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NIAB USING NEXT GENERATION SEQUENCING

Last Updated on 4th September, 2024
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Description

NIAB USING NEXT GENERATION SEQUENCING

Source: Hindu

Disclaimer: Copyright infringement not intended.

Context

  • National Institute of Animal Biotechnology (NIAB) is working to decode the genetic blueprints for conservation of indigenous cattle breeds.
  • NIAB is using Next Generation Sequencing (NGS) data to establish molecular signatures for registered cattle breeds.

Details

About NIAB

  • It is an Indian autonomous research establishment of the Department of Biotechnology, Ministry of Science and Technology.
  • The NIAB is set up in Hyderabad under the leadership of Prof. Pallu Reddanna.
  • "The state of the art of Animal Biotechnology and Transgenics institute" is housed in the NIAB Campus in Gachibowli.
  • The primary mandate of NIAB is towards the development of sustainability and globally competitive livestock (farm animals) for public and industry through innovative and cutting edge technology.

Focus Area

Feature

Conservation and Health Initiatives

Preserve indigenous cattle breeds using NGS.

New-generation vaccines against livestock diseases (e.g., brucellosis).

Industry Collaboration and Innovation

 

Boost livestock economy through food security, vaccines, and diagnostics.

Develop bio-scaffolds and a 3D model for TB research.

R&D

Identify TB resistance in cattle; explore CRISPR for productivity.

Promote sustainable protein and bacteriophage alternatives.

NIAB’s R&D efforts align with the BioE3 policy to boost bio-manufacturing and position India as a global leader in biotechnology.

Diagnostics and Sustainable Farming

 

Develop kits for brucellosis, mastitis, and hormone detection.

MILAN (Meeting of Livestock Farmers) showcases sustainable farming.

Use aquatic weed and yeast protein to cut emissions.

Alternative Nutrition and Feed

 

Aquatic Weed: Being introduced as potential livestock feed.

Yeast-Derived Protein: Substitution in regular feed formulations to boost productivity.

About Next-Generation Sequencing

  • Next-Generation Sequencing (NGS)is a modern technology that helps scientists study the DNA or RNA sequences of organisms.
  • It was introduced in 2005.
  • This method was initially called “massively-parallel sequencing”, because it enabled the sequencing of many DNA strands at the same time, instead of one at a time as with traditional Sanger sequencing by capillary electrophoresis (CE).

Steps in the NGS Workflow

Disclaimer: Copyright infringement not intended.

 

NGS Library Preparation

  • DNA Fragmentation:The DNA sample is broken into smaller pieces (100–800 base pairs long).
  • Adaptor Ligation:Special sequences called adaptors are added to the ends of these fragments.
  • There are special methods for library preparation:
      • Paired-End Libraries:DNA fragments are sequenced from both ends, which helps in mapping the reads more accurately.
      • Mate-Pair Libraries:Used for sequencing much larger DNA fragments (over 2,000 base pairs). This method is useful for studying complex genetic structures.

Clonal Amplification

The DNA fragments in the library need to be multiplied to ensure a strong enough signal during sequencing.

Sequencing

All DNA fragments are sequenced at the same time using an NGS machine.

Data Analysis

  • Primary Analysis:Converts raw signals from the sequencing machine into digital data (FASTQ files), which contain the sequences of DNA bases.
  • Secondary Analysis:The data is filtered for quality, aligned to a reference genome, or assembled into new genomes. The main output here is a BAM file..
  • Tertiary Analysis:This is the final step, where scientists interpret the data to find meaningful patterns and insights, such as identifying genetic mutations related to diseases.

Read basics of Genome Sequencing: https://www.iasgyan.in/daily-current-affairs/10000-genome-project-completed#:~:text=Importance%3A%20Genome%20sequencing%20enables%20scientists,diseases%2C%20and%20develop%20personalized%20treatments.

Read about DNA: https://www.iasgyan.in/daily-current-affairs/human-genome-project

Comparison of DNA sequencing methods

Category

Whole Genome Sequencing (WGS)

Targeted Sequencing: Exome Sequencing

Targeted Sequencing: Gene or Region-Specific Panels

 

Description

Sequencing the entire genome

Sequencing only exons (protein-coding regions)

Sequencing regions of interest such as disease-associated genes or genomic hotspots

 

Pros

Most comprehensive genome coverage

Detects widest range of features: indels, structural and copy number variants, regulatory elements

No bias from PCR amplification or probe hybridization

Best for discovery research

1% of human genome, less data to analyze than WGS

Faster workflow than WGS

Multiplexing small number of samples

Medium sample input (50 ng–1 μg depending on library prep method)

