Library quantification is a key stage in Illumina’s next generation sequencing (NGS) workflow. Scientists from Beckman Coulter have recently described an automation method that enables 196 NGS library samples to be quantified in triplicate, with low variability. As well as determining library quantification, the method also checks for cross-contamination. A 384-well set up was automated for the KAPA Biosystems Library Quantification Kit for Illumina® Sequencing Platforms on the Biomek® FXP (BFXP) Workstation with Multichannel Pipettor and Span-8 Pod.
Biomek® FXP Laboratory Automation Workstation
Forty-eight replicate samples of a single NGS library preparation were analyzed in a checkerboard pattern. After placing the library plate on the deck of the BFXP, the Span-8 Pod added library dilution buffer to three dilution plates and the Multichannel Pod diluted the samples 1:50 three times for a total dilution factor of 1:125,000. The Span-8 Pod moved the KAPA master mix and standards from 2 ml tubes to the 384-well plate and the Multichannel Pod stamped the diluted libraries/blanks into the first three quadrants of the qPCR plate.
Using the settings recommended by the KAPA kit, the qPCR plate was processed on a 7900HT Fast Real-Time PCR System. The Ct data generated for all triplicate samples showed that, of these wells, the lowest Ct was 33, corresponding to 0.002% of the library sample concentrations. Therefore, even if these readings corresponded with cross-contamination rather than background signals such as primer dimers, any effect on sample quantification would be negligible.
Analysis of the CVs for individual triplicates of the 48 samples showed that they all gave a CV at or below 8.3%, with the CV falling below 5% for all but four samples. Determination of the average library concentration and variability between the 48 samples showed that the concentration of the starting library was 13.29nM and variability across the plate was low, as indicated by a CV of 4.1%.
The BFXP is a fast and reliable instrument for automation of the KAPA Biosystems Library Quantification Kit for Illumina Sequencing Platforms in a 384-well format. Using this method, the operator can walk away from the equipment and leave it to quantify up to 96 samples in triplicate, which can be achieved in just 45 minutes.
For more information on this automation solution, please access the following link: Biomek Automated 384-well qPCR Bulletin
About Beckman Coulter
Beckman Coulter develops, manufactures and markets products that simplify, automate and innovate complex biomedical tests. More than a quarter of a million Beckman Coulter instruments operate in laboratories around the world, supplying critical information for improving patient health and reducing the cost of care.
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