Whole-Genome Amplification—Surveying Yield, Reproducibility, and Heterozygous Balance, Reported by STR-Targeting MIPs

Whole-genome amplification is a crucial first step in nearly all single-cell genomic analyses, with the following steps focused on its products. Bias and variance caused by the whole-genome amplification process add numerous challenges to the world of single-cell genomics. Short tandem repeats are sensitive genomic markers used widely in population genetics, forensics, and retrospective lineage tracing. A previous evaluation of common whole-genome amplification targeting ~1000 non-autosomal short tandem repeat loci is extended here to ~12,000 loci across the entire genome via duplex molecular inversion probes. Other than its improved scale and reduced noise, this system detects an abundance of heterogeneous short tandem repeat loci, allowing the allelic balance to be reported. We show here that while the best overall yield is obtained using RepliG-SC, the maximum uniformity between alleles and reproducibility across cells are maximized by Ampli1, rendering it the best candidate for the comparative heterozygous analysis of single-cell genomes

Summary of sequencing results and enrichment factors.

<p>The sequencing results of the input module from several hundred purified devices from Dam deficient bacteria are summarized and presented as the experimental enrichment factor of the device and several controls. Devices with activated and inactivated input modules (on the right and left, respectively) were tested with activated and inactivated Selection modules (blue and red, respectively). See text for explanations of activated and inactivated modules. They were then clone sequenced and their enrichment factor is presented on the Y axis. The results (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047795#pone-0047795-t001" target="_blank">table 1</a>) were normalized so that enrichment observed in experiments was compared to the results of the no-annealing, no-selection experiment.</p

Comparative summary of experimental results from various devices.

<p>The table describes four comparisons (rows 1–4) between the enrichment factor resulting from devices with various combinations of activated and inactivated selection and input modules.</p

Simulation of the enrichment landscape following re-annealing mediated activation of the input module.

<p><b>A.</b> The enrichment factor is computed as the ratio of correct (i.e. of the sequence we are enriching for) to total (correct+erroneous) homo-duplexes before re-annealing of the input module divided by the same ratio after its re-annealing. It is plotted as a function of the initial fraction of correct molecules and the number of unique erroneous molecules. <b>B.</b> magnified view of the specific zone in the enrichment space presented in A.</p

Programmable In Vivo Selection of Arbitrary DNA Sequences

<div><p>The extraordinary fidelity, sensory and regulatory capacity of natural intracellular machinery is generally confined to their endogenous environment. Nevertheless, synthetic bio-molecular components have been engineered to interface with the cellular transcription, splicing and translation machinery in vivo by embedding functional features such as promoters, introns and ribosome binding sites, respectively, into their design. Tapping and directing the power of intracellular molecular processing towards synthetic bio-molecular inputs is potentially a powerful approach, albeit limited by our ability to streamline the interface of synthetic components with the intracellular machinery in vivo. Here we show how a library of synthetic DNA devices, each bearing an input DNA sequence and a logical selection module, can be designed to direct its own probing and processing by interfacing with the bacterial DNA mismatch repair (MMR) system <em>in vivo</em> and selecting for the most abundant variant, regardless of its function. The device provides proof of concept for programmable, function-independent DNA selection <em>in vivo</em> and provides a unique example of a logical-functional interface of an engineered synthetic component with a complex endogenous cellular system. Further research into the design, construction and operation of synthetic devices <em>in vivo</em> may lead to other functional devices that interface with other complex cellular processes for both research and applied purposes.</p> </div

Overview of structure and operation principle of the synthetic device.

<p>A. The synthetic device is assembled <i>in vitro</i> using a 3-step process (top). It is then transformed to <i>E. coli</i> and processed <i>in vivo</i> by the MMR system according to the device's operating principles (middle). Finally, the output of the process is analyzed <i>in vitro</i> by purifying the devices out of bacteria and DNA sequencing them (bottom) <b>B.</b> Description of device components: the device library consists of (1) an input module containing many different variants of the same gene (green) and (2) a Selection module (blue) integrated within an Amp resistance gene (gray). The selection module contains a loop on its coding strand which frame-shifts (dark gray) and stops the translation (red stop codon) of the Amp gene. The device also bears a Kan resistance gene for noise reduction purposes. <b>C.</b> Schematic flow of device operation <i>in vivo</i>: if no mismatch is detected by the MMR (right, error free input scenario) no repair takes place, both strands are replicated and the heterozygous bacteria will live. Otherwise, if a mismatch is detected by the MMR (left, erroneous input scenario) repair synthesis spans the mismatch and hemi-methylated Dam site using the methylated, disrupted Amp strand as template and results in cell death.</p