Highly flexible, customizable designs

Data is focused specifically on regions/genes of interest

Lowest sample input (10 ng)

Multiplexing large numbers of samples

Better for detecting rare alleles

 

 

Cons

Generates a lot of potentially unnecessary data from non-coding/non-functional regions

Data is very complicated

Multiplexing usually not possible

Only provides data on exons (may miss functionally relevant variants)

May include extra data not needed for small gene studies

Only provides data on targeted regions (may miss relevant variants if not in design)

Speed/Return of Results

Slowest

Medium

Fastest

 

Cost

$$$

$$

$

 

Data Volume

Largest

Medium

Smallest

 

When to Use

Complete coverage of genome needed

De novo assembly

Discovery of unknown genomic variants causing a disease

Aneuploidy detection (preimplantation genetic testing)

Disease-specific research projects

Clinical sequencing

 

 

Clinical sequencing

Disease-specific research projects

Inherited disease

Oncology

Immune repertoire

Liquid biopsy

RNA sequencing methods

What is RNA?

  • Ribonucleic acid (RNA) molecules are crucial for gene coding, decoding, regulation, and expression.
  • In molecular biology, DNA's genetic information is transcribed into messenger RNA (mRNA), which is then translated into proteins, allowing cells to produce multiple proteins from a single gene.
  • mRNA makes up only 1-4% of total RNA, while the majority is noncoding RNA (ncRNA), which is not translated into proteins.
  • Types of ncRNA include ribosomal RNA (rRNA), transfer RNA (tRNA), long noncoding RNA (lncRNA), and microRNA (miRNA).
  • rRNA is the most abundant, comprising 80-95% of total RNA, while other ncRNAs are present in smaller amounts, often requiring larger samples or enrichment for study.

RNA type

Function

Messenger RNA

Codes for protein

Ribosomal RNA

Translation

Transfer RNA

Translation

Small nuclear RNA

Splicing and other functions

Small nucleolar RNA

Nucleotide modification of RNAs

Small Cajal body-specific RNA

Type of snoRNA; nucleotide modification of RNAs

Long noncoding RNA

Regulation of gene transcription; epigenetic regulation

MicroRNA

Gene regulation

Piwi-interacting RNA

Transposon defense

Small interfering RNA

Gene regulation

Transcriptome and RNA-Seq

  • The transcriptome refers to the complete set of RNA molecules, including all forms of RNA, present in a single cell or a population of cells.
  • Understanding the transcriptome is vital for uncovering the dynamics of gene expression, particularly how it changes over time or in response to external factors.
  • RNA-Seq (RNA Sequencing) is a method that utilizes next-generation sequencing (NGS) to analyze and quantify RNA present in a biological sample at a specific moment.
  • Advantages:
  • RNA-Seq allows for comprehensive analysis of the entire transcriptome without needing specific probes.
  • NGS technology in RNA-Seq provides the ability to detect differentially expressed genes with precise quantification, even for rare transcripts within the transcriptome.
  • RNA-Seq can identify novel variants, such as alternative splice sites and new isoforms.

RNA sequencing method

Description and benefits

Total RNA
Whole transcriptome

Whole transcriptome analysis to examine coding and noncoding RNA simultaneously; suitable for novel discovery.

More throughput intensive to achieve high enough coverage for discovery.

Potential inefficiencies and bias due to different sequencing lengths.

mRNA sequencing

Able to identify novel and known content

smRNA sequencing

Isolation of small RNA to focus study on noncoding RNA to identify novel and known content such as microRNA (miRNA)

Targeted RNA sequencing

Sequencing specific transcripts of interest to focus efforts and lower cost to analyze specific genes of interest.

Sources:

Hindu

PRACTICE QUESTION

Q: Consider the following statements regarding types of RNA:

  1. Piwi-interacting RNA can prevent transposition.
  2. microRNA is the name of a family of molecules that helps cells control the kinds and amounts of proteins they make.

Which of the above statements is/are correct?

a) 1 only
b) 2 only
c) both 1 and 2
d) neither 1 nor 2

 

Answer: c) 

Explanation:

1st statement is correct: PIWI-interacting RNAs (piRNAs) of 21–35 nucleotides in length silence transposable elements, regulate gene expression and fight viral infection.

2nd statement is correct: microRNA is the name of a family of molecules that helps cells control the kinds and amounts of proteins they make. That is, cells use microRNA to help control gene expression. Molecules of microRNA are found in cells and in the bloodstream.

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