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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