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Updated: Mar 20, 2026

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq

Published on: May 28, 2021

Simultaneous Pathway Activity Inference and Gene Expression Analysis Using RNA Sequencing.

Daniel J O'Connell¹, Raivo Kolde², Matthew Sooknah¹

¹Program in Medical and Population Genetics, Broad Institute, Cambridge, MA 02142, USA.

|May 24, 2016

Summary

This summary is machine-generated.

We developed transcription factor activity sequencing (TF-seq) for high-throughput analysis of signaling pathways. TF-seq reveals dynamic pathway activities and transcriptional changes in immune responses and drug mechanisms.

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Area of Science:

Systems biology
Molecular biology
Immunology

Background:

Traditional reporter gene assays have limitations in throughput and complexity for studying endogenous signaling networks.
A systems-level understanding of cellular signaling requires advanced methods to measure pathway activity and gene expression concurrently.

Purpose of the Study:

To introduce transcription factor activity sequencing (TF-seq), a novel parallel reporter assay for high-throughput analysis of signaling pathways.
To demonstrate TF-seq's capability in capturing dynamic pathway activities and transcriptional changes in primary cells.
To utilize TF-seq for investigating the mechanisms of action of small molecules.

Main Methods:

Development of TF-seq utilizing synthetic DNA enhancer elements for parallel measurements.
Application of TF-seq in Myd88(-/-) macrophages to study innate immune response.
Integration of TF-seq with global gene expression profiling.

Main Results:

TF-seq enabled parallel measurement of transcriptome and transcription factor activity across over 40 signaling pathways.
Dynamic pathway activity changes were captured during the innate immune response in Myd88(-/-) macrophages.
TF-seq identified NF-κB activation and STAT1 coordination in response to halofuginone in macrophages.

Conclusions:

Simultaneous TF-seq and gene expression profiling offer an integrative approach for mechanistic insights into signaling pathways.
TF-seq provides a powerful tool for dissecting complex biological responses to genetic and pharmacological perturbations.
This method advances the study of endogenous signaling networks and drug mechanisms.