Correct Sequence of Events in NGS-Based Whole Genome Sequencing

Choose the correct sequence of events in a next generation sequencing technology-based whole genome sequencing project.
A. DNA extraction shearing → library preparation sequencing → assembly → finishing → annotation submission to Genbank.
B. DNA extraction → library preparation sequencing → assembly → annotation finishing → submission to Genbank,
C. DNA extraction → shearing → adapter ligation → library amplification → sequencing → assembly finishing annotation → submission to Genbank.
D. DNA extraction adapter ligation → library amplification shearing sequencing finishing assembly annotation → submission to Genbank

Next-generation sequencing (NGS) has revolutionized the field of genomics, allowing for high-throughput and accurate whole genome sequencing. Understanding the correct sequence of events in an NGS-based whole genome sequencing project is essential for students preparing for CSIR NET Life Science, IIT JAM, GATE Biotechnology, and DBT JRF.

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Correct Answer: (C) DNA extraction → shearing → adapter ligation → library amplification → sequencing → assembly → finishing → annotation → submission to Genbank

The correct sequence in an NGS-based whole genome sequencing project follows a well-defined set of steps, ensuring accuracy and comprehensive genome coverage.

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Keyphrase: Whole Genome Sequencing Steps

The process of whole genome sequencing involves DNA extraction, library preparation, sequencing, assembly, annotation, and submission.

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What is Whole Genome Sequencing?

Whole genome sequencing (WGS) is a technique used to determine the complete DNA sequence of an organism’s genome. It involves fragmenting the DNA, attaching adapters, and sequencing the fragments using high-throughput sequencing platforms such as Illumina, PacBio, or Oxford Nanopore.

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Steps in Whole Genome Sequencing

1. DNA Extraction

• The first step is isolating high-quality genomic DNA from the sample.
• The extracted DNA should be free from contaminants like proteins and RNA.

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2. DNA Shearing

• The extracted DNA is fragmented into smaller pieces using physical or enzymatic methods.
• Sonication and nebulization are common shearing techniques.

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3. Adapter Ligation

• Short double-stranded DNA sequences (adapters) are attached to the fragmented DNA.
• Adapters provide a recognition site for sequencing primers and facilitate fragment amplification.

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4. Library Amplification

• The adapter-ligated DNA is amplified using PCR.
• This step ensures that enough DNA is available for sequencing.

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5. Sequencing

• The DNA library is sequenced using high-throughput sequencing platforms (e.g., Illumina, PacBio).
• Millions of short reads are generated, which are then assembled to reconstruct the genome.

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6. Assembly

• The short reads are aligned and stitched together to form a complete genome sequence.
• De novo assembly or reference-guided assembly methods are used.

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7. Finishing

• Gaps and ambiguous regions are resolved through additional sequencing or bioinformatics approaches.
• Ensures that the final genome sequence is of high accuracy.

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8. Annotation

• Functional elements such as genes, regulatory regions, and repeats are identified.
• Bioinformatics tools are used to assign biological functions to genome regions.

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9. Submission to GenBank

• The final assembled and annotated genome is submitted to public databases like GenBank.
• This allows researchers worldwide to access and analyze the genome data.

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Why Following the Correct Sequence Matters

• Ensures high-quality and accurate genome assembly.
• Reduces sequencing errors and improves genome coverage.
• Facilitates reproducibility and global data sharing.

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Importance of Whole Genome Sequencing

1. Identifying Genetic Variations

• Detects single nucleotide polymorphisms (SNPs), insertions, deletions, and structural variations.

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2. Studying Evolutionary Relationships

• Whole genome data helps in understanding phylogenetic relationships and evolutionary history.

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3. Clinical Applications

• Used in identifying genetic mutations associated with diseases.
• Helps in personalized medicine and targeted drug development.

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4. Microbial Genome Studies

• Used to study antibiotic resistance and microbial diversity.
• Aids in vaccine development and infectious disease control.

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Challenges in Whole Genome Sequencing

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Comparison of Whole Genome Sequencing Platforms

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Applications of Whole Genome Sequencing

1. Cancer Genomics

• Identifies mutations linked to cancer progression and drug resistance.

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2. Infectious Disease Surveillance

• Tracks viral and bacterial evolution and spread.

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3. Agricultural Biotechnology

• Improves crop yields and disease resistance through genetic analysis.

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4. Personalized Medicine

• Tailors drug therapies based on an individual’s genetic profile.

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Why NGS-Based Sequencing is Important for CSIR NET and Other Exams

Whole genome sequencing is a key topic in:

• CSIR NET Life Science
• IIT JAM
• GATE Biotechnology
• DBT JRF

Understanding the correct sequencing workflow and platforms is essential for answering related questions accurately in competitive exams.

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Conclusion

Whole genome sequencing using NGS is a powerful tool for studying genetic variations, evolutionary relationships, and disease mechanisms. Following the correct sequence of steps—from DNA extraction to GenBank submission—ensures high-quality and accurate genome assembly.

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FAQs

Q1. Why is DNA shearing important in whole genome sequencing?
Shearing generates smaller fragments that can be efficiently sequenced and assembled.

Q2. What is the purpose of adapter ligation?
Adapters allow the fragments to bind to the sequencing platform and enable primer recognition.

Q3. What is the difference between de novo assembly and reference-guided assembly?

• De novo assembly – Used when no reference genome exists.
• Reference-guided assembly – Aligns reads to an existing reference genome.

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This article was prepared with insights from Let’s Talk Academy, the top institute for life science competitive exams.