TY - JOUR
T1 - An improved method for including upper size range plasmids in metamobilomes
AU - Norman, Anders
AU - Riber, Leise
AU - Luo, Wenting
AU - Li, Lili
AU - Hansen, Lars H.
AU - Sørensen, Søren Johannes
PY - 2014
Y1 - 2014
N2 - Two recently developed isolation methods have shown promise when recovering pure community plasmid DNA (metamobilomes/plasmidomes), which is useful in conducting culture-independent investigations into plasmid ecology. However, both methods employ multiple displacement amplification (MDA) to ensure suitable quantities of plasmid DNA for high-throughput sequencing. This study demonstrates that MDA greatly favors smaller circular DNA elements (<10 Kbp), which, in turn, leads to stark underrepresentation of upper size range plasmids (>10 Kbp). Throughout the study, we used two model plasmids, a 4.4 Kbp cloning vector (pBR322), and a 56 Kbp conjugative plasmid (pKJK10), to represent lower- and upper plasmid size ranges, respectively. Subjecting a mixture of these plasmids to the overall isolation protocol revealed a 34-fold over-amplification of pBR322 after MDA. To address this bias, we propose the addition of an electroelution step that separates different plasmid size ranges prior to MDA in order to reduce size-dependent competition during incubation. Subsequent analyses of metamobilome data from wastewater spiked with the model plasmids showed in silica recovery of pKJK10 to be very poor with the established method and a 1,300-fold overrepresentation of pBR322. Conversely, complete recovery of pKJK10 was enabled with the new modified protocol although considerable care must be taken during electroelution to minimize cross-contamination between samples. For further validation, non-spiked wastewater metamobilomes were mapped to more than 2,500 known plasmid genomes. This displayed an overall recovery of plasmids well into the upper size range (median size: 30 kilobases) with the modified protocol. Analysis of de novo assembled metamobilome data also suggested distinctly better recovery of larger plasmids, as gene functions associated with these plasmids, such as conjugation, was exclusively encoded in the data output generated through the modified protocol. Thus, with the suggested modification, access to a large uncharacterized pool of accessory elements that reside on medium-to-large plasmids has been improved.
AB - Two recently developed isolation methods have shown promise when recovering pure community plasmid DNA (metamobilomes/plasmidomes), which is useful in conducting culture-independent investigations into plasmid ecology. However, both methods employ multiple displacement amplification (MDA) to ensure suitable quantities of plasmid DNA for high-throughput sequencing. This study demonstrates that MDA greatly favors smaller circular DNA elements (<10 Kbp), which, in turn, leads to stark underrepresentation of upper size range plasmids (>10 Kbp). Throughout the study, we used two model plasmids, a 4.4 Kbp cloning vector (pBR322), and a 56 Kbp conjugative plasmid (pKJK10), to represent lower- and upper plasmid size ranges, respectively. Subjecting a mixture of these plasmids to the overall isolation protocol revealed a 34-fold over-amplification of pBR322 after MDA. To address this bias, we propose the addition of an electroelution step that separates different plasmid size ranges prior to MDA in order to reduce size-dependent competition during incubation. Subsequent analyses of metamobilome data from wastewater spiked with the model plasmids showed in silica recovery of pKJK10 to be very poor with the established method and a 1,300-fold overrepresentation of pBR322. Conversely, complete recovery of pKJK10 was enabled with the new modified protocol although considerable care must be taken during electroelution to minimize cross-contamination between samples. For further validation, non-spiked wastewater metamobilomes were mapped to more than 2,500 known plasmid genomes. This displayed an overall recovery of plasmids well into the upper size range (median size: 30 kilobases) with the modified protocol. Analysis of de novo assembled metamobilome data also suggested distinctly better recovery of larger plasmids, as gene functions associated with these plasmids, such as conjugation, was exclusively encoded in the data output generated through the modified protocol. Thus, with the suggested modification, access to a large uncharacterized pool of accessory elements that reside on medium-to-large plasmids has been improved.
U2 - 10.1371/journal.pone.0104405
DO - 10.1371/journal.pone.0104405
M3 - Journal article
C2 - 25116381
AN - SCOPUS:84905918111
SN - 1932-6203
VL - 9
JO - PLoS ONE
JF - PLoS ONE
IS - 8
M1 - e104405
ER -