The resulting completed libraries can be pooled into a single sequencing run that is then “demultiplexed” during data analysis.įor specific information about how to prepare a sequencing library for the immune repertoire, see our page on immune repertoire sequencing. If multiple samples are to be sequenced in a single sequencing run, a unique identifier, or barcode, is additionally ligated to the amplicon. The adapter ligation step essentially bookends the amplified DNA or cDNA fragments, called amplicons, with specific oligonucleotide sequences that will interact with the surface of a sequencing flow cell. The primers used in library preparation are designed based on the sequences of interest, which range from a whole genome to particular RNA transcripts. PCR amplification yields a collection of specifically sized DNA fragments – called a library – that are compatible with the sequencing system to be used. If RNA is the starting template, an additional step is needed in which the RNA is first converted to cDNA by reverse transcription. Preparation of a sequencing library from your RNA or DNA sample involves two basic steps: 1) amplification to yield a pool of appropriately sized target sequences, and 2) the addition of sequencing adapters that will later interact with the NGS platform. To learn more about sample extraction for immune repertoire sequencing, see our sample preparation guide Step 2: Library preparation “Pure” DNA generally reports an A260/280 reading of 1.8, whereas RNA is closer to 2.0 The purity of a nucleic acid sample is typically expressed as an A260/280 value, which can be determined using a spectrophotometer such as Thermo Scientific’s Nanodrop (Cat. Various extraction protocols based on the starting material are available, and generally each extraction method has been optimized to yield the highest quality and largest amount of nucleic acid from the respective sample type. Following extraction, the amount and quality of DNA or RNA should be determined, as high-quality starting material is critical for successful sequencing. NGS can be performed on any sample that yields DNA or RNA (e.g., cell cultures, fresh-frozen tissues, formalin-fixed paraffin-embedded (FFPE) tissues, blood, saliva, and bone marrow ). The entire NGS workflow can be broken down into four steps: sample extraction, library preparation, sequencing, and analysis Step 1: Sample extraction Next generation sequencing (NGS) has become a powerful tool to identify genetic variants and variable gene expression patterns that correlate with disease state and provide clinically-relevant mechanistic insights.
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