Short-read sequencing

Sequencing by synthesis with reversible terminators, from benchtop instruments (millions of reads) to production instruments (billions).

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Illumina

Sequencing by synthesis with reversible terminators, from benchtop instruments (millions of reads) to production instruments (billions).

In this lesson · about 1 min read

Sequencing by synthesis
What the instrument needs to work
Single-end vs paired-end
Key terms
When to use it
Sub-techniques

1Sequencing by synthesis

Library molecules carry adapters that let them hybridise to oligos on a glass flow cell. Each molecule is copied in place into a cluster of ~1000 identical copies (bridge or exclusion amplification).

K/mm²

Cluster density

Too few clusters waste the flow cell; too many overlap and can't be told apart.

Q30

Base quality

Q = −10·log₁₀(error). Q30 means one error in 1000 bases. Runs are judged on % of bases ≥ Q30.

% PF

Passing filter

Clusters with clean enough signal in the first cycles to be kept.

2What the instrument needs to work

An accurately quantified, denatured library loaded at the right concentration (qPCR counts only molecules that can form clusters).
Base diversity: in every cycle the instrument expects all four colours across clusters. Amplicon libraries that start with the same primer sequence need PhiX spiked in (10–20%) to supply diversity.
No adapter dimers: short fragments cluster efficiently and steal reads.

3Single-end vs paired-end

Single-end runs read one end of each fragment. Paired-end runs turn the molecules around and read the other end too, which doubles the data per cluster, improves mapping and lets you detect insertions, deletions and rearrangements.

Key terms

Practise these as flashcards →

Library

DNA fragments with sequencing adapters and indexes attached.

Index (barcode)

A short sequence identifying the sample, so many libraries can be pooled and separated afterwards (demultiplexing).

Cluster

A clonal group of copies of one library molecule on the flow cell.

Phred quality (Q)

Log-scaled probability that a base call is wrong: Q20 = 1%, Q30 = 0.1%.

PhiX

A well-characterised viral library spiked in as a control and to add base diversity.

FASTQ

Text format holding each read's sequence and per-base quality scores.

When to use it

Almost every genomics assay: whole genomes, exomes, RNA-seq, ChIP-seq, ATAC-seq, amplicon and single-cell libraries.
When you need many reads at low cost per base and reads of 50–300 bases are enough.
When accuracy per base matters (short reads are ~99.9% accurate).