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Abstract BackgroundModern plant breeding strategies rely on the intensive use of advanced genomic tools to expedite the development of improved crop varieties. Genomic DNA extraction from crop seeds eliminates the need to grow plants in contrast to fresh leaf tissue; however, it can still be a bottleneck due to the presence of stored compounds and the complexity of the matrix. The interaction of environmentally benign choline-based ionic liquids (ILs) with DNA offers an innovative approach to enhance the quality of extracted DNA from seeds. While prior IL-based plant DNA extraction workflows have primarily supported polymerase chain reaction (PCR) and quantitative PCR-based applications, their suitability for high-throughput sequencing (HTS) remained largely unexplored. This study explores the efficacy of IL-assisted method for genomic DNA extraction from soybean (Glycine max) seeds, addressing the limited application of ILs in HTS. ResultsThe optimized DNA extraction method, utilizing 25% (w/v) choline formate, enabled the recovery of high-purity DNA with abundant fragment sizes > 20 kb, suitable for downstream applications including PCR, whole genome amplification (WGA), simple sequence repeat (SSR) amplification, and high-throughput Illumina sequencing. The IL-method was benchmarked against a silica-binding method using cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) as lysis agents using a commercial plant DNA extraction kit in terms of DNA yield, purity, abundant DNA fragment size distribution, and integrity. In addition, DNA isolated from this method demonstrated successful PCR amplification of markers from both the nuclear and plastid genomes and yielded > 99% whole genome coverage with Illumina (PE150) sequencing reads. ConclusionsThis is the first known instance of a whole genome sequence generated from DNA extracted with ILs. These findings mark a significant milestone in establishing ILs as promising alternatives to conventional methods for seed DNA extraction, with potential utility in third generation (long-read) sequencing experiments.
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Rapid Identification of DNA Fragments through Direct Sequencing with Electro‐Optical Zero‐Mode Waveguides
Abstract In contrast to sequence‐specific techniques such as polymerase chain reaction, DNA sequencing does not require prior knowledge of the sample for surveying DNA. However, current sequencing technologies demand high inputs for a suitable library preparation, which typically necessitates DNA amplification, even for single‐molecule sequencing methods. Here, electro‐optical zero‐mode waveguides (eZMWs) are presented, which can load DNA into the confinement of zero‐mode waveguides with high efficiency and negligible DNA fragment length bias. Using eZMWs, highly efficient voltage‐induced loading of DNA fragments of various sizes from ultralow inputs (nanogram‐to‐picogram levels) is observed. Rapid DNA fragment identification is demonstrated by burst sequencing of short and long DNA molecules (260 and 20 000 bp) loaded from an equimolar picomolar‐level concentration mixture in just a few minutes. The device allows further studies in which low‐input DNA capture is essential, for example, in epigenetics, where native DNA is required for obtaining modified base information.
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- Award ID(s):
- 1931777
- PAR ID:
- 10391783
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Materials
- Volume:
- 34
- Issue:
- 9
- ISSN:
- 0935-9648
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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