In-depth comparison of library pooling strategies for multiplexing bacterial species in NGS

• Published in: Diagnostic microbiology and infectious disease Vol. 95; no. 1; pp. 28 - 33
• Main Authors: Muller, B.H., Mollon, P., Santiago-Allexant, E., Javerliat, F., Kaneko, G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2019
• Subjects: Bacteria - classification; Bacteria - genetics; Bacterial genome size polymorphism; Coverage depth; Cross Infection - microbiology; DNA, Bacterial - genetics; Genome Size; Genome, Bacterial - genetics; Genomic investigation; High-Throughput Nucleotide Sequencing; Humans; Library pooling; Multi-species multiplexing; Multiplex Polymerase Chain Reaction; Sequence Analysis, DNA; Whole-genome sequencing; WGS; Bacterial genome size polymorphism; Library pooling; bp; HAI; Multi-species multiplexing; Whole-genome sequencing; kb; Genomic investigation; Mbp; EqSa; NGS; Coverage depth; gDNA
• ISSN: 0732-8893; 1879-0070
• DOI: 10.1016/j.diagmicrobio.2019.04.014

For bacterial genome sequencing, libraries from different strains are usually multiplexed in a single run. Normalized libraries are most often pooled in equal volumes, as recommended by next-generation sequencing platform manufacturers. This equal-volume strategy is well suited for multiplexing isolates from the same species. However, for runs involving multiple microbial species, an equimolar library pooling is more adapted because of the variation in bacterial genome size. To demonstrate its utility in clinical microbiology, we compared both equal-volume and equimolar strategies using a menu comprising 13 bacterial species involved in healthcare-associated infections. We show that equimolar pooling limits the retesting risk due to insufficient coverage depth, particularly when interspecies genome size difference is more than 2-fold. The use of this alternative strategy for multiplexing pathogenic bacteria should lead to more cost effective whole-genome sequencing applications in clinical microbiology.