Paired-End vs. Single-Read Sequencing

What are the differences between paired-end and single-read sequencing?

Single-read sequencing involves sequencing DNA from only one end, and is the simplest way to utilize Illumina sequencing. Unlike single-read seqeuncing, 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.

In addition to producing twice the number of reads for the same time and effort in library preparation, sequences aligned as read pairs enable more accurate read alignment and the ability to detect insertion-deletion (indel) variants, which is more difficult with single-read data.1 All Illumina next-generation sequencing (NGS) systems are capable of paired-end sequencing.

Paired-End Sequencing

Paired-end vs. single-end sequencing advantages

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 genomic DNA or cDNA sequencing
  • Broad range of applications: Does not require methylation of DNA or restriction digestion; can be used for bisulfite sequencing
  • Simple data analysis: Enables high-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.
Single-read sequencing
  • Cost-effective uses: This solution delivers large volumes of high-quality data, rapidly and economically
  • Specific applications: Single-read sequencing can be a good choice for certain methods such as small RNA-Seq or chromatin immunoprecipitation sequencing (ChIP-Seq)

Genomic and transcriptomic paired-end sequencing

Paired-end DNA sequencing

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

Paired-end RNA sequencing

Paired-end RNA sequencing (RNA-Seq) enables discovery applications such as detecting gene fusions in cancer and characterizing novel splice isoforms.2

For paired-end RNA-Seq, Illumina offers kits with an alternate fragmentation protocol, followed by standard Illumina paired-end cluster generation and sequencing.

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  1. Nakazato T, Ohta T, Bono H. Experimental design-based functional mining and characterization of high-throughput sequencing data in the sequence read archive. PLoS One. 2013;8(10):e77910.
  2. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10:57–63.