Improved library quantification for High Throughput Sequencing at the Wellcome Trust of Human Genetics Oxford

Paolo Piazza
Wellcome Trust Centre for Human Genetics, United Kingdom

Recent advancements in sequencing technologies (Next Generation Sequencing) developed for the Illumina and Life Technology platforms have driven down significantly the costs per base sequenced. Lower costs coupled with significant improvements in data quality have opened the door to sequencing projects that had previously been far too expensive to complete. As a result we are witnessing an increasing demand for high quality libraries. Significant effort has been invested at the WTCHG in improving the library preparation workflows through the introduction of new equipment and automation to remove the identified process bottlenecks. Adequate quality control of each of the many different type of libraries produced in our lab is crucial to the success of the projects that we support. In order to maximize the data output and quality, cluster generation on Illumina platforms requires accurate quantification. Alternative kits for library quantification were evaluated on a Mx3005 qPCR instrument. The performance of the QPCR NGS Library Quant Kit was compared to that of other SYBR® Green based kits. Since we have introduced the Agilent qPCR instrument and reagents in our workflow, we observed that approximately 85% of the library sequenced at the WTCHG produced cluster densities within 20% from the expected value. Consumables for sequencing or unusual library types have been often the cause for suboptimal cluster generation. All the changes and improvements introduced in our lab have contributed to lowering the costs and accelerate the turnaround time for projects due to reduced failure rates, however, the rapid and continuous changes in technology will require constant adjustments to fully utilize it.

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