DNA/RNA concentration normalisation using mosquito low-volume liquid handlers
Downstream analysis of DNA samples, including library prep protocols for NGS, often requires the concentration of DNA or RNA to be normalised. Patient samples are often precious with only small volumes available for each reaction. Therefore being able to perform the normalisation step of the samples at very low volumes would be essential.
case study: S. Quake, Stanford – low-volume and fast normalisation of DNA samples
A Nextera XT sample prep kit (Illumina, San Diego, USA) is commonly used to prepare DNA libraries. During this process, it is essential to have a precise, accurate ratio of tagmentation enzyme to DNA sample to obtain fragments of the correct size. In this study at Professor Stephen Quake lab (Stanford University, Stanford, CA) the differences between macrophages associated with healthy and abnormal mouse tissues are shown using single-cell RNAseq. mosquito X1 was used for automated miniaturised DNA normalisation post cDNAs synthesis using the C1 Autoprep system (Fluidigm, South San Francisco, USA). Nextera XT libraries were then prepared using mosquito HTS, with only 80pg of cDNA in 4 µL total reaction volume.
The figure shows the percentage of mappability of reads from 96 different macrophages to unique mouse reference genome (exons, introns and translation start sites). All the reads mapped over 78% at 4 µL, indicating reliability of the data.