Optimization of high-throughput 16S rRNA gene amplicon sequencing: an assessment of PCR pooling, mastermix use and contamination.

16S rRNA gene sequencing is widely used to characterize human and environmental microbiomes. Sequencing at scale facilitates better powered studies but is limited by cost and time. We identified two areas in our 16S rRNA gene library preparation protocol where modifications could provide efficiency gains, including (1) pooling of multiple PCR amplifications per sample to reduce PCR drift and (2) manual preparation of mastermix to reduce liquid handling. Using nasal samples from healthy human participants and a serially diluted mock microbial community, we compared alpha and beta diversity, and compositional abundance where the PCR amplification was conducted in triplicate, duplicate or as a single reaction, and where manually prepared or premixed mastermix was used. One hundred and fifty-eight 16S rRNA gene sequencing libraries were prepared, including a replicate experiment. Comparing PCR pooling strategies, we found no significant difference in high-quality read counts and alpha diversity, and beta diversity by Bray-Curtis index clustered by replicate on principal coordinate analysis (PCoA) and non-metric dimensional scaling (NMDS) analysis. Choice of mastermix had no significant impact on high-quality read and alpha diversity, and beta diversity by Bray-Curtis index clustered by replicate in PCoA and NMDS analysis. Importantly, we observed contamination and variability of rare species (<0.01 %) across replicate experiments; the majority of contaminants were accounted for by removal of species present at <0.1 %, or were linked to reagents (including a primer stock). We demonstrate no requirement for pooling of PCR amplifications or manual preparation of PCR mastermix, resulting in a more efficient 16S rRNA gene PCR protocol.

Extended early meets late for assessment of conduction block along an ablation line.

PURPOSE: The purpose of the study was to investigate the usefulness of the extended early meets late (EEML) feature of the HD Coloring software for confirmation of bidirectional block along an ablation line. METHODS: A single-center prospective observational study of consecutive patients with atypical atrial flutter or persistent atrial fibrillation submitted to catheter ablation including linear lesions from January 2019 to June 2020, with confirmation of bidirectional block across ablation lines assessed with the feature EEML and a multipolar catheter was conducted. Patients were divided into two groups - those with versus those without bidirectional block - and different EEML thresholds were analyzed to assess which one had the better sensitivity and specificity to predict block. RESULTS: During the 24-month enrollment period, a total of 94 patients were included (50% males, mean age of 64 ± 10 years, 60% with structural heart disease) - 55 patients with versus 39 patients without confirmed a bidirectional block. Activation maps were performed during atrial pacing, with a median number of 1340 (interquartile range 1135-2060) points acquired in 11 ± 3 min, and a mean mapped cycle length of 287 ± 51 ms. The EEML feature was highly useful in detecting bidirectional block along ablation lines, with a 25% threshold value showing the highest discriminative performance (area under the curve of 0.93), 95% sensitivity and 92% specificity in predicting block. CONCLUSIONS: The EEML feature of the HD Coloring software is a useful addition to the CARTO mapping system for assessment of block across an ablation line. The threshold value of 25% is the most accurate.