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Strand-wise and bait-assisted assembly of nearly-full rrn operons applied to assess species engraftment after faecal microbiota transplantation


Date: 11th September 2020 | Source: bioRxiv

Authors: Alfonso Benítez-Páez, Annick V. Hartstra, Max Nieuwdorp, Yolanda Sanz.

Effective methodologies to accurately identify members of the gut microbiota at the species and strain levels are necessary to unveiling more specific and detailed host-microbe interactions and associations with health and disease.

MinION™ MkIb nanopore-based device and the R9.5 flowcell chemistry were used to sequence and assemble dozens of rrn regions (16S-ITS-23S) derived from the most prevalent bacterial species in the human gut microbiota. As a method proof-of-concept to disclose further strain-level variation, we performed a complementary analysis in a subset of samples derived from an faecal microbiota transplantation (FMT) trial aiming amelioration of glucose and lipid metabolism in overweight subjects with metabolic syndrome.

The resulting updated rrn database, the data processing pipeline, and the precise control of covariates (sequencing run, sex, age, BMI, donor) were pivotal to accurately estimate the changes in gut microbial species abundance in the recipients after FMT. Furthermore, the rrn methodology described here demonstrated the ability to detect strain-level variation, critical to evaluate the transference of bacteria from donors to recipients as a consequence of the FMT. At this regard, we showed that our FMT trial successfully induced donors’ strain engraftment of e.g. Parabacteroides merdae species in recipients by mapping and assessing their associated single nucleotide variants (SNV).

We developed a methodology that enables the identification of microbiota at species- and strain-level in a cost-effective manner. Despite its error-prone nature and its modest per-base accuracy, the nanopore data showed to have enough quality to estimate single-nucleotide variation. This methodology and data analysis represents a cost-effective manner to trace genetic variability needed for better understanding the health effects of the human microbiome.

Trial registration The study was prospectively registered at the Dutch Trial registry - NTR4488 (

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