Optimized PCR conditions minimizing the formation of chimeric DNA molecules from MPRA plasmid libraries

Abstract

Background Massively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of various DNA regulatory elements and their mutant variants. The assays are based on construction of highly diverse plasmid libraries containing two variable fragments, a region of interest (a sequence under study; ROI) and a barcode (BC) used to uniquely tag each ROI, which are separated by a constant spacer sequence. The sequences of BC–ROI combinations present in the libraries may be either known a priori or not. In the latter case, it is necessary to identify these combinations before performing functional experiments. Typically, this is done by PCR amplification of the BC–ROI regions with flanking primers, followed by next-generation sequencing (NGS) of the products. However, chimeric DNA molecules formed on templates with identical spacer fragment during the amplification process may substantially hamper the identification of genuine BC–ROI combinations, and as a result lower the performance of the assays. Results To identify settings that minimize formation of chimeric products we tested a number of PCR amplification parameters, such as conventional and emulsion types of PCR, one- or two-round amplification strategies, amount of DNA template, number of PCR cycles, and the duration of the extension step. Using specific MPRA libraries as templates, we found that the two-round amplification of the BC–ROI regions with a very low initial template amount, an elongated extension step, and a specific number of PCR cycles result in as low as 0.30 and 0.32% of chimeric products for emulsion and conventional PCR approaches, respectively. Conclusions We have identified PCR parameters that ensure synthesis of specific (non-chimeric) products from highly diverse MPRA plasmid libraries. In addition, we found that there is a negligible difference in performance of emulsion and conventional PCR approaches performed with the identified settings. Electronic supplementary material The online version of this article (10.1186/s12864-019-5847-2) contains supplementary material, which is available to authorized users.

The genotype‐phenotype landscape of an allosteric protein

“…An additional barcode count threshold was applied, keeping only DNA barcodes with a total read count (across all 24 environments and 4 time points) greater than 2,000. A small number (139) of DNA barcodes were identified as likely chimeras with forward and reverse barcodes combined from different plasmid templates (Smyth et al, 2010; Schlecht et al, 2017; Omelina et al, 2019). The likely chimera barcodes were not used in further analysis.…”

Measuring Transcription Factor Binding and Gene Expression using Barcoded Self-Reporting Transposon Calling Cards and Transcriptomes

“…1A, site 2). This strategy would also include a long stretch of shared sequence present in all amplicons that would lead to extensive barcode swapping during the SRT amplification PCR step in library preparation 6,7 . Therefore, we sought to introduce a barcode directly into the TR itself (Fig.…”

“…The SRT consists of a promoter driving a selectable reporter flanked by the transposon terminal repeat sequences (TR). Introducing a barcode between the reporter gene and the TR, an approach used in our previous inverse PCR based DNA protocols 1 , would no longer be compatible with our SRT recovery protocol because the barcode would be ~300 bases away from the transposon-genome junction in the final PCR and having a long stretch of shared sequence present in all amplicons would lead to extensive barcode swapping 6,7 . Therefore, we sought to introduce a barcode directly into the TR itself, as close to the transposon-genome junction as possible to minimize the risks of barcode swapping.…”

Optimization of On-Bead Emulsion Polymerase Chain Reaction Based on Single Particle Analysis

“…We tested the effect on yield and percentage of saturated beads by raising each component's concentration as stated by another group of researchers who successfully developed an on-bead ePCR protocol for aptamer selection. [19] Since prior ePCR users reported using concentrations that are much higher than conventional PCR, we tested both higher and conventional concentrations. [23][24][25][26] Most ePCR applications increase the concentrations of each of these components by 4-to 20-fold compared with conventional PCR without documenting the change in outcome.…”

“…However, previous studies using end-point, ensemble-level analyses of the purified PCR products failed to elucidate these characteristics. [18][19][20][21] Thus, optimization of ePCR would be difficult According to Poisson statistics, there is a 'sweet spot' where the number of single-template drops is maximized with minimal number of multi-template droplets. This condition results in only ~20% of emulsion drops containing a template molecule.…”