Next Generation Sequencing
NGS is a new technology for DNA and RNA sequencing and variant/mutation detection. This technology combines the advantages of unique sequencing chemistries, different sequencing matrices, and bioinformatics technology. Such a combination allows a massive parallel sequencing of various lengths of DNA or RNA sequences or even whole genome within a relatively short period of time. It is a revolutionary sequencing technology after Sanger sequencing. NGS involves several major steps in sequencing. For example, DNA NGS involves DNA fragmentation, library preparation, massive parallel sequencing, bioinformatics analysis, and variant/mutation annotation and interpretation.
NGS TECHNOLOGY STEPS:
1. DNA fragmentation:
DNA fragmentation is used to break the targeted DNA into many short segments, usually 100–300 bp in length. Different methods can be used to achieve this. DNA can be fragmented using mechanical methods, enzymatic digestion6, or other methods. For example, sonication can be used to break DNA into short segments. The short segments relevant to the targeted DNA sequences are pulled out using specific complementary probes of different designs7,8. This method is usually referred to as hybridization capture assay. Another method involves polymerase chain reaction (PCR) amplification. In this method, many pairs of primers are used to amplify the targeted DNA segments using PCR. The PCR products serve as short segments of targeted DNA. This method is usually called amplicon assay9,10. The DNA segments are then used for library preparation.
2. Library preparation :
Library preparation is a process by which DNA segments are modified so that each DNA sample can have a sample-specific index like sample identification which helps to identify the patient from whom DNA sequencing was performed. This process also allows the sequencing adaptors to be added to the DNA segments. Such modification allows the sequencing primers to bind to all the DNA segments and enables massive parallel sequencing later.
Massive parallel sequencing is performed using an NGS sequencer. The library is uploaded onto a sequencing matrix in a certain sequencer. Different sequencers have different sequencing matrices. For example, Illumina NGS sequencer uses flow cells and Ion Torrent NGS sequencer uses sequencing chips. However, its goal is the same, which is to allow massive parallel sequencing of all the DNA segments at the same time. The sequence information generated from such massive parallel sequencing is analyzed using bioinformatics software.
4. Bioinformatics analysis and data interpretation:
Bioinformatics analysis is a process involving base calling, read alignment, variant identification, and variant annotation11. During this process, the sequence information is compared to a human genome reference sequence to identify whether there are any variants/mutations in the targeted sequences. All information from each sequenced segment is pieced together to generate final sequencing results for the full length of the targeted DNA. The final sequencing results are sent back to the user for interpretation.
The annotation and interpretation processes are set to identify each variant and their possible biological/clinical significance. Read more
NGS CLINICAL APPLICATIONS:
- Genetic Disease Diagnosis
- Cancer Genomics
- Infectious Disease Diagnosis
- Prenatal Screening and Diagnosis
- Microbial Genomics
- HLA Typing and Transplantation
The library dilution buffer contains components that aid in the stability and integrity of the nucleic acid fragments while facilitating their handling and subsequent processing.
The library dilution buffer include:
- Tris-HCl or phosphate buffer
- EDTA: Ethylenediaminetetraacetic acid (EDTA)
The Library Quantification Kit for Illumina is a specialized kit designed for the quantification and quality assessment of libraries prepared for Illumina sequencing platforms. Illumina is a leading provider of NGS technologies, and their library quantification kits are specifically tailored to work with their sequencing systems. These kits help researchers ensure that the libraries they prepare contain the appropriate amount of DNA fragments and meet the quality standards necessary for successful sequencing.
The Library Quantification Kit for Illumina typically includes the following components:
- Library Quantification Assay
- Library Quantification Standards
- PCR Master Mix
- Buffer and Diluents
- Fluorescence Detection or qPCR Instrumentation
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