(1175-D) Miniaturization of DNA library preparation for Illumina sequencing using automated positive displacement liquid handling

Advances in next generation sequencing have led to huge increases in throughput with associated decreases in costs. As a result, the process of manual library preparation has become even more of a financial and time constraint to high throughput sequencing core facilities, including the Centre for Genomic Research (CGR) at the University of Liverpool, UK.

Automation, in the form of liquid handling robots, has been able to alleviate some of these bottlenecks. One such system, mosquito® HV by SPT Labtech, pipettes liquids using positive displacement via a spool of disposable tips containing a small stainless steel rod inserted into each tip. This mechanism means that unlike traditional liquid handlers, mosquito can accurately pipette nanoliter volumes without a requirement for specialist liquid classes being assigned by the instrument control software.

This study describes the application of mosquito HV at CGR to successfully implement the preparation of DNA libraries for Illumina sequencing in one tenth the volume of the original manual protocol. The NEBNext Ultra II FS DNA method was chosen because it uses an enzymatic fragmentation that negates the requirement for a physical fragmentation of the DNA at the start of the library preparation.

This work demonstrates that NEBNext Ultra II FS DNA libraries prepared at 1 in 10 performed as well as the full volume manually prepared libraries, whilst providing significant cost savings through miniaturization of reaction volumes. The percentage duplicates, mapping levels, fold coverage and GC skew were comparable down to 1 ng of input DNA. The full workflow can be completed within a day enabling larger projects with a greater number of samples, with less technical bias.