Sterile paper points as a bacterial DNA-contamination source in microbiome profiles of clinical samples

2013 ◽  
Vol 41 (12) ◽  
pp. 1297-1301 ◽  
Author(s):  
Joyce van der Horst ◽  
Mark J. Buijs ◽  
Marja L. Laine ◽  
Daniël Wismeijer ◽  
Bruno G. Loos ◽  
...  
Keyword(s):  
Clinical Samples ◽  
Contamination Source ◽  
Bacterial Dna ◽  
Dna Contamination

scholarly journals Managing Contamination and Diverse Bacterial Loads in 16S rRNA Deep Sequencing of Clinical Samples: Implications of the Law of Small Numbers

mBio ◽  
2021 ◽  
Author(s):  
Ruben Dyrhovden ◽  
Martin Rippin ◽  
Kjell Kåre Øvrebø ◽  
Randi M. Nygaard ◽  
Elling Ulvestad ◽  
...  
Keyword(s):  
Infectious Disease ◽  
16S Rrna ◽  
Deep Sequencing ◽  
Gradual Increase ◽  
Clinical Samples ◽  
Materials Management ◽  
Bacterial Dna ◽  
Dna Contamination ◽  
Sequencing Studies ◽  
The Law

There has been a gradual increase in 16S deep sequencing studies on infectious disease materials. Management of bacterial DNA contamination is a major challenge in such diagnostics, particularly in low biomass samples.

scholarly journals Molecular diagnosis of orthopaedic device infection direct from sonication fluid by metagenomic sequencing

2017 ◽  
Author(s):  
Teresa L. Street ◽  
Nicholas D. Sanderson ◽  
Bridget L. Atkins ◽  
Andrew J. Brent ◽  
Kevin Cole ◽  
...  
Keyword(s):  
Diagnostic Information ◽  
Species Level ◽  
Metagenomic Sequencing ◽  
Tissue Samples ◽  
Bacterial Dna ◽  
Dna Contamination ◽  
Prosthetic Joint ◽  
Device Infection ◽  
Fluid Culture ◽  
Orthopaedic Device

AbstractCulture of multiple periprosthetic tissue samples is the current gold-standard for microbiological diagnosis of prosthetic joint infections (PJI). Additional diagnostic information may be obtained through sonication fluid culture of explants. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of complete bacterial DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI.We compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other orthopaedic device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 samples (derivation set), we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and validated our findings in 47 independent samples.Compared to sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69(88%,95%CI 77-94%) (derivation samples 35/38[92%,79-98%]; validation 26/31[84%,66-95%]), and genus-level sensitivity was 64/69(93%,84-98%). Species-level specificity, adjusting for plausible fastidious causes of infection, species found in concurrently obtained tissue samples, and prior antibiotics, was 85/97(88%,79-93%) (derivation 43/50[86%,73-94%], validation 42/47[89%,77-96%]). High levels of human DNA contamination were seen despite use of laboratory methods to remove it. Rigorous laboratory good practice was required to prevent bacterial DNA contamination.We demonstrate metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings combined with increasing availability of portable, random-access sequencing technology offers the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI.

scholarly journals Control of artefactual variation in reported inter-sample relatedness during clinical use of a Mycobacterium tuberculosis sequencing pipeline

2018 ◽  
Author(s):  
David H Wyllie ◽  
Nicholas Sanderson ◽  
Richard Myers ◽  
Tim Peto ◽  
Esther Robinson ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Read Depth ◽  
Pairwise Distance ◽  
Contact Tracing ◽  
Clinical Samples ◽  
Bacterial Dna ◽  
Consensus Sequences ◽  
Minor Variant ◽  
Validation Set ◽  
Genomic Regions

