Technical comment on ligation-based vs tagmentation-based (BLT) DNA Library prep
The Dresden-concept Genome Center (DcGC) offers state-of-the-art NGS library preparation methods. We continuously evaluate and update our methods portfolio to offer highly efficient, high quality and cost-effective workflows to meet your project’s needs.
Here we present an assessment of two methods used for preparation of sequencing libraries applied at the DcGC: Library preparation based on fragmentation and adapter ligation, or tagmentation-based library preparation.
The first approach is based on physical fragmentation of the DNA to a desired range of fragment length distribution, and subsequent adapter addition based on DNA ligation. For the latter approach, Illumina has spearheaded the development of bead-linked transposomes short BLT. This protocol allows simultaneous fragmentation and addition of PCR adapters in one enzymatic step. At the same time, due to the physical arrangement of the transposome on bead surfaces, this workflow enables considerable control of resulting library fragment sizes - independent on DNA input quantity and quality.
Overall, both workflows result in conceptually evenly distributed DNA shotgun sequencing data. However, transposome-based fragmentation could be biased by sequence pattern preference of the used enzyme, resulting in under-representation of genomic regions and subsequently compromised library complexity. While indeed an enriched sequence pattern on the first 10-15 bases of sequencing reads has been found (own observation and (Gunasekera et al. 2021)), multiple reports have shown that these do not adversely affect library complexity or genome coverage in a wide variety of species and sequence contexts (Gunasekera et al. 2021; Ribarska et al. 2022; Bruinsma et al. 2018; Shen et al. 2023). On the contrary, comparative studies of a wide variety of library preparation methods showed that the most important factor for library complexity and even genome coverage is the library fragment size, and that Illumina BLT methods reproducibly produce more homogenous fragment sizes when compared to ligation-based methods and to competitive products (Ribarska et al. 2022). Also in the clinical diagnostics perspective, BLT workflows perform on par if not better in SNP and CNV calling on medical samples – including GC-rich mitochondria sequences (Shen et al. 2023).
Based on our own experiences and due to the published evaluations of library complexity and data quality (see references), we decided to use per default BLT workflows for all requested DNA-Sequencing application. The BLT workflow allows a faster processing of samples and thus lower costs for clients, as adjustments on sample input quantity and quality can (partially) be omitted (Bruinsma et al. 2018). We still maintain the ligation-based upon request and after counselling of your specific project. You can contact us for in-depth counselling or further questions remaining under genomecenter@tu-dresden.de.