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Taxonomy of anaerobic digestion microbiome reveals biases associated with the applied high throughput sequencing strategies.

ABSTRACT: In the past few years, many studies investigated the anaerobic digestion microbiome by means of 16S rRNA amplicon sequencing. Results obtained from these studies were compared to each other without taking into consideration the followed procedure for amplicons preparation and data analysis. This negligence was mainly due to the lack of knowledge regarding the biases influencing specific steps of the microbiome investigation process. In the present study, the main technical aspects of the 16S rRNA analysis were checked giving special attention to the approach used for high throughput sequencing. More specifically, the microbial compositions of three laboratory scale biogas reactors were analyzed before and after addition of sodium oleate by sequencing the microbiome with three different approaches: 16S rRNA amplicon sequencing, shotgun DNA and shotgun RNA. This comparative analysis revealed that, in amplicon sequencing, abundance of some taxa (Euryarchaeota and Spirochaetes) was biased by the inefficiency of universal primers to hybridize all the templates. Reliability of the results obtained was also influenced by the number of hypervariable regions under investigation. Finally, amplicon sequencing and shotgun DNA underestimated the Methanoculleus genus, probably due to the low 16S rRNA gene copy number encoded in this taxon.

SUBMITTER: Campanaro S

PROVIDER: S-EPMC5792648 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Taxonomy of anaerobic digestion microbiome reveals biases associated with the applied high throughput sequencing strategies.

Campanaro Stefano S Treu Laura L Kougias Panagiotis G PG Zhu Xinyu X Angelidaki Irini I

Scientific reports 20180131 1

In the past few years, many studies investigated the anaerobic digestion microbiome by means of 16S rRNA amplicon sequencing. Results obtained from these studies were compared to each other without taking into consideration the followed procedure for amplicons preparation and data analysis. This negligence was mainly due to the lack of knowledge regarding the biases influencing specific steps of the microbiome investigation process. In the present study, the main technical aspects of the 16S rRN ...[more]

PMID: 29386622

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Project description:BackgroundPart-stream low-frequency ultrasound (LFUS) was one of the common practices for sludge disintegration in full-scale anaerobic digestion (AD) facilities. However, the effectiveness of part-stream LFUS treatment and its effect on AD microbiome have not been fully elucidated.MethodsHere we testified the effectiveness of part-stream LFUS pretreatment by treating only a fraction of feed sludge (23% and 33% total solid of the feed sludge) with 20 Hz LFUS for 70 s. State-of-the-art metagenomic and metatranscriptomic analysis was used to investigate the microbial process underpinning the enhanced AD performance by part-stream LFUS pretreatment.ResultsBy pretreating 33% total solid of the feed sludge, methane yield was increased by 36.5%, while the volatile solid reduction ratio remained unchanged. RNA-seq of the microbiome at stable stage showed that the continuous dosage of easy-degradable LFUS-pretreated feed sludge had gradually altered the microbial community by selecting Bacteroidales hydrolyzer with greater metabolic capability to hydrolyze cellulosic biomass without substrate attachment. Meanwhile, Thermotogales with excellent cell mobility for nutrient capturing was highly active within the community. Foremost proportion of the methanogenesis was contributed by the dominant Methanomicrobiales via carbon dioxide reduction. More interestingly, a perceivable proportion of the reverse electron flow of the community was input from Methanoculleus species other than syntrophic acetate-oxidizing bacteria. In addition, metagenomic binning retrieved several interesting novel metagenomic-assembled genomes (MAGs): MAG-bin6 of Alistipes shahii showed exceptional transcriptional activities towards protein degradation and MAG-bin11 of Candidatus Cloacimonetes with active cellulolytic GH74 gene detected.ConclusionsIn summary, despite the unchanged sludge digestibility, the applied part-stream LFUS pretreatment strategy was robust in adjusting the microbial pathways towards more effective substrate conversion enabled by free-living hydrolyser and beta-oxidation-capable methanogens.

Project description:Next-generation sequencing workflows, using either metabarcoding or metagenomic approaches, have massively contributed to expanding knowledge of the human gut microbiota, but methodological bias compromises reproducibility across studies. Where these biases have been quantified within several comparative analyses on their own, none have measured inter-laboratory reproducibility using similar DNA material. Here, we designed a multicenter study involving seven participating laboratories dedicated to partial- (P1 to P5), full-length (P6) metabarcoding, or metagenomic profiling (MGP) using DNA from a mock microbial community or extracted from 10 fecal samples collected at two time points from five donors. Fecal material was collected, and the DNA was extracted according to the IHMS protocols. The mock and isolated DNA were then provided to the participating laboratories for sequencing. Following sequencing analysis according to the laboratories' routine pipelines, relative taxonomic-count tables defined at the genus level were provided and analyzed. Large variations in alpha-diversity between laboratories, uncorrelated with sequencing depth, were detected among the profiles. Half of the genera identified by P1 were unique to this partner and two-thirds of the genera identified by MGP were not detected by P3. Analysis of beta-diversity revealed lower inter-individual variance than inter-laboratory variances. The taxonomic profiles of P5 and P6 were more similar to those of MGP than those obtained by P1, P2, P3, and P4. Reanalysis of the raw sequences obtained by partial-length metabarcoding profiling, using a single bioinformatic pipeline, harmonized the description of the bacterial profiles, which were more similar to each other, except for P3, and closer to the profiles obtained by MGP. This study highlights the major impact of the bioinformatics pipeline, and primarily the database used for taxonomic annotation. Laboratories need to benchmark and optimize their bioinformatic pipelines using standards to monitor their effectiveness in accurately detecting taxa present in gut microbiota.

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Taxonomy of anaerobic digestion microbiome reveals biases associated with the applied high throughput sequencing strategies.

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Taxonomy of anaerobic digestion microbiome reveals biases associated with the applied high throughput sequencing strategies.

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