ABSTRACTContact tracing requires reliable identification of closely related bacterial isolates. When we noticed the reporting of artefactual variation between M. tuberculosis isolates during routine next generation sequencing of Mycobacterium spp, we investigated its basis in 2,018 consecutive M. tuberculosis isolates. In the routine process used, clinical samples were decontaminated and inoculated into broth cultures; from positive broth cultures DNA was extracted, sequenced, reads mapped, and consensus sequences determined. We investigated the process of consensus sequence determination, which selects the most common nucleotide at each position. Having determined the high-quality read depth and depth of minor variants across 8,006 M. tuberculosis genomic regions, we quantified the relationship between the minor variant depth and the amount of non-Mycobacterial bacterial DNA, which originates from commensal microbes killed during sample decontamination. In the presence of non-Mycobacterial bacterial DNA, we found significant increases in minor variant frequencies of more than 1.5 fold in 242 regions covering 5.1% of the M. tuberculosis genome. Included within these were four high variation regions strongly influenced by the amount of non-Mycobacterial bacterial DNA. Excluding these four regions from pairwise distance comparisons reduced biologically implausible variation from 5.2% to 0% in an independent validation set derived from 226 individuals. Thus, we have demonstrated an approach identifying critical genomic regions contributing to clinically relevant artefactual variation in bacterial similarity searches. The approach described monitors the outputs of the complex multi-step laboratory and bioinformatics process, allows periodic process adjustments, and will have application to quality control of routine bacterial genomics.

Comparison of the utility of five commercial kits for extraction of DNA from Aspergillus fumigatus spores.

2010 ◽  
Vol 57 (4) ◽  
Author(s):  
Urszula Nawrot ◽  
Katarzyna Wlodarczyk ◽  
Magdalena Wrobel ◽  
Anita Wasik ◽  
Tadeusz Dobosz
Keyword(s):  
Dna Extraction ◽  
Enzymatic Digestion ◽  
Cell Disruption ◽  
Clinical Samples ◽  
Rrna Gene ◽  
28S Rrna ◽  
Bacterial Dna ◽  
Blood Samples ◽  
Dna Yield ◽  
Commercial Kits

The aim of this study was to compare the efficiency of DNA extraction from water as well as from blood samples spiked with A. fumigatus spores, using selected commercial kits. Extraction of DNA according to manufacturer's protocols was preceded by blood cells lysis and disruption of fungal cells by enzymatic digestion or bead beating. The efficiency of DNA extraction was measured by PCR using Aspergillus-specific primers and SYBR Green I dye or TaqMan probes targeting 28S rRNA gene. All methods allowed the detection of Aspergillus at the lowest tested density of water suspensions of spores (10¹ cells/ml). The highest DNA yield was obtained using the ZR Fungal/Bacterial DNA kit, YeastStar Genomic DNA kit, and QIAamp DNA Mini kit with mechanical cell disruption. The ZR Fungal/Bacterial DNA and YeastStar kits showed the highest sensitivity in examination of blood samples spiked with Aspergillus (100 % for the detection of 10² spores and 75 % for 10¹ spores). Recently, the enzymatic method ceased to be recommended for examination of blood samples for Aspergillus, thus ZR Fungal/Bacterial DNA kit and QIAamp DNA Mini kit with mechanical cell disruption could be used for extraction of Aspergillus DNA from clinical samples.

scholarly journals A Novel Eukaryote-Made Thermostable DNA Polymerase Which Is Free from Bacterial DNA Contamination

2011 ◽  
Vol 49 (9) ◽  
pp. 3316-3320 ◽  
Author(s):  
Hideki Niimi ◽  
Masashi Mori ◽  
Homare Tabata ◽  
Hiroshi Minami ◽  
Tomohiro Ueno ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Bacterial Dna ◽  
Dna Contamination ◽  
Thermostable Dna Polymerase

Overcoming bacterial DNA contamination in real-time PCR and RT-PCR reactions for LacZ detection in cell therapy monitoring

2004 ◽  
Vol 18 (6) ◽  
pp. 437-441 ◽  
Author(s):  
Sylvie Tondeur ◽  
Onnik Agbulut ◽  
Marie-Laurence Menot ◽  
Jérôme Larghero ◽  
Denise Paulin ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Real Time ◽  
Real Time Pcr ◽  
Therapy Monitoring ◽  
Rt Pcr ◽  
Bacterial Dna ◽  
Dna Contamination

Identification of mixed bacterial DNA contamination in broad-range PCR amplification of 16S rDNA V1 and V3 variable regions by pyrosequencing of cloned amplicons

2003 ◽  
Vol 219 (1) ◽  
pp. 87-91 ◽  
Author(s):  
Niclas Grahn ◽  
Margaretha Olofsson ◽  
Katarina Ellnebo-Svedlund ◽  
Hans-Jürg Monstein ◽  
Jon Jonasson
Keyword(s):  
16S Rdna ◽  
Pcr Amplification ◽  
Variable Regions ◽  
Bacterial Dna ◽  
Dna Contamination

scholarly journals A Rapid Method for Detecting and Quantifying Bacterial DNA in Rust Fungal DNA Samples

