RESUMO
The next-generation DNA sequencing workflows require an accurate quantification of the DNA molecules to be sequenced which assures optimal performance of the instrument. Here, we demonstrate the use of qPCR for quantification of DNA libraries used in next-generation sequencing. In addition, we find that qPCR quantification may allow improvements to current NGS workflows, including reducing the amount of library DNA required, increasing the accuracy in quantifying amplifiable DNA, and avoiding amplification bias by reducing or eliminating the need to amplify DNA before sequencing.
Assuntos
Biblioteca Gênica , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , Hibridização de Ácido Nucleico , Sensibilidade e EspecificidadeAssuntos
Carcinoma Ductal Pancreático/classificação , Perfilação da Expressão Gênica/métodos , MicroRNAs/genética , Neoplasias Pancreáticas/classificação , RNA Mensageiro/genética , Adenocarcinoma Mucinoso/classificação , Adenocarcinoma Mucinoso/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Papilar/classificação , Carcinoma Papilar/metabolismo , Análise por Conglomerados , Sondas de DNA/genética , Humanos , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/metabolismo , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , TranscriptomaRESUMO
We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software. The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 mul. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.