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Introduction

Previously, only a small fraction of all bacteria had been isolated and characterized, and analysis of any bacterial community was severely limited by the available technology and the shortage of reference genomes (1,2). The development of next generation sequencing provided an excellent platform to explore the association between humans and microbiota (3–6).

The human body harbors bacterial, viral and eukaryotic communities in the skin, nasopharynx, oral cavity, respiratory tract, gastrointestinal tract and female reproductive tract (7–10), and the microbes have profound implications on humanmetabolism, immunity and the gut-brain axis (3–6). As it has been demonstrated, the human intestine exists in symbiosis with hundreds of trillions of microbes, and there is an increasing awareness that the bacteria residing within the gut has a significant influence on host health (7). In addition, microbial dysbiosis caused by the altered intestinal microbes has been proved to contribute to the onset of several disorders, and it is important to identify the key bacteria that serve a direct or indirect effect on human health.

Previously, analysis of microbial diversity in specific environments was severely limited by available technologies and referenced genomes. Technological advances in next generation sequencing have enabled the elucidation of the pleiotropic effects of microorganisms on the human host, and the high-throughput sequencing can detect almost all the DNA signatures of microbes within specific environments, even those bacteria present in low numbers (2). However, a number of studies have only focused on the examination of microbial diversity using high-throughput sequencing technology, which provided little information on the potential and limitations of this approach in microbial ecology studies (11–13).

Sample handling, DNA extraction, amplification efficiency, run processing and downstream analyses have seriously affected the generation of high quality data (2,14–17). Therefore, in the present study, a number of known bacteria were mixed together at a certain percentage, and extracted DNAs were subjected high throughput sequencing to evaluate the accuracy and sensitivity of high-throughput sequencing technology, providing basic data to help researchers to better investigate the relationship between microorganisms and host health.

Materials and methods

Bacterial activation and culture

A total of seven common bacterial species, including Escherichia coli (no. 44102; isolated from the donated human feces to screen the bacteria in human intestines, and stored in the authors' lab), Lactobacillus plantarum (no. HM218749; isolated from sourdough and stored in the author's lab), Streptococcus thermophilus [no. 19258; American Type Culture Collection (ATCC), Manassas, VA, USA], Bifidobacterium bifidum (no. WBIN03; isolated from yogurt and stored in the authors' lab), Bacillus subtilis (no. 14416; ATCC), Enterococcus faecalis (no. HM218543; isolated from the donated human feces to screen the bacteria in human intestines, and stored in the authors' lab) and Salmonella typhimurium (no. 14028; ATCC), were selected in the present studyand divided into five groups (Table I). All bacteria were propagated in corresponding media three times before DNA extraction (Table I).

Table I. Bacterial composition in groups A, B, C, D and E.

Table I.

Bacterial composition in groups A, B, C, D and E.

	Group, CFU/ml
Bacteria	A	B	C	D	E
E. coli (44102)	1×106	1×104	1×103	1×102	1×10
L. plantarum (HM218749)	1×106	1×104	1×103	1×102	1×10
S. thermophilus (ATCC 19258)	1×106	1×106	1×106	1×106	1×106
B. bifidum (WBIN03)	1×106	1×106	1×106	1×106	1×106
B. subtilis (ATCC 14416)	1×106	1×106	1×106	1×106	1×106
E. faecalis (HM218543)	1×106	1×106	1×106	1×106	1×106
S. typhimurium (ATCC 14028)	1×106	1×106	1×106	1×106	1×106

[i] CFU, colony-forming units.

Extraction of genomic DNA and high-throughput sequencing

Genomic DNA from each sample was extracted using a TIANamp Genomic DNA kit (Tiangen Biotech Co., Ltd., Beijing, China) combined with bead beating, as previously described (3). Subsequently, the genomic DNA was sent to a high-throughput sequencing company (Biomarker Technologies Corporation, Beijing, China) for high-throughput sequencing and analysis.

