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Related Experiment Video

Updated: May 22, 2026

Comprehensive Spatial Profiling of Species-agnostic Transcriptomes via Stereo-seq

Comprehensive Spatial Profiling of Species-agnostic Transcriptomes via Stereo-seq

Published on: October 31, 2025

Multiple insert size paired-end sequencing for deconvolution of complex transcriptomes.

Lisa M Smith¹, Lisa Hartmann, Philipp Drewe

¹Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany.

|May 23, 2012

Summary

This summary is machine-generated.

This study introduces a modified Illumina protocol for strand-specific, paired-end (SS-PE) sequencing, enhancing transcriptomic analysis. The new method significantly improves the detection of alternative splicing events and natural antisense transcripts.

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Area of Science:

Genomics
Molecular Biology
Bioinformatics

Background:

Deep sequencing of transcriptomes is crucial for analyzing RNA species, including expression levels, splicing variants, and transcriptional start/stop sites.
Standard Illumina protocols lack strand-specificity and multiple insert sizes, limiting comprehensive transcriptomic analysis, particularly for alternative splicing in eukaryotes.

Purpose of the Study:

To modify the Illumina RNA ligation protocol for strand-specific, paired-end (SS-PE) sequencing.
To generate parallel libraries with varying insert sizes to improve the deconvolution of alternative splicing events.
To characterize natural antisense transcription in C. elegans.

Main Methods:

Computational modeling to demonstrate the benefits of multiple insert sizes and SS-PE sequencing.
Modification of the Illumina RNA ligation protocol using a custom pre-adenylated 3' adaptor for SS-PE sequencing.
Generation of parallel libraries with differing insert sizes for transcriptomic analysis.

Main Results:

The modified protocol enables SS-PE sequencing, increasing transcriptomic information gained.
The number of annotated intron retention and exon skipping events in C. elegans increased by 127% and 121%, respectively.
The protocol demonstrates competitive library quality compared to established benchmarks.

Conclusions:

Parallel libraries with a range of insert sizes enhance transcriptomic information obtained through sequencing.
The developed SS-PE sequencing protocol offers improved detection of alternative splicing and natural antisense transcripts.
This advancement provides a more comprehensive analysis of transcriptomes, particularly in complex organisms.