Comparison of six commercial kits to extract bacterial chromosome and plasmid DNA for MiSeq sequencing.
We compared commercial kits for extraction of genomic DNA from the Gram-negative bacterium Klebsiella pneumoniae for subsequent Miseq sequencing. Purification of DNA was based on matrix binding (silica or anion exchange resin) or differential precipitation (salting out), respectively. The choice of extraction kit had little effect on sequencing quality and coverage across drastically different replicons, except for an apparent depletion of small plasmids (<5 kb) during precipitation-based extractions. Sequencing coverage provided copy-number estimates for small plasmids that were consistently higher than those from quantitative real-time PCR.
Robert Koch Institute, FG13 Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Wernigerode, Germany.
Laura
L
Becker
Leibniz Institute DSMZ, Braunschweig, Germany.
Matthias
M
Steglich
Robert Koch Institute, FG13 Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Wernigerode, Germany.
Stephan
S
Fuchs
Robert Koch Institute, FG13 Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Wernigerode, Germany.
Guido
G
Werner
Leibniz Institute DSMZ, Braunschweig, Germany.|German Centre of Infection Research (DZIF), Partner Site Hannover-Braunschweig, Braunschweig, Germany.
Ulrich
U
Nübel
D000837:Anion Exchange Resins / Q000737:chemistry; D011232:Chemical Precipitation; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000592:standards; D007711:Klebsiella pneumoniae / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D011786:Quality Control; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards; D012822:Silicon Dioxide / Q000737:chemistry
D000837:Anion Exchange Resins; D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic; D012822:Silicon Dioxide