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Comparing library preparation methods

article detailing sequencing library preparation for different sequencing technologies
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Library preparation is the first step of next-generation sequencing (NGS), preparing your samples to allow them to bind to the flow cell and be identified. This article will briefly guide you through standard library preparation for NGS using Illumina and Oxford Nanopore Technologies (ONT) sequencing, comparing the differences and similarities between these two methods.

The steps for ligation-based library preparation of any NGS are broadly the same. These consist of the following (Figure 1):

Schematic workflow of a general NGS library preparation. Detailed description in the main text

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Figure 1 – General next-generation sequencing ligation-based library preparation workflow. Diagram created using

Sample preparation

The first step for COVID-19 library preparation is the reverse transcription of extracted viral RNA to cDNA.

First quality check and quantification

Starting material should always be quality-checked first before beginning the library preparation. This quality check should include quantification to ensure sufficient starting material, and investigating, any signs of contamination as prior clean-up may be required.

End-repair and dA tailing

DNA fragments are end-repaired and dA-tailed. This, therefore, requires converting fragmented DNA to blunt-end DNA containing a 5’-phosphate and 3’-hydroxyl groups via a polymerase, and adding a tailing module to prepare fragments for subsequent ligation steps.

Addition of adapters

A ligase enzyme covalently links the adapter and end-repaired DNA fragments, producing DNA fragments that can be loaded to a flow cell. These adapters serve multiple functions; they predominantly attach the sequences to the flow cell to allow sequencing, and they can also include barcodes to identify samples and permit multiplexing.

Size selection

Another step shared between the protocols is size selection. Although performed at different stages, this step involves including or excluding fragments of DNA most of interest in relation to their size. This is either done using sample purification beads, or can be done simply by gel electrophoresis and band excision.

Second quality check and quantification

A final important step which remains constant in both protocols is quantification and validation of the library prior to flowcell loading, thereby ensuring there is sufficient DNA for sequencing, and checks for contamination of controls and samples.

Where Illumina and Nanopore differ is in Illumina’s inability to read long fragments, meaning that Illumina sequencing samples are first fragmented into uniform pieces to make them amenable for sequencing. This is often done by nebulisation, but other methods include sonication, chemical or enzyme-based fragmentation. Generally, this will mean Illumina libraries are subsequently PCR amplified, which also allows for a significantly lower DNA input.

Both methodologies allow for the multiplexing of samples, which is regularly used when performing high throughput sequencing of COVID-19 samples. This step is often incorporated with the adapter ligation stage for Illumina library preparation (Figure 2). For ONT, barcoding is a separate step prior to adaptor ligation with separate subsequent clean-up steps required.

The shared methodologies of Illumina and ONT library preparation are depicted in Figures 2 and 3 below.

Fragmented DNA sample (0-12000bp) > End-paired DNA > End-repaired dA tailed DNA > Adapter-ligated fragments > PCR-amplified library > Single-stranded DNA library > clonal DNA clusters on flowcell surface > Single-stranded clusters ready for sequencing > Incorporation of fluorescent reversible terminator nucleotides > Cleavage of fluorophores and blocking groups.”>

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Figure 2 – Illumina sequencing workflow. Diagram created using Adapted from Nature Methods.

End-repaired dA-tailed DNA > Optionally barcoded samples > Adapter-ligated fragments > Library read through pores on flowcell > DNA bases cause changes in electrical current.”>

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Figure 3 – Oxford Nanopore Technologies sequencing workflow. Diagram created using


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