RESUMEN
Next-generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR-based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG-seq primer set (MIG-seq being a PCR method enabling library construction with low-quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops. This innovation, named degenerate oligonucleotide primer MIG-seq (dpMIG-seq), enabled a streamlined protocol for constructing dpMIG-seq libraries from unpurified DNA, which was implemented stably in several crop species, including fruit trees. Furthermore, dpMIG-seq facilitated efficient lineage selection in wheat and enabled linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean without necessitating DNA concentration adjustments. These findings underscore the potential of the dpMIG-seq protocol for advancing genetic analyses across diverse plant species.
Asunto(s)
Técnicas de Genotipaje , Secuenciación de Nucleótidos de Alto Rendimiento , Reacción en Cadena de la Polimerasa , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reacción en Cadena de la Polimerasa/métodos , Técnicas de Genotipaje/métodos , Cartilla de ADN/genética , Sitios de Carácter Cuantitativo/genética , Oryza/genética , Triticum/genética , Solanum lycopersicum/genética , Mapeo Cromosómico , ADN de Plantas/genética , Glycine max/genética , Biblioteca de Genes , Polimorfismo Genético , Productos Agrícolas/genética , GenotipoRESUMEN
As a key supporting technology in the fields of life sciences and medicine, high-throughput sequencing has developed rapidly and become increasingly mature. The workflow of this technology can be divided into nucleic acid extraction, library construction, sequencing, and data analysis. Among these, library construction is a pivotal step that bridges the previous and subsequent stages. The effectiveness of library construction is contingent on the quality of upstream samples and also impacts the data analysis following sequence data output. The selection and implementation of library construction quality control techniques are crucial for enhancing the reliability of results and reducing errors in sequencing data. This review provides an in-depth discussion of library construction quality control techniques, summarizing and evaluating their principles, advantages and disadvantages, and applicability. It also discusses the selection of relevant technologies in practical application scenarios. The aim is to offer theoretical foundations and references for researchers, disease prevention and control personnel, and others when choosing library quality control techniques, thereby promoting the quality and efficiency of high-throughput sequencing work.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reproducibilidad de los Resultados , Biblioteca de Genes , Clonación Molecular , Control de Calidad , Análisis de Secuencia de ADN/métodosRESUMEN
This chapter describes the most practical manual and automated methods of isolating genomic DNA and RNA from different sources. It also summarizes currently popular methods of quality control (QC) for genomic DNA, RNA, and next generation sequencing (NGS) libraries.