Higher off-target amplicon detection rate in MiSeq v3 compared to v2 reagent kits in the context of 16S-rRNA-sequencing.

One of the most widely used techniques in microbiota research is 16S-rRNA-sequencing. Several laboratory processes have been shown to impact sequencing results, especially in low biomass samples. Low biomass samples are prone to off-target amplification, where instead of bacterial DNA, host DNA is erroneously amplified. Knowledge on the laboratory processes influencing off-target amplification and detection is however scarce. We here expand on previous findings by demonstrating that off-target amplification is not limited to invasive biopsy samples, but is also an issue in low bacterial biomass respiratory (mucosal) samples, especially when below 0.3 pg/µL. We show that off-target amplification can partly be mitigated by using gel-based library purification methods. Importantly, we report a higher off-target amplicon detection rate when using MiSeq reagent kit v3 compared to v2 (mean 13.3% vs 0.1% off-target reads/sample, respectively), possibly as a result of differences in reagents or sequencing recipes. However, since after bioinformatic removal of off-target reads, MiSeq reagent kit v3 still results in a twofold higher number of reads when compared to v2, v3 is still preferred over v2. Together, these results add to the growing knowledge base on off-target amplification and detection, allowing researchers to anticipate this problem in 16S-rRNA-based microbiome studies involving low biomass samples.

Benchmarking laboratory processes to characterise low-biomass respiratory microbiota.

The low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available laboratory protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community profile, we amplified the 16S rRNA gene of respiratory samples using various bacterial loads and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 or V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community profile of low biomass samples. Purification methods and MiSeq reagent kits provided nearly similar microbiota profiles (paired Bray-Curtis dissimilarity median: 0.03 and 0.05, respectively). While profiles of positive controls were significantly influenced by the type of dilution solvent, the theoretical profile of the Zymo mock was most accurately analysed when the Zymo mock was diluted in elution buffer (difference compared to the theoretical Zymo mock: 21.6% for elution buffer, 29.2% for Milli-Q, and 79.6% for DNA/RNA shield). Microbiota profiles of DNA blanks formed a distinct cluster compared to low biomass samples, demonstrating that low biomass samples can accurately be distinguished from DNA blanks. In summary, to accurately characterise the microbial community composition we recommend 1. amplification of the obtained microbial DNA with 30 PCR cycles, 2. purifying amplicon pools by two consecutive AMPure XP steps and 3. sequence the pooled amplicons by V3 MiSeq reagent kit. The benchmarked standardized laboratory workflow presented here ensures comparability of results within and between low biomass microbiome studies.

Roux-Y Gastric Bypass and Sleeve Gastrectomy directly change gut microbiota composition independent of surgery type.

Bariatric surgery in morbid obesity, either through sleeve gastrectomy (SG) or Roux-Y gastric bypass (RYGB), leads to sustainable weight loss, improvement of metabolic disorders and changes in intestinal microbiota. Yet, the relationship between changes in gut microbiota, weight loss and surgical procedure remains incompletely understood. We determined temporal changes in microbiota composition in 45 obese patients undergoing crash diet followed by SG (n = 22) or RYGB (n = 23). Intestinal microbiota composition was determined before intervention (baseline, S1), 2 weeks after crash diet (S2), and 1 week (S3), 3 months (S4) and 6 months (S5) after surgery. Relative to S1, the microbial diversity index declined at S2 and S3 (p < 0.05), and gradually returned to baseline levels at S5. Rikenellaceae relative abundance increased and Ruminococcaceae and Streptococcaceae abundance decreased at S2 (p < 0.05). At S3, Bifidobacteriaceae abundance decreased, whereas those of Streptococcaceae and Enterobacteriaceae increased (p < 0.05). Increased weight loss between S3-S5 was not associated with major changes in microbiota composition. No significant differences appeared between both surgical procedures. In conclusion, undergoing a crash diet and bariatric surgery were associated with an immediate but temporary decline in microbial diversity, with immediate and permanent changes in microbiota composition, independent of surgery type.

Molecular surveillance of nasopharyngeal carriage of Streptococcus pneumoniae in children vaccinated with conjugated polysaccharide pneumococcal vaccines.

Following the introduction of pneumococcal conjugate vaccines (PCVs) for infants, surveillance studies on Streptococcus pneumoniae carriage have proven valuable for monitoring vaccine effects. Here, we compared molecular versus conventional diagnostic methods in prospective cross-sectional surveillances in vaccinated infants in the Netherlands. Nasopharyngeal samples (n = 1169) from 11- and 24-month-old children, collected during autumn/winter 2010/2011 and 2012/2013, were tested by conventional culture for S. pneumoniae. DNA extracted from all culture-plate growth was tested by qPCR for pneumococcal-specific genes (lytA/piaB) and selected serotypes (including PCV13-serotypes). qPCR significantly increased the number of carriers detected compared to culture (69% vs. 57%, p < 0.0001). qPCR assays targeting vaccine-serotypes 4 and 5 proved non-specific (results excluded). For serotypes reliably targeted by qPCR, the number of serotype-carriage events detected by qPCR (n = 709) was 1.68× higher compared to culture (n = 422). There was a strong correlation (rho = 0.980; p < 0.0001) between the number of serotypes detected using qPCR and by culture. This study demonstrates the high potential of molecular methods in pneumococcal surveillances, particularly for enhanced serotype detection. We found no evidence of a hidden circulation of vaccine-targeted serotypes, despite vaccine-serotypes still significantly contributing to invasive pneumococcal disease in unvaccinated individuals, supporting the presence of a substantial S. pneumoniae reservoir outside vaccinated children.