Molecular biology / next-generation sequencing · BEGINNER LESSON
Sequencing library
A sequencing library is a prepared collection of DNA or RNA molecules made compatible with a sequencing system. It is the material that gets read by a sequencer, and its preparation usually includes adding adapter sequences and often measuring how much duplicate or incomplete material is present.
What it is and what is measured
A sequencing library is the sample placed into a sequencing instrument after the target nucleic acid has been converted into a form the platform can read. In practice, the library is measured indirectly by the sequence reads it produces, including read count, read length, coverage, duplicate-read rate, and the fraction of useful molecules versus artifacts such as adapter dimers. Coverage means how many times a base is read on average. Read means one continuous stretch of sequence returned by the instrument. [0,1,2]
[1] Deep sequencing in library selection projects: what insight does it ... [2] Library construction for next-generation sequencing: Overviews [3] Explanatory Chapter: Next Generation Sequencing - PMC
How sequencing libraries are made
Library preparation starts with DNA or RNA and usually includes fragmentation, end-repair, adding a phosphate to 5′ ends, adding A bases to 3′ ends, ligating adapters, and sometimes PCR, which is a copying step used to enrich molecules that have adapters on both ends. Some systems use special workflows, such as tagmentation, where a transposase both cuts DNA and inserts adapters at the same time. Adapter sequences are required so the sequencer can recognize and amplify the library molecules. [1,2]
[2] Library construction for next-generation sequencing: Overviews [3] Explanatory Chapter: Next Generation Sequencing - PMC
How library design affects sequencing results
The main goal is to reduce bias, meaning a systematic distortion caused by the design or handling of the sample. Library complexity can be estimated from duplicate reads, because many duplicates suggest fewer unique starting molecules. Read length and sequencing depth trade off against each other: platforms that read longer stretches often produce fewer reads, while platforms with very high throughput may read shorter fragments. [0,1]
[1] Deep sequencing in library selection projects: what insight does it ... [2] Library construction for next-generation sequencing: Overviews
Why library choice matters in different studies
Different library types are suited to different questions. Fragment libraries are useful for detecting small sequence differences because overlapping reads can cover the same region many times. Mate-paired libraries use larger DNA fragments and help connect distant sequence pieces. In antibody or binding-protein studies, sequencing libraries can be used to trace enriched clones and estimate how much diversity remains after selection. [0,2]
[1] Deep sequencing in library selection projects: what insight does it ... [3] Explanatory Chapter: Next Generation Sequencing - PMC
Limitations and pitfalls
A library can be misleading if it contains adapter dimers, low input material, amplification artifacts, or sequencing errors. Errors are especially important when trying to estimate the true diversity of a large unselected library, because the full set of possible molecules may exceed the capacity of deep sequencing. Incomplete reads can also prevent full reconstruction of longer molecules. [0,1,2]
[1] Deep sequencing in library selection projects: what insight does it ... [2] Library construction for next-generation sequencing: Overviews [3] Explanatory Chapter: Next Generation Sequencing - PMC
SEE THE IDEA
From sample to sequencing results
Starting nucleic acid from the sample
Fragmentation or tagmentation, then adapter addition and often PCR
Prepared molecules that the instrument can read
Read count, coverage, duplicates, and sequence variation
The target nucleic acid is converted into a library, and the library is then read to generate sequence data and quality measures.
A sequencing library is the bridge between a biological sample and the sequence data used for analysis.
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Sources and further reading
This general lesson was generated from public source material. It explains a concept; it does not interpret a private paper or provide medical advice.