9 Sequencing library preparation

Sequencing library preparation is a crucial step in the process of DNA sequencing. It involves the conversion of fragmented DNA molecules into a format that is compatible with the sequencing platform. The goal of library preparation is to create a collection of DNA fragments, each with sequencing adapters attached, which enables high-throughput sequencing of the DNA molecules. This process ensures that the genetic information contained in the DNA sample can be accurately and efficiently read by the sequencing instrument.

Sequencing strategies and platforms

Library features are specific to each sequencing platform, which requires selecting in advance the sequencing strategy to be employed. Pure nucleic acid sequencing-based strategies can be broadly divided in two groups. Short-read sequencing (SRS) platforms provide large amounts of data yet with short sequencing reads (typically 150 nucleotides). In contrast, long-read sequencing (LRS) platforms yield much longer sequences (thousands or even million of nucleotides), yet with a lower throughput, and typically lower sequence quality. The SRS market is dominated by two main companies with proprietary platforms, namely Illumina and BGI, although PacBio recently released their own SRS platform called ONSO. The LRS market is also dominated by two different companies with proprietary technologies, which are Oxford Nanopore (ONT) and Pacific Biosciences (Pacbio).

Sequencing enterprises, as well as auxiliary biotechnological companies, provide library preparation kits that can be more or less customised for different purposes.

Technology Platforms Sequencing type Company
Sequencing by synthesis (SBS) MiSeq, NovaSeq Short-read sequencing Illumina
Combinatorial probe-anchor synthesis (cPAS) DNBSeq Short-read sequencing BGI
Sequencing by binding (SBB) technology Onso Short-read sequencing PacBio
Single Molecule Real-Time sequencing (SMRT) Sequel, Revio Long-read sequencing PacBio
Nanopore sequencing MinION, GridION, PromethION Long-read sequencing Oxford Nanopore

Some of the most widely used sequencing technologies and platforms.

PCR-based vs. PCR-free library preparation

Sequencing library preparation procedures can be split into two main groups depending on whether they PCR-amplify or not the DNA templates. Unlike in the case of targeted amplicon sequencing, in which the objective is to amplify a specific target region, the aim of including a PCR step in shotgun-based library preparation is to increase the molarity of the library and/or to attach indices (see below) to the adaptors.

Learn more about PCR-based and PCR-free library preparation in this article by Jones et al. [30].

Indices and multiplexing

Usually, library preparation also entails tagging molecules with unique sample identifiers known as indices, which enable pooling molecules derived from multiple samples in a single sequencing run. This can be achieved in PCR-free protocols by using adaptors containing unique indices per sample, or by using indexed amplification primers in PCR-based library preparation protocols.

Learn more about indices and multiplexing in this article by Kircher et al. [31].

Unique molecular identifiers (UMIs)

Unique molecular identifiers (UMIs) are a type of molecular barcoding that provides error correction and increased accuracy during sequencing by uniquely tag each molecule (rather than each pool of molecules derived from a sample) in a sample library. UMIs are used for a wide range of sequencing applications, many around PCR duplicates in DNA and cDNA. UMI deduplication is also useful for RNA-seq gene expression analysis and other quantitative sequencing methods.

Learn more about unique molecular identifiers in this article by Kivioja et al. [32].

Contents of this section were created by Antton Alberdi.


30. Jones MB, Highlander SK, Anderson EL, Li W, Dayrit M, Klitgord N, et al. Library preparation methodology can influence genomic and functional predictions in human microbiome research. Proc Natl Acad Sci U S A. 2015;112:14024–9.
31. Kircher M, Sawyer S, Meyer M. Double indexing overcomes inaccuracies in multiplex sequencing on the illumina platform. Nucleic Acids Res. 2012;40:e3.
32. Kivioja T, Vähärautio A, Karlsson K, Bonke M, Enge M, Linnarsson S, et al. Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods. 2011;9:72–4.