MAUI-seq: Metabarcoding using amplicons with unique molecular identifiers to improve error correction

10.21203/rs.2.21630/v1 ◽

2020 ◽

Author(s):

Bryden Fields ◽

Sara Moeskjær ◽

Ville-Petri Friman ◽

Stig U. Andersen ◽

J. Peter W. Young

Keyword(s):

Genetic Diversity ◽

16S Rrna ◽

Error Correction ◽

High Throughput ◽

Environmental Dna ◽

Amplicon Sequencing ◽

Efficient Elimination

Abstract Background Sequencing and PCR errors are a major challenge when characterising genetic diversity using high-throughput amplicon sequencing (HTAS). Results We have developed a multiplexed HTAS method, MAUI-seq, which uses unique molecular identifiers (UMIs) to improve error correction by exploiting variation among sequences associated with a single UMI. We show that two main advantages of this approach are efficient elimination of chimeric and other erroneous reads, outperforming DADA2 and UNOISE3, and the ability to confidently recognise genuine alleles that are present at low abundance or resemble chimeras. Conclusions The method provides sensitive and flexible profiling of diversity and is readily adaptable to most HTAS applications, including microbial 16S rRNA profiling and metabarcoding of environmental DNA.

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High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

Nucleic Acids Research ◽

10.1093/nar/gkz569 ◽

2019 ◽

Vol 47 (18) ◽

pp. e103-e103 ◽

Cited By ~ 58

Author(s):

Benjamin J Callahan ◽

Joan Wong ◽

Cheryl Heiner ◽

Steve Oh ◽

Casey M Theriot ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Amplicon Sequencing ◽

Full Length ◽

Rrna Gene ◽

Single Nucleotide ◽

Full Complement ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

AbstractTargeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

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Exploring the roles of and interactions among microbes in dry co-digestion of food waste and pig manure using high-throughput 16S rRNA gene amplicon sequencing

Biotechnology for Biofuels ◽

10.1186/s13068-018-1344-0 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 10

Author(s):

Yan Jiang ◽

Conor Dennehy ◽

Peadar G. Lawlor ◽

Zhenhu Hu ◽

Matthew McCabe ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Food Waste ◽

Amplicon Sequencing ◽

Pig Manure ◽

Rrna Gene

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Synthetic spike-in standards for high-throughput 16S rRNA gene amplicon sequencing

Nucleic Acids Research ◽

10.1093/nar/gkw984 ◽

2016 ◽

pp. gkw984 ◽

Cited By ~ 23

Author(s):

Dieter M. Tourlousse ◽

Satowa Yoshiike ◽

Akiko Ohashi ◽

Satoko Matsukura ◽

Naohiro Noda ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Amplicon Sequencing ◽

Rrna Gene

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Barcoded Primers Used in Multiplex Amplicon Pyrosequencing Bias Amplification

Applied and Environmental Microbiology ◽

10.1128/aem.05220-11 ◽

2011 ◽

Vol 77 (21) ◽

pp. 7846-7849 ◽

Cited By ~ 327

Author(s):

David Berry ◽

Karim Ben Mahfoudh ◽

Michael Wagner ◽

Alexander Loy

Keyword(s):

Genetic Diversity ◽

Restriction Fragment Length Polymorphism ◽

Fragment Length Polymorphism ◽

Environmental Dna ◽

Amplicon Sequencing ◽

Pcr Primers ◽

Amplification Bias ◽

Amplicon Pyrosequencing ◽

Dna Template ◽

Restriction Fragment Length

ABSTRACT“Barcode-tagged” PCR primers used for multiplex amplicon sequencing generate a thus-far-overlooked amplification bias that produces variable terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing data from the same environmental DNA template. We propose a simple two-step PCR approach that increases reproducibility and consistently recovers higher genetic diversity in pyrosequencing libraries.

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rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

10.21203/rs.2.9507/v2 ◽

2019 ◽

Author(s):

Jean-Claude OGIER ◽

Sylvie Pagès ◽

Maxime Galan ◽

Matthieu Barret ◽

Sophie Gaudriault

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Amplicon Sequencing ◽

Taxonomic Resolution ◽

Taxonomic Structure ◽

Rrna Gene ◽

Specificity And Sensitivity ◽

The 16S Rrna Gene ◽

Mock Communities

Abstract Background Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene. Results We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii. Conclusions Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

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rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

10.21203/rs.2.9507/v1 ◽

2019 ◽

Author(s):

Jean-Claude OGIER ◽

Sylvie Pagès ◽

Maxime Galan ◽

Matthieu Barret ◽

Sophie Gaudriault

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Amplicon Sequencing ◽

Taxonomic Resolution ◽

Taxonomic Structure ◽

Rrna Gene ◽

Specificity And Sensitivity ◽

The 16S Rrna Gene ◽

Mock Communities

Abstract Background Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene. Results We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii. Conclusions Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

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High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

10.1101/392332 ◽

2018 ◽

Cited By ~ 5

Author(s):

Benjamin J Callahan ◽

Joan Wong ◽

Cheryl Heiner ◽

Steve Oh ◽

Casey M Theriot ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Amplicon Sequencing ◽

Full Length ◽

Rrna Gene ◽

Single Nucleotide ◽

Full Complement ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

AbstractTargeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate.In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowedE. colistrains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in severalE. colistrains.There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

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High throughput amplicon sequencing to assess within- and between-host genetic diversity in plant viruses

10.1101/168773 ◽

2017 ◽

Cited By ~ 1

Author(s):

Sylvain Piry ◽

Catherine Wipf-Scheibel ◽

Jean-François Martin ◽

Maxime Galan ◽

Karine Berthier

Keyword(s):

Genetic Diversity ◽

High Throughput ◽

Variant Calling ◽

Amplicon Sequencing ◽

Plant Viruses ◽

Sequencing Error ◽

List Type ◽

Data Generating Process ◽

Host Genetic ◽

Robust Variant

AbstractMolecular epidemiology approaches at the landscape scale require to study the genetic diversity of viral populations from numerous hosts and to characterize mixed infections. In such a context, high-throughput amplicon sequencing (HTAS) techniques create interesting opportunities as they allow identifying distinct variants within a same host while simultaneously genotyping a high number of samples. Validating variants produced by HTAS may, however, remain difficult due to biases occurring at different steps of the data-generating process (e.g. environmental contaminations and sequencing error). Here, we focused on Endive necrotic mosaic virus (ENMV), a member of family Potyviridae, genus Potyvirus to develop an HTAS approach and to characterize the genetic diversity at the intra- and inter-host levels from 430 samples collected over an area of 1660 km2 located in south-eastern France. We demonstrated how it is possible, by incorporating various controls in the experimental design and by performing independent sample replicates, to estimate potential biases in HTAS results and to implement an automated and robust variant calling procedure.HighlightsHigh-throughput amplicon sequencing to assess plant virus genetic diversityEstimating bias in high throughput amplicon sequencing resultsAutomated variant calling procedure for robust high throughput amplicon sequencing

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