DNARNASequenzierungsmethoden

DNA and RNA sequencing are set of technologies used to determine the order of nucleotides in genetic material and to measure the transcripts present in a cell or tissue. DNA sequencing focuses on the genome, revealing genetic variation, structure, and evolution. RNA sequencing, or RNA-Seq, analyzes the transcriptome to assess gene expression, splice variants, and RNA editing.

Historically, Sanger sequencing established the basic method for reading DNA sequences but is limited to relatively short reads. The rise of next-generation sequencing (NGS) introduced massively parallel methods capable of producing millions to billions of short reads in a single run, with Illumina sequencing by synthesis being the most widely used platform. Other NGS technologies include Ion Torrent and SOLiD. Third-generation sequencing emphasizes single-molecule, real-time approaches and long reads, notably PacBio’s SMRT sequencing and Oxford Nanopore Technologies, which improve genome assembly and detection of structural variants despite higher per-read error rates.

RNA-Seq workflows typically begin with RNA extraction, conversion to complementary DNA (cDNA), library preparation, and sequencing. This enables quantitative measurement of gene expression, discovery of novel transcripts, and analysis of alternative splicing. Long-read RNA sequencing is increasingly used to resolve complex isoforms.

Data analysis involves quality control, alignment to reference genomes, and various downstream analyses. Common outputs include FASTQ, BAM/CRAM, and VCF files. Tools exist for read alignment, transcript quantification, variant calling, and differential expression analysis.

Applications span basic biology, medical genetics, oncology, infectious disease, and evolutionary studies. Limitations include sequencing biases, error profiles, data storage needs, and interpretation challenges. Ethical considerations and data sharing practices are important in clinical and population contexts.