Tissue to Library Preparation on the Apollo 324 System for Clinical Sequencing

Over the past decade, next generation sequencing (NGS) technologies have revolutionized genetic and biological research. The sensitivity, speed, throughput and continuing reductions in cost per sample of NGS technologies have stimulated broad interest in clinical applications. Successful adoption of NGS technologies for routine and widespread clinical applications requires consistent and reliable sample processing from raw tissue through nucleic acid extraction, size fractionation, and library preparation. We have explored automation of library preparation from mammalian brain tissue samples for analysis on the Genome Analyzer IIx (Illumina Inc.). Rabbit brain tissue lysate was prepared by grinding frozen tissue with buffer in a mortar and pestle. The process of DNA isolation, enzymatic fragmentation and library preparation was automated on the Apollo 324 system (IntegenX Inc.). In our streamlined automated workflow, we isolated DNA from the tissue lysate by digestion with Proteinase K (New England BioLabs), followed by DNA purification with paramagnetic beads, enzymatic fragmentation using NEBNext dsDNA Fragmentase® (New England BioLabs) and library preparation with the PrepX ILM Library Preparation Kit reagents (IntegenX Inc.). The library was enriched by amplification in a commercial thermocyler and sequenced on the Genome Analyzer IIx. Our automated method produces consistent libraries from eight tissue samples in three hours. We obtained no GC bias, 80% of confidently mapped reads from libraries prepared from 1 μg of brain tissue sample. The automation of all downstream library processing steps after homogenization will be directly relevant to the development of clinical NGS systems.