Liquid drop of DNA libraries reveals total genome information

Abstract

Conventional “bulk” PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.

Implementation of High-Throughput Sequencing (HTS) in Aptamer Selection Technology

“…oPCR failed to amplify approximately 67% of the sequences with a strong tendency to lose GC-rich ones [67]. Similar results were reported in the extensive study of the PCR amplification process of randomized RNA [63] and DNA [66] libraries using HTS. Interestingly, the selection of DNA aptamers for ovarian cancer biomarker CA125 resulted in the isolation of four sequences that were characterized as aptamer candidates [65].…”

“…A similar HTS experiment compared non-targeted and targeted selections against different mammary carcinoma cell lines using an RNA library with a 51 nt length of the random region [66]. The changes in the nucleotide and dinucleotide distribution detected in this experiment also indicated the presence of bias in non-targeted selection, though the favor was exhibited for the accumulation of pyrimidines, while the A content decreased.…”

“…The changes in the nucleotide and dinucleotide distribution detected in this experiment also indicated the presence of bias in non-targeted selection, though the favor was exhibited for the accumulation of pyrimidines, while the A content decreased. This difference can be attributed to the nature of the initial library and influence of primer binding sites [66]. Both experiments indicated the non-targeted propagation of certain sequences.…”

Compartmentalized CRISPR Reactions (CCR) for High-Throughput Screening of Guide RNA Potency and Specificity

“…This too requires transcribing, purifying, and combining gRNAs individually with Cas9. Assessing large numbers of spacer/target/off-target combinations presents many practical difficulties [40, 41] for the purpose of determining most effective and most precise gRNA for a given application.…”

DNA storage in thermoresponsive microcapsules for repeated random multiplexed data access

“…Thus current strategies for retrieval of multiple DNA files are based on physically separating reactions and amplifying each individual file using multiple singleplex PCR reactions 8,35,36 . Parallel amplification in a single reaction vessel has been realized using emulsion PCR, which segregates DNA templates using water-in-oil droplets and prevents the formation of artifacts 37,38 . Although emulsion PCR has been employed in DNA data retrieval 8,34 , the elaborate workflow, destructive nature and large quantities of organic solvents makes it unattractive for large-scale adoption.…”