Combining 16S rRNA gene variable regions enables high-resolution microbial community profiling View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2018-01-26

AUTHORS

Garold Fuks, Michael Elgart, Amnon Amir, Amit Zeisel, Peter J. Turnbaugh, Yoav Soen, Noam Shental

ABSTRACT

BACKGROUND: Most of our knowledge about the remarkable microbial diversity on Earth comes from sequencing the 16S rRNA gene. The use of next-generation sequencing methods has increased sample number and sequencing depth, but the read length of the most widely used sequencing platforms today is quite short, requiring the researcher to choose a subset of the gene to sequence (typically 16-33% of the total length). Thus, many bacteria may share the same amplified region, and the resolution of profiling is inherently limited. Platforms that offer ultra-long read lengths, whole genome shotgun sequencing approaches, and computational frameworks formerly suggested by us and by others all allow different ways to circumvent this problem yet suffer various shortcomings. There is a need for a simple and low-cost 16S rRNA gene-based profiling approach that harnesses the short read length to provide a much larger coverage of the gene to allow for high resolution, even in harsh conditions of low bacterial biomass and fragmented DNA. RESULTS: This manuscript suggests Short MUltiple Regions Framework (SMURF), a method to combine sequencing results from different PCR-amplified regions to provide one coherent profiling. The de facto amplicon length is the total length of all amplified regions, thus providing much higher resolution compared to current techniques. Computationally, the method solves a convex optimization problem that allows extremely fast reconstruction and requires only moderate memory. We demonstrate the increase in resolution by in silico simulations and by profiling two mock mixtures and real-world biological samples. Reanalyzing a mock mixture from the Human Microbiome Project achieved about twofold improvement in resolution when combing two independent regions. Using a custom set of six primer pairs spanning about 1200 bp (80%) of the 16S rRNA gene, we were able to achieve ~ 100-fold improvement in resolution compared to a single region, over a mock mixture of common human gut bacterial isolates. Finally, the profiling of a Drosophila melanogaster microbiome using the set of six primer pairs provided a ~ 100-fold increase in resolution and thus enabling efficient downstream analysis. CONCLUSIONS: SMURF enables the identification of near full-length 16S rRNA gene sequences in microbial communities, having resolution superior compared to current techniques. It may be applied to standard sample preparation protocols with very little modifications. SMURF also paves the way to high-resolution profiling of low-biomass and fragmented DNA, e.g., in the case of formalin-fixed and paraffin-embedded samples, fossil-derived DNA, or DNA exposed to other degrading conditions. The approach is not restricted to combining amplicons of the 16S rRNA gene and may be applied to any set of amplicons, e.g., in multilocus sequence typing (MLST). More... »

PAGES

17

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/s40168-017-0396-x

DOI

http://dx.doi.org/10.1186/s40168-017-0396-x

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1100667179

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/29373999


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31 schema:description BACKGROUND: Most of our knowledge about the remarkable microbial diversity on Earth comes from sequencing the 16S rRNA gene. The use of next-generation sequencing methods has increased sample number and sequencing depth, but the read length of the most widely used sequencing platforms today is quite short, requiring the researcher to choose a subset of the gene to sequence (typically 16-33% of the total length). Thus, many bacteria may share the same amplified region, and the resolution of profiling is inherently limited. Platforms that offer ultra-long read lengths, whole genome shotgun sequencing approaches, and computational frameworks formerly suggested by us and by others all allow different ways to circumvent this problem yet suffer various shortcomings. There is a need for a simple and low-cost 16S rRNA gene-based profiling approach that harnesses the short read length to provide a much larger coverage of the gene to allow for high resolution, even in harsh conditions of low bacterial biomass and fragmented DNA. RESULTS: This manuscript suggests Short MUltiple Regions Framework (SMURF), a method to combine sequencing results from different PCR-amplified regions to provide one coherent profiling. The de facto amplicon length is the total length of all amplified regions, thus providing much higher resolution compared to current techniques. Computationally, the method solves a convex optimization problem that allows extremely fast reconstruction and requires only moderate memory. We demonstrate the increase in resolution by in silico simulations and by profiling two mock mixtures and real-world biological samples. Reanalyzing a mock mixture from the Human Microbiome Project achieved about twofold improvement in resolution when combing two independent regions. Using a custom set of six primer pairs spanning about 1200 bp (80%) of the 16S rRNA gene, we were able to achieve ~ 100-fold improvement in resolution compared to a single region, over a mock mixture of common human gut bacterial isolates. Finally, the profiling of a Drosophila melanogaster microbiome using the set of six primer pairs provided a ~ 100-fold increase in resolution and thus enabling efficient downstream analysis. CONCLUSIONS: SMURF enables the identification of near full-length 16S rRNA gene sequences in microbial communities, having resolution superior compared to current techniques. It may be applied to standard sample preparation protocols with very little modifications. SMURF also paves the way to high-resolution profiling of low-biomass and fragmented DNA, e.g., in the case of formalin-fixed and paraffin-embedded samples, fossil-derived DNA, or DNA exposed to other degrading conditions. The approach is not restricted to combining amplicons of the 16S rRNA gene and may be applied to any set of amplicons, e.g., in multilocus sequence typing (MLST).
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39 Computationally
40 DNA
41 Drosophila melanogaster microbiome
42 Earth
43 Human Microbiome Project
44 MUltiple Regions Framework
45 Microbiome Project
46 PCR
47 SMURF
48 amplicon length
49 amplicons
50 analysis
51 approach
52 bacteria
53 bacterial biomass
54 bacterial isolates
55 biological samples
56 biomass
57 cases
58 cases of formalin
59 coherent profiling
60 common human gut bacterial isolates
61 community
62 community profiling
63 computational framework
64 conditions
65 convex optimization problem
66 coverage
67 current techniques
68 custom set
69 depth
70 different PCR
71 different ways
72 diversity
73 downstream analysis
74 efficient downstream analysis
75 fast reconstruction
76 formalin
77 fossil-derived DNA
78 fragmented DNA
79 framework
80 gene sequences
81 gene variable regions
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83 genes
84 genome shotgun sequencing approaches
85 gut bacterial isolates
86 harsh conditions
87 high resolution
88 high-resolution microbial community profiling
89 high-resolution profiling
90 human gut bacterial isolates
91 identification
92 improvement
93 increase
94 independent regions
95 isolates
96 knowledge
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98 length
99 little modification
100 low bacterial biomass
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107 microbial diversity
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112 modification
113 multilocus sequence typing
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129 rRNA gene
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155 shortcomings
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157 silico simulations
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160 standard sample preparation protocols
161 subset
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163 today
164 total length
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