#transcriptomics

Sequencing

RNA-seq provides insight into:

DNA polymerase synthesizes DNA strictly in 5' to 3' direction by adding nucleotides to the free 3' hydroxyl group of the growing chain. In Sanger sequencing, labeled primer anneals to template and polymerase extends it; each termination product represents a strand made 5' to 3'. Consequently, when fragments are ordered by size from smallest to largest after electrophoresis, sequence read corresponds to 5' to 3' synthesis product, complementary to template strand oriented 3' to 5'. Synthesis never proceeds 3' to 5' enzymatically, and reading is directional not random.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Sequencing

Which step in RNA-seq distinguishes novel transcripts?

Distinguishing novel transcripts from annotated ones depends heavily on post-sequencing alignment analysis. After sequencing cDNA libraries, reads are mapped with splice-aware aligners such as STAR or HISAT2 that can split alignments across introns. Reads spanning previously unannotated exon-exon junctions, retained introns or alternative splice sites emerge only during alignment. Reverse transcription and library preparation create cDNA fragments, sequencing depth improves sensitivity, but computational alignment reveals exon connectivity, fusion genes and novel isoforms. Hence alignment is step that identifies transcript novelty and structural variation.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Sequencing

RNA-seq was first introduced in:

The concept of massively parallel cDNA sequencing for transcriptome profiling, now called RNA-seq, was demonstrated in pioneering studies published in 2008. Groups led by Wold, Grimmond and others used Illumina Genome Analyzer and 454 platforms to sequence yeast, mouse and human transcriptomes, showing superior dynamic range compared to microarrays for expression quantification and discovery of novel transcripts. The technology became feasible only after next-generation sequencers became robust. 2005 corresponds to early 454 sequencing, while 2010 and 2012 marked expansion into stranded, single-cell and long-read RNA-seq applications.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Sequencing

What is the primary goal of RNA sequencing?

RNA sequencing aims to capture the transcriptome, the complete set of RNA molecules transcribed from genome at a given time. After isolating RNA and converting to cDNA library, deep parallel sequencing provides quantitative and qualitative information: abundance of transcripts, alternative splicing isoforms, allele-specific expression, fusion transcripts and non-coding RNAs. This enables comparison of gene expression across tissues, developmental stages or disease states. It is not designed for protein purification, direct DNA mutation screening independent of expression, or removal of splice sites. Transcriptome analysis is its primary biological purpose.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Transcriptome analysis

What is the role of EcoP151 in CAGE?

After cap-trapping and cDNA synthesis, CAGE adaptors containing the recognition motif for EcoP151, a type III restriction enzyme, are ligated upstream of the cDNA. EcoP151 cuts DNA at a fixed distance of 25 to 27 base pairs downstream of its site, independent of sequence context. This property generates uniform CAGE tags of approximately 27 nucleotides originating from the very 5′ end of transcripts. The length is optimal for unique genomic alignment while preserving TSS information. It does not label mRNA or ligate probes directly; its role is strictly tag definition.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Transcriptome analysis

Which transcriptomics technique uses 5′ cap as a feature?

CAGE exploits the unique 5′ cap structure of RNA polymerase II transcripts. In eukaryotes, a 7-methylguanosine is added co-transcriptionally to the first nucleotide. CAGE employs cap-trapping, where biotinylated cap is selected after cDNA synthesis, ensuring enrichment of full-length 5′ ends. Sequencing of these capped ends yields tags precisely marking transcription initiation sites and promoter boundaries. SAGE uses internal NlaIII sites, poly(A) profiling selects 3′ ends, and microarrays rely on hybridization without cap specificity, so only CAGE intrinsically depends on cap recognition for genome-wide promoter mapping.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Transcriptome analysis

Which enzyme is used in SAGE to remove ~12 bp fragments?

SAGE generates short positional tags that serve as identifiers for transcripts. After cDNA synthesis on oligo(dT) beads and cleavage with anchoring enzyme NlaIII, adaptors containing recognition sites for the tagging enzyme are ligated. BsmFI is a type IIS restriction endonuclease that cleaves 10/14 bases downstream of its recognition site, releasing fragments containing approximately 10 to 14 base pair tags. These released tags are ligated into ditags for concatemerization and sequencing. AluI and EcoP151 have different roles, with EcoP151 used in CAGE, not classical SAGE tag release.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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