The extracted genomic DNAs extracted from these seven common bacteria were used as the templates, and the universal primer pair 338F/806R with the respective barcode (supplied by BiomarkerTechnologiesCorporation, Beijing, China) for ease of identification (Table II) were used to to amplify the V3-V4 region of 16S ribosomal (r)RNA genes of all samples. Polymerase chain reaction (PCR), pyrosequencing of the PCR amplicons and quality control of raw data were performed as described previously with minor modifications as presented in the ‘Bioinformatics and multivariate statistics’ section below (18).

Table II. Primer barcodes.

Table II.

Primer barcodes.

No.	Sequence
A1	AGGGTCAATGAACCTT
A2	AGGGTCAAAGTCAACA
A3	AGGGTCAACTCTCTAT
B1	AGGGTCAAAGAGTAGA
B2	AGGGTCAAGTAAGGAG
B3	AGGGTCAAACTGCATA
C1	AGGGTCAAAAGGAGTA
C2	AGGGTCAACTAAGCCT
C3	AGGAGTGGTGAACCTT
D1	AGGAGTGGAGTCAACA
D2	AGGAGTGGCTCTCTAT
D3	AGGAGTGGAGAGTAGA
E1	AGGAGTGGGTAAGGAG
E2	AGGAGTGGACTGCATA
E3	AGGAGTGGAAGGAGTA

Bioinformatics and multivariate statistics

Low-quality sequences were eliminated from the analysis based on the following criteria: i) Raw reads shorter than 400 bp; ii) a sequence producing >8 homopolymers; iii) >2 mismatches in the primers; and iv) 1 or more mismatches in the barcode. Pyrosequenced amplicons were removed using the PyroNoise algorithm in Mothur (version 1.33.3) (19). Bioinformatics analysis was implemented using the Quantitative Insights Into Microbial Ecology (QIIME) platform (version 1.8.0) (20). Briefly, 16S rRNA operational taxonomic units (OTUs) were clustered using an open-reference OTU picking protocol based on 97% nucleotide similarity with the UCLUST algorithm (21). ChimeraSlayer was employed to remove chimeric sequences (22). The relative abundance of each OTU was determined as a proportion of the sum of sequences for each sample. Taxonomic relative abundance profiles (including at the phylum, class, order, family and genus levels) were generated based on OTU annotation. The microbial community structure (i.e., species richness, evenness and between-sample diversity) of bacterial samples was estimated by biodiversity. Shannon index, phylogenetic diversity, chao1 index and the observed number of species were used to evaluate α diversity, and the weighted and unweighted UniFrac distances were used to evaluate β diversity.

All of these indices (α and β diversity) were calculated via the QIIME pipeline.

Statistical analysis

Statistical analysis was implemented using the R platform. Principal coordinate analysis (PCoA) was performed using the ‘ape’ package based on the UniFrac distances between samples. The difference among groups was further assessed using analysis of similarities and multi-response permutation planning methodswith Metastats software (http://metastats.cbcb.umd.edu/) as described previously, and statistical significance was set at P<0.05 for correction of multiple comparisons (23).

Results

Sequencing coverage

To compare the microbes in each sample, 16S rRNA amplicon sequencing analysis was used to sequence the V3-V4 hypervariable region, the sequencing data was filtered to obtain the valid data, and the effective tags of all samples were clustered, and those sequences with >97% similarity were considered to beone OTU. In total, 2,032,484 filtered clean tags (135,498.9 tags/sample) and 1,840 OTUs were obtained from all the samples with an average of 122.7 OTUs per group (Table III). The chao1 index was almost saturated and the rarefaction curve of every sample could enter the plateau phase (data not shown).

Table III. Number of raw tags, clean tags, average bp, OTUs and actual bacterial composition in groups A, B, C, D and E by high-throughput sequencing.

Table III.

Number of raw tags, clean tags, average bp, OTUs and actual bacterial composition in groups A, B, C, D and E by high-throughput sequencing.

