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Related Concept Videos

Next-generation Sequencing03:00

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
RNA-seq03:21

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...
Sanger Sequencing01:57

Sanger Sequencing

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
Maxam-Gilbert Sequencing01:05

Maxam-Gilbert Sequencing

In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
Challenges of the Maxam-Gilbert Method
The...

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Related Experiment Video

Updated: May 29, 2026

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments
09:14

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

Published on: January 27, 2016

Titration-free 454 sequencing using Y adapters.

Zongli Zheng1, Abdolreza Advani, Öjar Melefors

  • 1Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. zhengzongli@gmail.com

Nature Protocols
|September 3, 2011
PubMed
Summary
This summary is machine-generated.

This study presents a rapid protocol for preparing sequencing libraries for emulsion PCR (emPCR). It optimizes library quantification and quality control, enabling efficient downstream sequencing applications.

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Area of Science:

  • Molecular Biology
  • Next-Generation Sequencing

Background:

  • Emulsion PCR (emPCR) is crucial for next-generation sequencing (NGS) platforms.
  • Accurate library quantification and quality control are essential for successful emPCR.
  • Existing methods for library preparation and quantification can be laborious and time-consuming.

Purpose of the Study:

  • To develop an efficient and reliable protocol for constructing and quantifying libraries for emPCR.
  • To optimize the template-to-bead ratio for emPCR using a novel quantification method.
  • To ensure high-quality libraries with minimal adapter dimers and optimal length distribution.

Main Methods:

  • Library construction using customized Y adapters.
  • Quantification of effective libraries using TaqMan-MGB probe-based quantitative PCR (qPCR).
  • Calculation of optimal template-to-bead ratio based on Poisson statistics.

Main Results:

  • The protocol enables rapid preparation of barcoded libraries (∼7 h for eight libraries).
  • TaqMan-MGB probe-based qPCR accurately quantifies effective libraries in molar concentration without specialized equipment.
  • A single quality control step ensures libraries are free of adapter dimers and have optimal length distribution.

Conclusions:

  • The presented protocol streamlines library preparation for emPCR-based sequencing.
  • This method avoids laborious titration assays and improves efficiency.
  • The protocol is valuable for analyzing precious samples and for amplification-free metatranscriptomics.