The Introduction and Workflow of Pre-made Library Sequencing

Introduction to Pre-made Library Sequencing

For next-generation sequencing, fully automated sequencing runs at a lower cost per base and faster assay times are available with lately introduced high throughput and benchtop instruments. As a result, an important bottleneck has been detected in the complex and time-consuming library preparation process, which begins with isolated nucleic acids and ends with amplified and barcoded DNA with sequencing adapters. Library preparation protocols are usually multistep processes that require expensive reagents and a lot of hands-on time. To guarantee robustness and reproducibility, a strong emphasis on standardization will be required.

Preparing Sequencing Libraries

DNA and RNA sequencing libraries

Starting with genomic DNA or RNA, a sequencing library can be created. The workflow for creating a DNA sequencing library is made up of three basic steps:

- Nucleic acid (DNA or RNA) fragmentation and sizing to acquire fragments of a predetermined length,

- Connection of adaptors (adapters) to the fragment extremities, and

- Library quantification.

There is an additional step in any RNA sequencing library: RNA conversion to cDNA. The fragmentation procedure can be performed either before or after the cDNA synthesis.

Fragmentation

Physical methods, enzymatic methods, and chemical methods can all be used to fragment nucleic acids (DNA, RNA, or cDNA). The most frequently used techniques are physical and enzymatic. Specifically, long fragments can be achieved for mate-pair libraries (6,000 to 20,000 bp).

Fragment sizing

The size of the fragments is extremely important. The ideal library fragment size is determined by the platform to be used and the scope of the analysis. On Illumina platforms, for example, fragments of up to 1,500 bp can be used In the case of exome sequencing, however, a maximum insert size of 200-250 bp is suggested. This is due to a human exon's average size of 200 base pairs.

Attachment of the adapters

The so-called adapters must be affixed to both extremities of each fragment once the DNA or RNA fragmentation is complete. By definition, a sequencing library is a collection of DNA fragments with adapters connected Adapters are made to work with a specific sequencing platform, such as the flow-cell surface or beads. Following the attachment of the adapters, a sizing phase occurs, during which all fragments of undesirable size and all adapter dimers are removed Adapter dimers form when adapters self-ligate without a library insert sequence, and they're especially common when the initial DNA quantity is low. It is critical to remove adapter dimers from the library because they can significantly reduce sequencing yield by consuming valuable flow cell space. A clean-up with magnetic beads can effectively erase the dimers.

Library quantification

The library quantification is a critical step that should be carried out using the most precise and practical method possible. PCR-based methods (digital PCR or quantitative PCR) are commonly used to quantify sequencing libraries.

The final quality of the sequencing library

When creating a sequencing library, it's critical to aim for the highest level of complexity possible. In other words, it is critical that the final library captures as much of the original material's uniqueness as possible. Limiting the number of segmental duplications is the first step toward achieving this result. The shorter the fragments are, the more likely they are to be less specific and align at multiple loci in the reference sequence. As a result, the percentage of duplicate reads in the sequencing data can be used to determine library complexity.

References:

1. Hess JF, Kohl TA, Kotrová M, et al. Library preparation for next generation sequencing: a review of automation strategies. Biotechnology advances. 2020 Jul 1;41.
2. Head SR, Komori HK, LaMere SA, et al. Library construction for next-generation sequencing: overviews and challenges. Biotechniques. 2014 Feb;56(2).
3. Robin JD, Ludlow AT, LaRanger R, et al. Comparison of DNA quantification methods for next generation sequencing. Scientific reports. 2016 Apr 6;6(1).