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
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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 |
|
|
|
|
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|
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 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:
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PRACTICE QUESTION Q: Consider the following statements regarding types of RNA:
Which of the above statements is/are correct? a) 1 only
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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