reducing the cost of Nextera XT library preparation for single cell RNA-seq
The rapid advance of NGS technologies has led to an increasing need for high-throughput, low-cost NGS library preparation. Single cell genomics, microbial genomics and other fields can strongly benefit from an increased sample throughput through cost-effective library preparation in a flexible SBS microplate format. TTP Labtech’s positive displacement pipetting technology enables automated, miniaturised library preparation. With mosquito® liquid handlers, Nextera® XT library preparation for Illumina® sequencing has been established and validated at up to 25-fold reduced volumes compared with the manufacturer’s standard protocol.
Watch our on-demand webinar: Miniaturization and Automation of Single-Cell RNAseq Library Preparation
In this free, on-demand tutorial, Dr Louise Laurent (UC San Diego) demonstrates how Nextera XT library preparation can be scaled down to as little as 2 µL total volume, using the accurate positive displacement pipetting technology of a mosquito liquid handler in an automated high-throughput environment. The workflow has been published in the Journal of Laboratory Automation.
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Dr. Soheila Vaezeslami talks about miniaturising next generation sequencing (NGS) reaction volumes within an automated, high throughput setting. – Keeping the same complexity of data with lower volumes.
genomics application areas for miniaturised liquid handling
TTP Labtech’s mosquito liquid handling robots (single, 8- or 16-channel; volume range 25 -1200 nL or 0.5 – 5 µL) can be used for miniaturising a wide range of genomics applications:
- Next-Generation Sequencing – Low-cost, low-volume library preparation, including magnetic bead clean-up and normalisation; validated with market leading kits including Illumina Nextera XT.
- Single Cell Genomics – miniaturised cDNA synthesis, whole genome amplification and library prep.
- Synthetic & molecular biology – High-throughput, miniaturised reactions for PCR, cloning and DNA assembly.
- Gene expression profiling – Cost-effective, high-throughput analysis of mRNA expression with rapid qPCR set-up.
At final volumes of 5 μL or less, miniaturised workflows can reduce sample preparation costs by 90% or more, while providing increased throughput, process control and standardisation.
TTP Labtech’s liquid handlers are not restricted to specific kits or processes. Being an open, fully programmable platform, these systems are commonly used in any enzymatic reactions that would benefit from lower reagent costs, standardised and reproducible high throughput set-up and/or reduced sample requirements. Moreover, our experts are available to help with different genomics applications, including but not limited to: NGS library prep, SPRI- bead clean-up and synthetic biology applications.
Find out how GeneMill (University of Liverpool) managed to increase their throughput of the standard synthetic biology workflow in our latest application note >> Get the app note here
the technology: positive displacement pipetting
Unlike traditional liquid handlers, the mosquito robot uses positive displacement liquid handling technology. This superior pipetting mechanism allows dispensing in the 25 nL – 5 µL range with high precision and accuracy, across the volume range. Using this technology to prepare lower reaction volumes results in dramatic cost savings while preserving data quality compared to standard reaction volumes.
Furthermore, the use of disposable mosquito tips ensures zero cross-contamination and gentle sample handling. Additionally, it offers the ability to pipette liquids with high viscosities (such as enzymes in 50% glycerol or genomic DNA) accurately and precisely. It provides a unique opportunity to maximise data output in a Genomics research laboratory or core facility setting.
Validated applications from major genomics collaborators include:
- Automated low-volume DNA normalisation and NGS library prep for single cell analysis (Prof Stephen Quake and Prof Irving Weissman lab, Stanford)
- Miniaturised Gateway cloning, PCR amplification and Nextera library generation (Novartis)
- Low-volume, single-cell RNA-seq library preparation using Nextera XT (UC San Diego)
Featured in JALA with Louise Laurent’s lab (UCSD): Miniaturization Technologies for Efficient Single-Cell Library Preparation for Next-Generation Sequencing publication.
Different stages of human pancreatic stem cells were studies using single cell RNA-seq (scRNA-seq). 2, 4 and 8 uL Nextera XT libraries were prepared with technical and biological replicates using mosquito. The libraries were also cleaned up with magnetic beads in 384-well format on mosquito. The data confirms that miniaturization of Nextera XT reaction volumes does not affect the scRNA-seq data quality. >> Read the publication
Featured in Cell by Irving Weissman’s lab (Stanford University School of Medicine): Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types..
Here, high-through-put scRNA-seq was used to systematically catalogue the diversity of intermediate cell states formed during differentiation, and the minimal combinations of positive and negative signals that were sufficient for differentiation between each of these intermediate states was tested. mosquito X1 was used for the normalisation of cDNA concentrations, and mosquito HTS was used for Nextera XT library prep at 4 uL total volume. >> Read the publication
Featured in Scientific Data: An atlas of transcriptional, chromatin accessibility, and surface marker changes in human mesoderm development.
Expanding on the methods section of the recent Cell publication from Irving Weissman’s lab at Stanford, this publication details the use of mosquito HTS and mosquito X1 in enabling a high throughput single cell SMART-seq workflow with tight process control, standardization and volume reductions. >> Read the publication
Featured in Cell: DNA damage signaling instructs polyploid macrophage fate in granulomas.
scRNA-seq with a modified CEL-seq2 protocol helped uncover the transcription events that produce polyploid macrophages in response to persistent inflammatory stimuli. With mosquito, researchers from the Max Planck Institute of Immunobiology and Epigenetics and the University of Freiburg were able to set up a robust high-throughput CEL-seq2 workflow, reducing reaction volumes by 5-fold. >> Read the publication