Advantages of paired-end and single-read sequencing

Understand the key differences between these sequencing read types

Paired-End vs. Single-Read Sequencing

Paired-end sequencing allows users to sequence both ends of a fragment and generate high-quality, alignable sequence data. Paired-end sequencing facilitates detection of genomic rearrangements and repetitive sequence elements, as well as gene fusions and novel transcripts.

Since paired-end reads are more likely to align to a reference, the quality of the entire data set improves. All Illumina next-generation sequencing (NGS) systems are capable of paired-end sequencing.

Paired-End Sequencing
  • Simple Paired-End Libraries: Simple workflow allows generation of unique ranges of insert sizes
  • Efficient Sample Use: Requires the same amount of DNA as single-read gDNA or cDNA sequencing
  • Broad Range of Applications: Does not require methylation of DNA or restriction digestion; can be used for bisulfite sequencing
  • Simplified Data Analysis: Higher quality sequence assemblies with short-insert libraries. A simple modification to the standard single-read library preparation process facilitates reading both the forward and reverse template strands of each cluster during one paired-end read. Both reads contain long-range positional information, allowing for highly precise alignment of reads.
Illumina Sequencing Introduction

This overview describes major sequencing technology advances, key methods, the basics of Illumina sequencing chemistry, and more.

Read Introduction

Paired-end DNA sequencing reads provide superior alignment across DNA regions containing repetitive sequences, and produce longer contigs for de novo sequencing by filling gaps in the consensus sequence. Paired-end DNA sequencing also detects rearrangements such as insertions, deletions, and inversions.

Methods for DNA Sequencing

DNA sequencing can be applied to small, targeted regions or the entire genome through a variety of methods.

Learn More

Paired-end RNA sequencing (RNA-Seq) enables discovery applications such as detecting gene fusions in cancer and characterizing novel splice isoforms.

For paired-end RNA-Seq, use the TruSeq RNA Library Prep Kits with an alternate fragmentation method, followed by standard Illumina paired-end cluster generation and sequencing.

For mRNA-Seq library prep, use:
For stranded total RNA library prep, use:
RNA-Seq Overview

This method offers a high-resolution view of coding and noncoding regions of the transcriptome for a deeper understanding of biology.

Learn More

Single-read sequencing involves sequencing DNA from only one end, and is the simplest way to utilize Illumina sequencing. By leveraging proprietary reversible terminator chemistry and a novel polymerase, this solution delivers large volumes of high-quality data, rapidly and economically.

Single-Read Sequencing Highlights
  • Simple Library Preparation: Follows standard molecular biology methods; compatible with robotics
  • Low Input DNA Requirements: As little as 100 ng genomic DNA or cDNA
  • Economical: 1/100th the cost of traditional Sanger sequencing
  • Simplified Data Analysis: High-quality sequence assemblies with short-insert libraries
Library Preparation

Innovative, comprehensive library prep solutions are a key part of the Illumina sequencing workflow.

Explore Library Prep
Interested in receiving newsletters, case studies, and information from Illumina based on your area of interest? Sign up now.
Sequencing Technology Video
Sequencing Technology Video

See SBS technology in action.

View Video
Sequencing Platform Selection Tool
Sequencing Platform Selection Tool

Compare the speed and throughput of Illumina sequencing systems to find the best platform for your lab.

Launch Tool