miniaturised next generation sequencing library preparation
True-positive displacement pipetting technology of mosquito liquid handlers provides fast, accurate and cross-contamination free sample prep for variety of NGS applications.
- very low sample input (as low as 20 pg or less)
- very low reaction volumes (as low as 1 µL total reaction volume), resulting in significant cost savings (see app note ‘A simple and automated solution for miniaturising reaction volumes for Nextera NGS sample preparation’)
- ease of use and therefore optimal for low, mid to high throughput applications
Nextera and Nextera XT sample preparation with mosquito HTS
Nextera and Nextera XT (Illumina, San Diego, USA) sample prep kits are commonly used to prepare DNA libraries. Both Nextera and Nextera XT, use an enzymatic reaction which fragments the DNA and ligates the adapters simultaneously. In order to obtain fragments of the correct size for the sequencing run, it is essential to have a precise and accurate ratio of tagmentation enzyme to DNA. Using mosquito HTS or mosquito HV, the tagmentation step can be both automated and miniaturised to sub-microliter levels, thus reducing the bottleneck in high throughput applications such as single cell analysis.
case study 1: S. Quake, Standford University
single cell DNA sequencing using miniaturised Nextera XT library prep
Researchers from Professor Stephen Quake’s laboratory at Stanford University, see poster, undertook sequencing of 384 cDNAs extracted from a mixture of 5 different bacteria species (6-20 kb); separated into single cells. Only 60 pg of input gDNA was used per each 4 µL library.
Key advantages of miniaturization of library prep reactions using mosquito HTS or mosquito HV liquid handler are:
- ultra low volume sample prep, reduces reagent cost and sample input (gDNA/DNA/RNA)
- positive displacement technology, essential in accurate and precise pipetting of small volumes and resulting in robust data
- disposable tips eliminate cross contamination
- easy to use software provides fast reaction set up for low to high throughput applications
- capable of full integration with other liquid handlers and ancillary equipment
4 µL Nextera XT libraries were prepared from a mixture of 5 different bacteria species (6-20 kb), separated into single cells. Only 60 pg of input gDNA was used per library.
Inferred insert length graph (above) showing insert sizes of ~300 bp for 4 µL libraries, as expected in standard libraries. The percentage of trimmed reads (right graph) show that well below 10% of the reads being rejected, confirming great data quality.
Data from Stephen Quake Lab
case study 2: D. Nickerson, UWGS
DNA sequencing using miniaturised Nextera library prep
Percent of the reads of 3 different large MW (over 10 kb, Coriell Institute) gDNA libraries prepared at 1 μL in 8 replicates, mapping to the given characteristic: human and unique. Only 1 ng of gDNA was used per library. Very low standard deviations show the great reproducibility of the miniaturised-volume Nextera libraries.
Scientists in Dr Debbie Nickerson’s laboratory at the University of Washington-Genome Sciences Center prepared 3 different large molecular weight (over 10 kb) gDNA libraries prepared at 1 μL in 8 replicates, using Nextera library prep kit, see table above and poster. Only 1 ng of gDNA was used per library and the sample prep cost was reduced by 50 fold as compared to the original 50 µL reactions.
Table above – Fraction of the reads of 3 libraries mapped to the given characteristic: human and unique. Low standard deviations and high mapping ability show the reproducibility of the miniaturised Nextera libraries at 1 μL.
Data from Debbie Nickerson Lab-UW Genome Sciences Center