Sample ID	Raw tags	Clean tags	Average length, bp	OTU	Actual bacterial number
A1	180,452	163,786	467	120	7
A2	167,355	147,971	467	82	7
A3	159,440	144,734	467	120	7
B1	135,586	118,439	467	131	7
B2	88,832	71,781	469	84	7
B3	134,956	117,370	468	136	7
C1	139,230	123,136	468	122	7
C2	163,421	144,130	467	130	7
C3	188,147	167,154	468	131	7
D1	187,451	162,980	468	132	7
D2	139,141	120,711	468	128	7
D3	147,408	129,402	467	129	7
E1	147,574	125,254	468	135	7
E2	166,163	147,835	468	131	7
E3	165,512	147,801	468	129	7
Average	154,044.5	135,498.9	467.7	122.7	7

[i] OTU, operational taxonomic units.

Shared genera in each sample

The Venn diagrams reflected the differences between all groups. As presented in Fig. 1, there were 138 (A), 138 (B), 137 (C), 140 (D) and 140 (E) OTUs in each group, and the percentage of common OTUs was 46.38 (64/138), 58.70 (81/138), 86.13 (118/137), 83.57 (117/140) and 89.29% (125/140), respectively.

Figure 1. Scalar-Venn representation of the microbiota among groups A, B, C, D and E.

β diversity of the microbial community

The overall graph of the microbial composition of the samples was obtained by using PCoA, based on the relative abundance profiles of bacterial taxa. As presentedin Fig. 2, the samples A1 and A3 clustered together on the right upper corner of the coordinate axis, samples A2, B1, B3, C1, C2, C3, D1, D2, D3, E1, E2 and E3 gathered together on the lower right corner of the coordinate axis and sample B2 scattered on the bottom left corner.

Figure 2. PCoA of β diversity index of groups A, B, C, D and E. PCoA, principle component analysis.

Scientific classification of bacterial communities in each sample

To further investigate the relative abundance of the known bacteria in each group, the identified bacterial abundance was compared at the phylum, class, order, family, genus and species levels. Fig. 3 demonstrated that all the known bacteria were detected at the phylumand class levels, although their percentages only occupied 41.66 and 28.10% of the total bacteria, respectively. In addition, certain bacteria, for example, Bifidobacteriales at the order level, Streptococcaceae and Bifidobacteriaceae at the family level, Streptococcus and Bifidobacterium at the genus level, and E. coli, L. plantarum, S. thermophilus, B. bifidum, B. subtilis and S. typhimurium at the species level, failed to be detected, and the identified bacteria only occupied 20.84, 20.98, 19.31 and 1.62% at the order, family, genus and species levels, respectively (Fig. 3).

Figure 3. Ratios of the known bacteria in groups A, B, C, D and E at the phylum, class, order, family, genus and species levels.

Relative abundance of the bacterial communities in each sample

To compare the relative abundances among different groups, the Metastats method was applied in the presentstudy. At the genus level, the relative abundances of Salmonella, Bacillus and Enterococcus were each lowered or enhanced in the groups, although their actual number in each group was the same (Table I). For E. coli, actual bacterial number in groups A, B, C, D and E, was 1×106, 1×104, 1×103, 1×102 and 1×101 CFU/ml, respectively, while the OTU numbers in groups C, D and E were significantly higher compared with groups A and B (Fig. 4). In addition, though the OTU number of L. plantarum in groups B, C, D and E was significantly decreased compared with group A (Fig. 4), there was no significant difference observed among groups B, C, D and E, and the actual bacterial number of L. plantarum in groups B, C, D and E was 1×104, 1×103, 1×102 and 1×101, respectively (Table I).

Figure 4. Relative microbial abundance of Bacillus, Enterococcus, Escherichia, Lactobacillus and Salmonella in groups A, B, C, D and E. **P<0.05 vs. group A.

Discussion

The world is dominated by prokaryotes. The total number of microbial cells on Earth is estimated to be 1×1030 and, in the human body alone, there are up to 100 trillion organisms, which approximately equates to ten times the number of our own human cells (24,25), and there are millions of prokaryotic species which may not be cultivated (15,26). In the humanbody, bacteria serve important roles in the modulation of digestive, endocrineand immune functions. With the discovery of more recent culture-independentsequencing-based methods, the composition and diversity of the human microbiome is being uncovered. However, sample handling, DNA extraction, amplification efficiency, run processing and downstream analyses may seriously affect the generation of high quality data (2,14–17) and, therefore, it is important to evaluate the accuracy and sensitivity of high-throughput sequencing technology.

