocol is shown in Figure 1.

We have prospectively collected stool samples from hospitalized patients with suspected infectious diarrhea. Those samples were submitted to the Clinical Microbiology Lab at the Sheba Medical Center between February and May 2015, as was previously described¹. Stool samples were subjected to conventional microbiolo...

Discussion

16S rRNA-amplicon and metagenomics shotgun sequencing have gained popularity in clinical microbiology applications^21,22,23. These techniques are advantageous in their increased ability to capture culturable and non-culturable taxa, providing data about the relative abundance of the pathogenic inoculum, and their ability to identify more precisely a polymicrobial infectious fingerprint²⁴. The advances in t...

Materials

Name	Company	Catalog Number	Comments
Primers	Integrated DNA Technologies (IDT)
Extraction solution	Sigma-Aldrich	E7526
Dilution solution	Sigma-Aldrich	D5688
Kapa HiFi HotStart ReadyMix PCR Kit	KAPABIOSYSTEMS	KK2601	PCR Master mix
Quant-iT PicoGreen dsDNA Reagent kit	Invitrogen	P7589	dsDNA quantify reagent
MinElute Gel extraction kit	Qiagen	28606
Agarose	Amresco	0710-250G
Ultra Pure Water Dnase and Rnase Free	Biological Industries	01-866-1A
Qubit dsDNA HS assay kit	Molecular probes	Q32854	dsDNA detecting kit
High Sensitivity D1000	Agilent Technologies	Screen Tape 5067-5582	separation and analysis
Screen Tape Assay	Agilent Technologies	Reagents 5067-5583	for DNA libraries
PhiX Control v3	Illumina	15017666	control library
MiSeq Reagent Kit v2 (500 cycle)	Illumina	MS-102-2003
Ethidium Bromide	Amresco	E406-10mL-TAM
2 mL collection tubes	SARSTEDT	72.695.400	Safe Seal collection tubes
Plastic stick swab in PP test tube	STERILE INTERIOR	23117
Name	Company	Catalog Number	Comments
Equipment
PCR Machine	Applied Biosystems	2720 Thermal Cycler
Sequncing Machine	Illumina	Miseq
PCR workstation	Biosan	UV-cleaner
scissors
vortexer	Scientific Industries	Vortex-Genie 2