DropSynth 2.0: high-fidelity multiplexed gene synthesis in emulsions

Abstract

Multiplexed assays allow functional testing of large synthetic libraries of genetic elements, but are limited by the designability, length, fidelity and scale of the input DNA. Here we improve DropSynth, a low-cost, multiplexed method which builds gene libraries by compartmentalizing and assembling microarray-derived oligos in vortexed emulsions. By optimizing enzyme choice, adding enzymatic error correction, and increasing scale, we show that DropSynth can build thousands of gene-length fragments at >20% fidelity. Main TextMultiplexed functional assays link gene function or regulation to activities that can be read by next-generation sequencing such as through enrichment screens (cellular growth 1 , cell sorting 2,3 , binding 4,5 ) or transcriptional reporters 6 . Multiplexed assays can functionally assess thousands of different sequences in a single pooled experiment, and are thus powerful approaches for understanding how sequence affects function 7 . The DNA sequences to test are produced by genome fragmentation 8 , mutagenesis of existing sequences 9 , or by direct synthesis of oligonucleotides (oligos) 10 . Direct oligo synthesis allows for testing controlled hypotheses against one another without the constraints of natural variation or mutagenesis. However, individual oligos are generally shorter than 200 nucleotides (nt), limiting potential applications. Gene synthesis from oligo libraries can be used to extend these lengths 11,12 , but the high cost of individual assembly and processing becomes prohibitive for large gene libraries.To address these concerns, we previously developed a low-cost, multiplexed method termed DropSynth, which is capable of building large gene libraries from microarray-derived oligos 13 .

High-throughput discovery and characterization of human transcriptional effectors

“…Future improvements in synthesis length can be expected to enable more complete domain libraries. Additionally, larger sequences could potentially be accessed with alternative cloning approaches, including using oligonucleotide assembly methods (Sidore et al, 2020).…”

Massively parallel phenotyping of variant impact in cancer with Perturb-seq reveals a shift in the spectrum of cell states induced by somatic mutations

“…These calculations account for a 65% rate of variant barcode detection (as observed in our study), 20 cells per variant (sufficient for detecting the largest effect sizes per variant), and would amount to 400 cells per amino acid position, allowing us to detect lower impact variants by transferring information across similar variants when modeling variant impact. As both large-scale single cell profiling and DNA synthesis technologies are rapidly increasing in scale, at lower costs (Datlinger et al, 2019; Ma et al, 2020; Sidore et al, 2019), such comprehensive experiments are now within reach.…”

A Multiplexed Bacterial Two-Hybrid for Rapid Characterization of Protein-Protein Interactions and Iterative Protein Design

“…Furthermore, for the investigator who is not interested in an all-against-all approach, synthesis allows the explicit pairings of only certain proteins. While we benefited from the short length of our proteins of interest, recent pooled gene synthesis techniques 28,29 can be used to interrogate much larger proteins. Deconvoluting library diversity has also been a challenge for other multiplexed assays.…”