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Related Experiment Video

Updated: Feb 18, 2026

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

Published on: February 24, 2015

Partial bisulfite conversion for unique template sequencing.

Vijay Kumar¹, Julie Rosenbaum¹, Zihua Wang¹

¹Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724 USA.

Nucleic Acids Research

|November 22, 2017

Summary

This summary is machine-generated.

We developed mutational sequencing (muSeq), a new method that uses unique mutation signatures to accurately count and assemble DNA molecules from short-read sequencing data, revealing hidden transcriptional diversity.

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Last Updated: Feb 18, 2026

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DNA Methylation: Bisulphite Modification and Analysis

DNA Methylation: Bisulphite Modification and Analysis

Published on: October 21, 2011

Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Short-read sequencing technologies are limited in their ability to accurately quantify DNA molecules and perform long-range assembly.
Existing methods struggle to resolve complex transcriptional landscapes due to inherent limitations in read length and error profiles.

Purpose of the Study:

To introduce and validate mutational sequencing (muSeq), a novel protocol for enhanced DNA molecule counting and long-range assembly.
To demonstrate muSeq's capability in reducing sequencing errors and improving copy number inference.
To showcase muSeq's utility in uncovering previously unobservable transcriptional diversity.

Main Methods:

Utilized sodium bisulfite to deaminate unmethylated cytosines at a controlled rate, creating unique mutation signatures on template molecules.
Developed a read clustering approach based on observed C-to-T/G-to-A conversion patterns to identify and count original template molecules.
Applied muSeq to profile restriction fragments and assemble cDNA sequences.

Main Results:

muSeq successfully generated unique mutation signatures present across all template copies and fragments.
Clustering reads by signature enabled accurate counting and long-range assembly of initial template molecules from short-read data.
Demonstrated improved copy number inference and significant reduction in sequencing error rates.
Achieved contiguous cDNA transcript clusters exceeding 3,000 bp, revealing substantial transcriptional diversity.

Conclusions:

muSeq offers a robust solution for accurate molecule counting and long-range assembly using short-read sequencing.
The protocol significantly enhances the resolution of genomic and transcriptomic analyses.
muSeq unlocks the potential to discover complex biological patterns previously obscured by sequencing limitations.