In the present study, seven common bacteria were mixed together to make a known microbiota, of which S. thermophilus, B. bifidum, B. subtilis, E. faecalis and S. typhimurium sustained a constant number of 1×106 CFU/ml in each sample, and the typical gram-negative bacteria E. coli and gram-positive bacteria L. plantarum were decreased between groups A and E. Following mixing of the known bacteria, DNA extraction was performed and the extracted genomic DNA was used as a template to amplify the V3-V4 region of 16S rRNA genes using primers of 338F/806R (18), and the results suggested that the DNA quality and PCR amplicons met the requirements for pyrosequencing (data not shown).

To evaluate tag quality, the raw tags and clean tags, average bp and OTUs per sample were compared. The mean number of 135,498.9 clean tags, average length of 467.7 bp, and saturated chao1 index and rarefaction curves ensured their reliability for future analysis. However, the average OTUs (122.7) in all groups indicated a 17.5-fold (122.7/7) increase compared with the actual microbial number in each group (7). To evaluate the consistency of the high-throughput sequencing, the Venn and PCoA methods were utilized and the results suggested that only 46.38% (64/138), 58.70% (81/138), 86.13% (118/137), 83.57% (117/140) and 89.29% (125/140) common OTUs were identified in groups A, B, C, D and E, of which the majority of OTUs did not belong to the added bacteria. However, the PCoA results demonstrated that the majority of samples clustered together, except for sample B2.

Compared with the relative abundance of known microbiota, all known bacteria were identified at the phylum and class levels; one or more bacteria was missed at the order, family and genus levels, and the known bacteria only occupied ~20% of the total OTUs at these levels. As microbiota are generally analyzed at the genus level, the statistical analysis in the present study was performed at the genus level, and Salmonella, Bacillus and Enterococcus, which existed at the same number in each group, exhibited a significant decreased or increase using high-throughput sequencing technology; whereas, the ten-fold dilution of Escherichia and Lactobacillus among groups C, D and E exhibited little alteration.

Microbial genomic DNA extraction and purification is the first step for library preparation however researchers indicated that there were significant differences in microbial composition when comparing microbiota diversity obtained from the same samples using different DNA extraction methods (27). To avoid the influence of DNA extraction, all the DNA samples were simultaneously extracted by the same researcher using the same DNA extraction kit. Therefore, the DNA extraction method is not the key factor for the misidentification of the known microbiota. In addition, all the DNA was amplified using the same primers of 338F/806R, as a result ofthe potential for amplification bias during PCR amplification reactions and the generation of chimeric amplification products that may exaggerate the bacterial number (28). Furthermore, the chimeric sequences, which are not identified by computational filtering software will lead to incorrect taxonomic identifications and an overestimated bacterial richness in the final microbiota profiling results (24,28).

In conclusion, the results of the present study suggested that the actual bacterial number in a specific environment maybe greatly exaggerated due to run processing and downstream analyses, and that DNA extraction and amplification efficiency may cause a reduction or exaggeration of certain bacteria. Therefore, certain measures, for example, adding the indicated bacteria/microbiota and analysing using more than three types of calculation software, are required to provide reasonable results. However, this result was only based on the present study, and the small size and lack of the comparison among various sequencing companies may not allowthe results of the present study to fully reflect the actual drawbacks of high-throughput sequencing. In the authors' future work, the microbial diversity in various environments will be tested using enlarged sample sizes and comparing the data quality among different sequencing companies. This will provide basic data for the improvement of high-throughput sequencing technology and benefit its applications in the monitoring of bacterial alterations during various diseases.

Acknowledgements

The present study was supported by grants from the National Natural Science Foundation of China (grant nos. 31560264, 81503364, 91639106, 81270202, and 91339113), the National Basic Research Program of China (grant no. 2013CB531103) and grants from Jiangxi Province (grant nos. 20171BCB23028 and 20175526).

References