Nanodroplets persisted in solution longer than microbubbles.

<p><b>A)</b> Flow chart outlining method for production of nanodroplets. <b>B)</b> A persistence study was performed in the ultrasonic bath to compare nanodroplets and microbubbles. An Accusizer particle sizing system (Particle Sizing Systems, Port Richey, FL) was used to measure the microbubble and nanodroplet concentrations at specific time points between 0 and 300 seconds (5 minutes). Nanodroplets maintained between 10–20% of their initial concentration as far out as 3 minutes into the sonication treatment, while the microbubble concentration dropped to 10% after 1 second.</p

Nanodroplet-mediated DNA fragmentation compared to a commercial method.

<p><b>(A)</b> Flow chart outlining method for comparing DNA fragmentation methods. <b>(B)</b> False gel picture from Agilent D1000 ScreenTape system showing DNA fragment size distribution in base pairs for samples fragmented in the Covaris E110 sonicator. Purple bars indicate the upper (1,500 bp) molecular weight marker and green bars indicate the lower (25 bp) molecular weight marker in each lane.</p

The use of nanodroplets allowed an ultrasonic water bath to fragment genomic DNA.

<p><b>(A)</b> Schematic showing the ultrasonic bath used for sonication. Samples were immobilized in the water bath using a stand with a tube rack attached. The circulating water chiller was optional. Water chilled to four degrees Centigrade can be added just prior to sonication, with no loss in DNA fragmentation efficiency. <b>(B)</b> A time-titration was performed with samples with and without nanodroplets. Following fragmentation, samples were run on a 1.5% agarose gel and visualized using SYBR green. DNA ladder sizes are indicated in base pairs. <b>(C)</b> Arrangement of DNA samples fragmented in the ultrasonic bath with and without samples to produce <b>(D)</b> an acoustic field map of the bath. The fragmentation ability (base pair size) is visualized with the s1color bar, where red indicates complete fragmentation in the 200–500 bp range.</p

DNA fragmentation in an ultrasonic water bath compared to a commercially available device.

<p><b>(A)</b> Flow chart outlining method for comparing DNA fragmentation methods.</p

Nanodroplets were an effective cavitation agent for use in DNA fragmentation.

<p><b>A)</b> The effectiveness of nanodroplets as a cavitation enhancement agent after multiple freeze-thaw cycles was tested. DNA ladder size is indicated in base pairs. Input is DNA prior to sonication with nanodroplets. <b>B)</b> Comparison of DNA fragmentation efficiency after two minutes in glass (Lanes 1–3) versus plastic (Lanes 4–6) tubes in the Covaris E110 sonicator. The addition of nanodroplets to Covaris microTUBES produces a DNA fragment size distribution comparable to the microTUBES used with the supplied rod (compare Lanes 1 and 3). DNA fragmented in glass microTUBES had a smaller DNA size distribution compared to plastic 0.2 mL PCR tubes (compare Lanes 3 and 5–6). DNA ladder size is indicated in base pairs.</p

Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath

<div><p>A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation.</p></div

<i>Saccharomyces cerevisiae</i> gDNA (BY4741) fragmented with nanodroplets in an ultrasonic bath was comparable in quality to DNA fragmented using a commercial method.

<p><b>(A)</b> Agilent D1000 ScreenTape system traces for DNA samples in microTUBES with the rod (left panel) or microTUBES with nanodroplets (right panel) that were subjected to sequencing. Average size is indicated in base pairs (bp). DNA size markers are denoted by Upper and Lower. <b>(B)</b> Traces showing similar size distribution of DNA after sequencing library preparation. Average size is indicated in base pairs (bp). DNA size markers are denoted by Upper and Lower. <b>(C)</b> Mapping sequencing reads to the <i>Saccharomyces cerevisiae</i> (S288c) reference genome is comparable in detection of single nucleotide variations, insertions, and deletions. Abundance and profile of relative errors in sequencing reads does not indicate a difference in the presence of error bias in the data.</p

<i>Saccharomyces cerevisiae</i> gDNA (BY4741) fragmented with nanodroplets in an ultrasonic water bath was comparable in quality to DNA fragmented in a commercially available device.

<p><b>(A)</b> Agilent D1000 ScreenTape data showing size distribution of DNA fragmented in tubes without (left panel) or tubes with nanodroplets (right panel). Average size is indicated in base pairs (bp). DNA size markers are denoted by Upper and Lower. <b>(B)</b> False gel picture indicating that DNA fragmented without nanodroplets had an average fragment size >1,500 bp. Purple bars indicate the upper (1,500 bp) molecular weight marker and green bars indicate the lower (25 bp) molecular weight marker in each lane. <b>(C)</b> Size distribution of DNA after sequencing library preparation. Average size is shown in base pairs (bp). DNA size markers are denoted by Upper and Lower. <b>(D)</b> Mapping sequencing reads to the <i>Saccharomyces cerevisiae</i> (S288c) reference genome is comparable in detection of single nucleotide variations and indels in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133014#pone.0133014.g004" target="_blank">Fig 4C</a>. Abundance and profile of relative errors in sequencing reads does not indicate a difference in the presence of error bias in the data compared to data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133014#pone.0133014.g004" target="_blank">Fig 4C</a>.</p

The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts

<div><p>Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01087307" target="_blank">NCT01087307</a></p></div

Structural changes detected in skin fibroblast clones D1-L-F1 and D2-L-F.

<p>(A) All genome changes detected in D1-L-F1 and D2-L-F clones. The tracks numbered from innermost are as follows: 1—structural changes. Green = deletions, black = duplications, blue = inversions and red = translocations. 2—deletions as detected by read-depth analyses. 3—amplifications as detected by read-depth analyses. 4—LOH events. 5—somatic SNVs, black dots are heterozygous SNVs and red dots are homozygous SNVs. (B) Schematic describing the chr19, chr20 translocation in D1-L-F1. Black rectangles depict region wherein translocation event was detected with a concomitant change in copy number.</p