PMID- 32076089
OWN - NLM
STAT- MEDLINE
DCOM- 20201120
LR  - 20210218
IS  - 2045-2322 (Electronic)
IS  - 2045-2322 (Linking)
VI  - 10
IP  - 1
DP  - 2020 Feb 19
TI  - DNA extraction of microbial DNA directly from infected tissue: an optimized 
      protocol for use in nanopore sequencing.
PG  - 2985
LID - 10.1038/s41598-020-59957-6 [doi]
LID - 2985
AB  - Identification of bacteria causing tissue infections can be comprehensive and, in 
      the cases of non- or slow-growing bacteria, near impossible with conventional 
      methods. Performing shotgun metagenomic sequencing on bacterial DNA extracted 
      directly from the infected tissue may improve time to diagnosis and targeted 
      treatment considerably. However, infected tissue consists mainly of human DNA 
      (hDNA) which hampers bacterial identification. In this proof of concept study, we 
      present a modified version of the Ultra-Deep Microbiome Prep kit for DNA 
      extraction procedure, removing additional human DNA. Tissue biopsies from 3 
      patients with orthopedic implant-related infections containing varying degrees of 
      Staphylococcus aureus were included. Subsequent DNA shotgun metagenomic 
      sequencing using Oxford Nanopore Technologies' (ONT) MinION platform and ONTs 
      EPI2ME WIMP and ARMA bioinformatic workflows for microbe and antibiotic 
      resistance genes identification, respectively. The modified DNA extraction 
      protocol led to an additional ~10-fold reduction of human DNA while preserving S. 
      aureus DNA. Including the DNA sequencing and bioinformatics analyses, the 
      presented protocol has the potential of identifying the infection-causing 
      pathogen in infected tissue within 7 hours after biopsy. However, due to low 
      number of S. aureus reads, positive identification of antibiotic resistance genes 
      was not possible.
FAU - Helmersen, Karin
AU  - Helmersen K
AD  - Akershus University Hospital, Department of Microbiology and Infection Control, 
      Lorenskog, 1478, Norway.
FAU - Aamot, Hege Vangstein
AU  - Aamot HV
AD  - Akershus University Hospital, Department of Microbiology and Infection Control, 
      Lorenskog, 1478, Norway. hege.vangstein.aamot@ahus.no.
AD  - Akershus University Hospital and University of Oslo, Department of Clinical 
      Molecular Biology (Epigen), Lorenskog, 1478, Norway. 
      hege.vangstein.aamot@ahus.no.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20200219
PL  - England
TA  - Sci Rep
JT  - Scientific reports
JID - 101563288
RN  - 0 (Anti-Bacterial Agents)
RN  - 0 (DNA, Bacterial)
RN  - 0 (Reagent Kits, Diagnostic)
SB  - IM
MH  - Anti-Bacterial Agents/pharmacology/therapeutic use
MH  - Biopsy
MH  - DNA, Bacterial/*isolation & purification
MH  - Drug Resistance, Bacterial/genetics
MH  - High-Throughput Nucleotide Sequencing
MH  - Humans
MH  - Metagenome/genetics
MH  - Metagenomics/*instrumentation
MH  - Nanopore Sequencing
MH  - Proof of Concept Study
MH  - Prosthesis-Related Infections/diagnosis/drug therapy/microbiology/pathology
MH  - *Reagent Kits, Diagnostic
MH  - Sequence Analysis, DNA
MH  - Staphylococcal Infections/*diagnosis/drug therapy/microbiology/pathology
MH  - Staphylococcus aureus/drug effects/genetics/*isolation & purification
PMC - PMC7031281
COIS- The authors declare no competing interests.
EDAT- 2020/02/23 06:00
MHDA- 2020/11/21 06:00
PMCR- 2020/02/19
CRDT- 2020/02/21 06:00
PHST- 2019/11/01 00:00 [received]
PHST- 2020/01/08 00:00 [accepted]
PHST- 2020/02/21 06:00 [entrez]
PHST- 2020/02/23 06:00 [pubmed]
PHST- 2020/11/21 06:00 [medline]
PHST- 2020/02/19 00:00 [pmc-release]
AID - 10.1038/s41598-020-59957-6 [pii]
AID - 59957 [pii]
AID - 10.1038/s41598-020-59957-6 [doi]
PST - epublish
SO  - Sci Rep. 2020 Feb 19;10(1):2985. doi: 10.1038/s41598-020-59957-6.