Related Experiment Video
Updated: May 26, 2026

3' End Sequencing Library Preparation with A-seq2
Published on: October 10, 2017
Sequencing by ligation variation with endonuclease V digestion and deoxyinosine-containing query oligonucleotides
Antoine Ho1, Maurice Murphy, Susan Wilson
1Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, New Mexico, USA.
Background:
Sequencing-by-ligation (SBL) is one of several next-generation sequencing methods that has been developed for massive sequencing of DNA immobilized on arrayed beads (or other clonal amplicons). SBL has the advantage of being easy to implement and accessible to all because it can be performed with off-the-shelf reagents. However, SBL has the limitation of very short read lengths.
Results:
To overcome the read length limitation, research groups have developed complex library preparation processes, which can be time-consuming, difficult, and result in low complexity libraries. Herein we describe a variation on traditional SBL protocols that extends the number of sequential bases that can be sequenced by using Endonuclease V to nick a query primer, thus leaving a ligatable end extended into the unknown sequence for further SBL cycles. To demonstrate the protocol, we constructed a known DNA sequence and utilized our SBL variation, cyclic SBL (cSBL), to resequence this region. Using our method, we were able to read thirteen contiguous bases in the 3' - 5' direction.
Conclusions:
Combining this read length with sequencing in the 5' - 3' direction would allow a read length of over twenty bases on a single tage. Implementing mate-paired tags and this SBL variation could enable > 95% coverage of the genome.
More Related Videos
05:412D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications
Published on: July 10, 2020
14:15Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing
Published on: November 18, 2014
Related Concept Videos
Next-generation Sequencing
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
Sanger Sequencing
Maxam-Gilbert Sequencing
Challenges of the Maxam-Gilbert Method
The...
RNA-seq
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...
DNA Isolation