2008 ◽  
Vol 98 (1) ◽  
pp. 115-119 ◽  
Author(s):  
C. W. Barnes ◽  
L. J. Szabo
Keyword(s):  
Real Time ◽  
Bacterial Contamination ◽  
Rust Fungus ◽  
Total Sample ◽  
Qpcr Assay ◽  
Bacterial Dna ◽  
Sequence Contigs ◽  
Dna Contamination ◽  
Fungal Dna ◽  
Contamination Levels

Bacterial DNA contamination of rust fungal DNA can be a significant problem for sequencing the rust fungus. Sequence assembly is much more difficult if the sequence contigs are mixed with bacterial sequence. A quantitative real-time polymerase chain reaction (qPCR) assay was developed to quantify bacterial DNA within rust fungal DNA samples and the results were compared with those obtained from traditional CFU counts. Real-time PCR showed higher values of DNA contamination than CFU. However, the ranking of samples from low to high for bacterial contamination was consistent between the methods. Reasons for the differences between the methods are discussed. The qPCR assay was tested by adding known quantities of Escherichia coli DNA to Puccinia graminis DNA samples. The assay reliably quantified bacterial contamination at ≥ 1.0% of the total sample DNA. When bacterial contamination was <1.0%, fungal DNA also occasionally was amplified, nullifying the quantification measurement. However, primer specificity was not simply the product of the ratio of bacterial DNA to fungal DNA. Bacterial contamination could be quantified below 1.0% if the bacterial DNA concentration was ≈70 pg/μl or greater. Therefore, spiking the fungal samples with a known concentration of E. coli bacterial DNA successfully eliminated the amplification of fungal DNA, making quantification of contaminating bacterial DNA possible for samples with low contamination levels.

scholarly journals Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples

Gut Pathogens ◽  
2016 ◽  
Vol 8 (1) ◽  
Author(s):  
Angela Glassing ◽  
Scot E. Dowd ◽  
Susan Galandiuk ◽  
Brian Davis ◽  
Rodrick J. Chiodini
Keyword(s):  
Bacterial Biomass ◽  
Bacterial Dna ◽  
Dna Contamination

scholarly journals Control of Artifactual Variation in Reported Intersample Relatedness during Clinical Use of a Mycobacterium tuberculosis Sequencing Pipeline

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
David H. Wyllie ◽  
Nicholas Sanderson ◽  
Richard Myers ◽  
Tim Peto ◽  
Esther Robinson ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Consensus Sequence ◽  
Read Depth ◽  
Pairwise Distance ◽  
Contact Tracing ◽  
Clinical Samples ◽  
Bacterial Dna ◽  
Content Type ◽  
Minor Variant ◽  
Genomic Regions

ABSTRACT Contact tracing requires reliable identification of closely related bacterial isolates. When we noticed the reporting of artifactual variation between Mycobacterium tuberculosis isolates during routine next-generation sequencing of Mycobacterium spp., we investigated its basis in 2,018 consecutive M. tuberculosis isolates. In the routine process used, clinical samples were decontaminated and inoculated into broth cultures; from positive broth cultures DNA was extracted and sequenced, reads were mapped, and consensus sequences were determined. We investigated the process of consensus sequence determination, which selects the most common nucleotide at each position. Having determined the high-quality read depth and depth of minor variants across 8,006 M. tuberculosis genomic regions, we quantified the relationship between the minor variant depth and the amount of nonmycobacterial bacterial DNA, which originates from commensal microbes killed during sample decontamination. In the presence of nonmycobacterial bacterial DNA, we found significant increases in minor variant frequencies, of more than 1.5-fold, in 242 regions covering 5.1% of the M. tuberculosis genome. Included within these were four high-variation regions strongly influenced by the amount of nonmycobacterial bacterial DNA. Excluding these four regions from pairwise distance comparisons reduced biologically implausible variation from 5.2% to 0% in an independent validation set derived from 226 individuals. Thus, we demonstrated an approach identifying critical genomic regions contributing to clinically relevant artifactual variation in bacterial similarity searches. The approach described monitors the outputs of the complex multistep laboratory and bioinformatics process, allows periodic process adjustments, and will have application to quality control of routine bacterial genomics.

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