Illustration of the process workflow.

<p>1.) Cells are diluted in alginate polymer to a concentration of approximately 10⁵ cells per microliter, resulting in a 10% occupancy rate in 100 μm microspheres. 2.) Cells are lysed using heat, and the bulk microsphere solids are mixed with reagents for a 2-step whole genome amplification reaction. 3.) After amplification, microspheres are diluted to extinction in a 384 well plate, and scanned for presence of single microspheres that fluoresce with PicoGreen DNA stain. 4.) An isolated microsphere is transferred to a fresh tube and the DNA products are recovered by dissolving the alginate matrix. These amplified products are submitted to further rounds of amplification to add sequencing adapters. 5.) The products are prepared for high throughput sequencing using the Illumina MiSeq platform. Intermediate steps include fluorescence microscopy and quantitative PCR for quality control.</p

Sequence coverage of the <i>Rhodobacter sphaeroides</i> genome after whole genome amplification.

<p>Sequence coverage of the <i>Rhodobacter sphaeroides</i> genome after whole genome amplification.</p

Results from Illumina MiSeq paired-end sequencing run, 2 × 150 bp.

<p>Results from Illumina MiSeq paired-end sequencing run, 2 × 150 bp.</p

Strategy for WGA in two steps.

<p>This diagram outlines the whole genome amplification strategy in 2 steps, plus an additional step for adding the full length sequencing adapters. In Step 1, primers with 2 different tag sequences are added in a 50:50 mix. The primers anneal to the template, and a thermophilic strand-displacing enzyme (Vent exo- polymerase) is used to generate a population of fragments. For Step 2, the tag sequences are used as primers to further amplify the population of fragments. Half of the fragments are expected to have two different primer sequences on each end. After isolation of a microsphere containing fluorescent products, the sample is processed in a third reaction for addition of the full length sequencing adapters, or tails.</p

A Simple Method for Encapsulating Single Cells in Alginate Microspheres Allows for Direct PCR and Whole Genome Amplification

<div><p>Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. <i>Rhodobacter sphaeroides</i> cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to <i>R. sphaeroides</i>. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk reactions, although further work must be done to improve the amplification coverage of single genomes.</p></div

Effects of cell loading rate, visualized before and after PCR.

<p>Microscope images of 100 μm alginate microspheres containing: (A) no cells, (B) one cell per microsphere, and (C) multiple cells per microsphere. After PCR or whole genome amplification (2-step method), the DNA is stained by GelGreen and visualized by fluorescence microscopy. Images show: (D) no amplification, (E) amplification that is characteristic for one cell per microsphere, and (F) amplification of many cells, showing multiple foci of amplification and “comet tails” from cells that are trapped in the border region of the microsphere.</p

Primers for 2-step PCR whole genome amplification, tailing, and qPCR.

<p>Primers for 2-step PCR whole genome amplification, tailing, and qPCR.</p