GENOMIC REVOLUTION: NEXT GENERATION SEQUENCING



Next Generation Sequencing (NGS) is a new and recent technology for sequencing hundreds and thousands of genomes of DNA and RNA in a very short time. It can also detect any variation or mutation in sequencing so ultimately helps in disease diagnosis and therapeutic decisions. It is a huge revolutionary technology after Sanger sequencing. It has various applications in many fields like genetic diseases, personalized medicines, clinical diagnostics, etc.

SANGER SEQUENCING vs NGS

The real difference between them is the amount of sequencing done by each technique. The Sanger can sequence one DNA fragment at a time while NGS can run millions of fragments at a time. The other difference is that NGS has high sensitivity even can detect very novel or rare mutations via deep sequencing whereas Sanger has low sensitivity.

NGS Method:

1) DNA Fragmentation

Firstly, the DNA is broken down into small fragments or segments into 100 to 300 bp by some techniques like mechanical breakdown or enzymatic digestion. Then the fragments of DNA are taken out by some complementary probes. PCR could also be done where different pairs of primers are used to amplify DNA fragments of the targeted DNA.

2) Library preparation

These small DNA segments are then used for library preparation. DNA modification takes place in library preparation by adding adopters on DNA fragments which helps primers for massive parallel sequencing.

3) Sequencing

Massive sequencing is done on the NGS sequencer. Different sequencers use different matrices but the overall function is the same i.e. sequencing of all DNA segments at the same time. For example, the Illumina NGS sequencer uses flow cells and the Ion Torrent NGS sequencer uses sequencing chips. For sequencing each time, a fluorescently labelled nucleotide is incorporated, the type of nucleotide (A, T, G, and C) can be detected by a specific type of fluorescent attached. Analyzation of information generated is taken place by bioinformatics software.

4) Bioinformatics

To identify whether there are any mutations/variants in the targeted sequences, they are compared with the human genome reference sequence. Information collected from each fragment of DNA is united together to generate the full length of the targeted DNA.

CLINIC APPLICATION

1) Testing of small biopsy specimens and multiple mutations by NGS

Biopsy specimens are becoming smaller and smaller in current medical practices. With traditional molecular tests available information related to mutation in these small biopsy samples is difficult so the NGS technique has been evolved to meet such needs. With NGS it has become very easy to test multiple samples and multiple targets at the same time.

It is investigated that multiple mutations may be involved in tumors but by traditional molecular assays multiple tests might run to test multiple mutations but by NGS results of hundreds or thousands of DNA can be obtained easily by performing only one test. For example, different mutations have been discovered in different hematopoietic leukemias. The mutations in NPM1 and CEBPA of a RUNX1 have been found in acute myeloid leukemia (AML) associated with different entities of AML subtypes.

2) NGS for Liquid Biopsy

Many solid tumors shed their DNA into bloodstream or any other body fluids. Here NGS technology comes into action it can detect variants/mutations from cell-free circulating DNA. The cell-free circulating DNA is also known as "liquid biopsy". As these fluids like plasma or other body fluids are easily assessable, the tumours which were impossible to biopsy, this technique is especially useful for them. The key issue in doing liquid biopsy is that DNA is shed differently by different tumors, a particular tumor at a different stage may shed DNA differently, so here comes the problem of test sensitivity as numerous liquid biopsy studies have to be carried out for different tumors. So, to improve NGS sensitivity, a molecular barcode is attached to label the originally targeted molecules.

3)NGS for Targeted Therapy

Next-generation sequencing has revolutionized the field of diagnosis and has been proven effective in the treatment of malignancy. Different molecular targeted drugs have been made over the past few years and there are more to come. the genes associated with melanoma therapy could include but are not limitedto, BRAF, KIT, NRAS, NF1, GNAQ, CDK4, MITF, and PD-1 and The genesassociated with lung cancer therapy could include but are not limited to, EGFR,KRAS, BRAF, NRAS, PIK3ca, ROS1, MEK, VEGFA, ALK, MET, ERBB2, andERBB4. So NGS has applications to identify different mutations for targeted therapy. Panels are designed according to the type of cancer and mutations are detected. The survival rate of cancer has increased tremendously because of awareness and biomarker testing. By targeted therapy, the targeted mutation is detected, and the specific pathway detects which roles in that cancer growth so a specific drug is used that targets that specific mutation ultimately it helps in killing the cancer.

4) Importance of NGS in drug resistance testing

When some drug-resistant TB patient showed no improvement even after analysing their drug resistance by GeneXpert or a drug sensitivity test NGS came and played its role it has more sensitivity than any other conventional drug resistance test.

Conventionally HIV drug resistance (HIVDR) genotyping is carried out by population-based Sanger sequencing technique but it has low sensitivity, it can detect a very limited number of variants present at intra-host frequencies below a threshold of approximately 20%. NGS is preferred due to its high sensitivity to detect low-abundance variants as it generates millions of sequencings reads from the input templates in parallel, and also due to the availability of commercial kits, and its decreasing cost.

The WHO has proposed an aggressive plan to act upon HIVDR, that includes periodic nationally representative surveillance of pre-treatment and acquired HIVDR with the help of standardized methods, with the support of a precisely named and nearly covered laboratory network to guarantee high- quality, reproducible, and similar HIVDR check reports worldwide.

 Examples of NGS Cost Per Sample

Accordingto Illumina, a biotechnology company which is a leading providers of tools and services for genomic research.

For the application of Targeted gene expression profiling estimated cost per sample is $23 USD. Cost per sample calculation is based on a run using:

  • MiniSeq System
  • 65 targets
  • 1000× coverage
  • 1 × 150 bp read length
  • TruSeq Targeted RNA Expression custom panel kit
  • MiniSeq Reagent High Output 75-cycle kit

And for the application of 16S metagenomic sequencing estimated cost per sample is $18 USD. Cost per sample calculation is based on a run using:

  • MiSeq System
  • 96 samples
  • 2 × 300 bp read length
  • Nextera XT index primers
  • MiSeq Reagent v3 600-cycle kit

 CONCLUSION

Next Generation Sequencing (NGS) has revolutionized the field of genetics and enabled genetic analysis, the rapid and comprehensive sequencing of DNA and RNA. Unlike Sanger sequencing, NGS can process millions of fragments simultaneously, offering high sensitivity for detecting variations and mutations. Its applications cover various fields, including genetic diseases, personalized medicine, and clinical diagnostics. By efficiently analysing small biopsy specimens, conducting liquid biopsies, facilitating targeted therapy, and enhancing drug resistance testing, NGS significantly advances our understanding and treatment of diseases. Its ability to provide detailed genetic insights with more speed and accuracy helps to enter a new era of healthcare and research.




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