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Question 12 of 20 Fundamentals Concept

What does paired-end sequencing add over single-read sequencing?

Short answer

Reading both ends of each fragment gives two linked reads with a known orientation and approximate distance. That improves alignment in repeats and makes indels, rearrangements, gene fusions and new isoforms easier to detect.

Why

In single-read sequencing, one end of each fragment is read. In paired-end sequencing, both ends are read, and the two reads are known to come from one fragment of roughly known length, facing each other. Illumina's own guidance lists the gains: more accurate alignment, better handling of repetitive sequence, and easier detection of insertions, deletions, inversions, gene fusions and novel transcripts.

The logic is that one read may fall in a repeat and map to many places, while its mate lands in unique sequence and anchors the pair. A pair that maps too far apart, in the wrong orientation or on two chromosomes is evidence of a structural change. The known pairing also helps duplicate marking, because two pairs with the same start positions for both reads are much more likely to be PCR copies than two single reads that share one start.

Single reads remain adequate and cheaper for counting applications, such as many gene expression or ChIP-seq studies, where the question is how many reads fall in a region rather than its exact structure.

A strong answer covers

  • Two reads from one fragment with known orientation and spacing
  • Mates anchor reads that fall in repeats
  • Discordant pairs reveal structural variants and fusions
  • Improves duplicate identification
  • Single reads can suffice for counting applications

Common mistakes

  • Treating the two mates as independent reads that double the evidence
  • Assuming paired-end is always worth the cost for simple counting studies
  • Ignoring insert size when interpreting overlapping mates