Two-Target Quantitative PCR To Predict Library Composition for Shallow Shotgun Sequencing

Cho, Matthew Y; Oliva, Marc; Spreafico, Anna; Chen, Bin; Xu, Wei; Choi, Yoojin; Kaul, Rupert; Siu, Lillian L.; Coburn, Bryan; Schneeberger, Pierre H. H.

doi:10.1128/msystems.00552-21

Cited by 5 publications

(3 citation statements)

References 17 publications

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“…However, CARD is widely used, updated on an approximately monthly basis, and is representative of known ARG diversity, especially for well-characterized pathogens such as E. coli ( 22 ). Human metagenomic samples often have human DNA that can account for a large proportion of the total sample, which impacts sequencing strategies ( 45 , 46 ). An understanding of the total genetic material contributed by human reads prior to sequencing would further inform sequencing effort required to maintain a minimum sequencing depth for AMR gene detection.…”

Section: Discussionmentioning

confidence: 99%

Performance Characteristics of Next-Generation Sequencing for the Detection of Antimicrobial Resistance Determinants in Escherichia coli Genomes and Metagenomes

et al. 2022

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Systematically determining Illumina sequencing performance characteristics for detection of ARGs in metagenomic samples is essential to inform study design and appraisal of human, animal, and environmental metagenomic antimicrobial resistance studies. In this study, we quantified the performance characteristics of ARG detection in E. coli genomes and metagenomes and established a benchmark of ~15× coverage for ARG detection for E. coli in metagenomes.

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Section: Discussionmentioning

confidence: 99%

Performance Characteristics of Next-Generation Sequencing for the Detection of Antimicrobial Resistance Determinants in Escherichia coli Genomes and Metagenomes

et al. 2022

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“…However, this would require having a reference sample (without depletion) for every patient sample, which might not be feasible in practice. Alternatively, targeted qPCR absolute quantification methods for both bacterial and host DNA can be used to predict library composition for WMS and help determine needed sequencing depth ( Cho et al, 2021 ). In our individual patient samples, 2 out of 6 samples did not yield sufficient material for both WMS library preparation and two qPCR reactions.…”

Section: Discussionmentioning

confidence: 99%

Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies

et al. 2022

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IntroductionLow microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization.MethodsThree depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples.ResultsThe only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness.DiscussionDespite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.

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“…Quantitative PCR (qPCR) using TaqMan Fast Advanced was used to confirm the presence and absolute abundance of S. maltophilia ( Fraser et al, 2019 ) and total-bacterial 16S rRNA ( Cho et al, 2021 ) following adapted protocols (full details in the Supplementary File ).…”

Section: Methodsmentioning

confidence: 99%

The airway microbiome of persons with cystic fibrosis correlates with acquisition and microbiological outcomes of incident Stenotrophomonas maltophilia infection

Bowron,

Acosta,

Thornton

et al. 2024

Front. Microbiol.

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RationaleChronic infection with Stenotrophomonas maltophilia in persons with cystic fibrosis (pwCF) has been linked to an increased risk of pulmonary exacerbations and lung function decline. We sought to establish whether baseline sputum microbiome associates with risk of S. maltophilia incident infection and persistence in pwCF.MethodspwCF experiencing incident S. maltophilia infections attending the Calgary Adult CF Clinic from 2010–2018 were compared with S. maltophilia-negative sex, age (+/−2 years), and birth-cohort-matched controls. Infection outcomes were classified as persistent (when the pathogen was recovered in ≥50% of cultures in the subsequent year) or transient. We assessed microbial communities from prospectively biobanked sputum using V3-V4 16S ribosomal RNA (rRNA) gene sequencing, in the year preceding (Pre) (n = 57), at (At) (n = 22), and after (Post) (n = 31) incident infection. We verified relative abundance data using S. maltophilia-specific qPCR and 16S rRNA-targeted qPCR to assess bioburden. Strains were typed using pulse-field gel electrophoresis.ResultsTwenty-five pwCF with incident S. maltophilia (56% female, median 29 years, median FEV1 61%) with 33 total episodes were compared with 56 uninfected pwCF controls. Demographics and clinical characteristics were similar between cohorts. Among those with incident S. maltophilia infection, sputum communities did not cluster based on infection timeline (Pre, At, Post). Communities differed between the infection cohort and controls (n = 56) based on Shannon Diversity Index (SDI, p = 0.04) and clustered based on Aitchison distance (PERMANOVA, p = 0.01) prior to infection. At the time of incident S. maltophilia isolation, communities did not differ in SDI but clustered based on Aitchison distance (PERMANOVA, p = 0.03) in those that ultimately developed persistent infection versus those that were transient. S. maltophilia abundance within sputum was increased in samples from patients (Pre) relative to controls, measuring both relative (p = 0.004) and absolute (p = 0.001). Furthermore, S. maltophilia abundance was increased in sputum at incident infection in those who ultimately developed persistent infection relative to those with transient infection, measured relatively (p = 0.04) or absolute (p = 0.04), respectively.ConclusionMicrobial community composition of CF sputum associates with S. maltophilia infection acquisition as well as infection outcome. Our study suggests sputum microbiome may serve as a surrogate for identifying infection risk and persistence risk.

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Two-Target Quantitative PCR To Predict Library Composition for Shallow Shotgun Sequencing

Abstract: When determining human microbiota composition, shotgun sequencing is a powerful tool that can generate large amounts of data. However, in sample compositions with low or variable microbial density, shallowing sequencing can negatively affect resulting data.

Cited by 5 publications

References 17 publications

Performance Characteristics of Next-Generation Sequencing for the Detection of Antimicrobial Resistance Determinants in Escherichia coli Genomes and Metagenomes

Performance Characteristics of Next-Generation Sequencing for the Detection of Antimicrobial Resistance Determinants in Escherichia coli Genomes and Metagenomes

Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies

The airway microbiome of persons with cystic fibrosis correlates with acquisition and microbiological outcomes of incident Stenotrophomonas maltophilia infection

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