Next-generation sequencing allows the profiling of sequences from transcriptomes and genomes and DNA-protein interaction. The techniques used are an integral component of the research process and discoveries in the field of biology. The capability to quickly create large volumes of sequence information offers various applications, speeding advancements in research and transforming our understanding of the human body’s health and diseases.
PREPARING SEQUENCING LIBRARIES FOR SEQUENCING
For DNA, or cDNA (synthesized using the RNA), to be sequenced, it must be separated, repaired at the end and put into libraries for sequencing. Put the term sequencing libraries refers to pools of DNA fragments containing adapter sequences that work with a particular sequencing platform and indexing barcodes that allow for identifying unique samples. The primary methods for library preparation include ligation-based library prep, segmentation-based library preparation and amplicon library preparation. The particular protocol you select depends on your sequencing platform and the downstream analysis. The most fundamental steps for library preparation include fragmentation, end repair, as well as the addition of adapters and (optional) Amplification of PCR:
- End repair and fragmentation Short-read sequencing techniques like Illumina cannot easily analyze very long DNA strands. As a result, DNA must be broken down into smaller, uniform pieces. After the fragmentation process, the DNA fragments are either repaired or polished at the end. A single Adenine base is added to create an overhang through the A-tailing process. This A overhang permits adapters with only a single thymine base to pair DNA fragments. When performing RNA-seq, RNA must first be transcribed into cDNA. The fragmentation process can be carried out prior to or following cDNA synthesizing.
- The addition of adapters occurs after fragmentation (or when it comes to library tagmentation preparation in conjunction with fragmentation). Adapters are permanently attached to the edges of DNA fragments. The adapters have multiple uses. They can transfer sequences into a flow cell, ensure compatibility with specific sequencing platforms, including barcodes (also known as indexes) to help identify the samples, and allow multiplexing in both target enrichment and sequencing. IDT has a range of options available in NGS adapters and indexing primers.
- Amplification of PCR (optional): The decision to amplify your libraries using PCR will depend on your research objectives and sample input, as well as whether you have to amplify a specific region(s) within the genome and the adapter type you choose to use. After PCR amplifying, any remaining oligonucleotides or small fragments should be eliminated. Clean-up of PCR can be accomplished by using magnetic beads or spin columns.
Applications for DOWNSTREAM
Many downstream applications work with NGS. The selection of the correct library preparation procedure can dramatically affect the accuracy and quality that you can get your data. Applications that are downstream include:
- Whole Genome Sequencing (WGS)
- PCR-amplified or PCR-free sequencing
- The detection of germline-inherited SNPs (SNPs) as well, Copy number variations (CNVs) as well as deletions or insertions (indels)
- Low-frequency somatic variation detection singular nucleotide variation (SNVs) CNVs and indels
- Target enrichment employs to capture hybridization or amplicon sequence of specific areas within the genome (e.g. transcriptions or exomes) of interest)specific areas in the genome (e.g. the transcripts or exomes of interest)
- RNA-seq
- Metagenomic sequencing
