In Sanger Single-Cell Pipeline, Echo Liquid Handler Reduces Library Prep Time and Cost

Dr. Stephan Lorenz

Senior Scientific Manager, Wellcome Trust Sanger Institute Single Cell Genomics Core Facility

In Sanger Single-Cell Pipeline, Echo Liquid Handler Reduces Library Prep Time and Cost

With the rise of single-cell genomics, scientists have a new opportunity to make breakthrough discoveries in cancer, aging, immunology, and much more. Enabling those breakthroughs is the mission of the Single Cell Genomics Core Facility at the Wellcome Trust Sanger Institute, where scientists provide deep technical expertise and a sophisticated sequencing pipeline for characterizing genomes and transcriptomes. Customers can also take advantage of novel protocols developed by the institute’s single-cell specialists to generate better results.

The core lab is headed up by Stephan Lorenz, who sees single-cell studies as “one of the most exciting and fast-growing fields in genomic science.” For his customers’ research, access to such precise resolution is a game-changer. “This technology allows us to look at each fundamental unit of life — a cell — and study any biological process that leaves its fingerprint on the genome or the transcriptome,” he says. “It provides a lot more insight and data about the biological process of interest rather than sequencing the average of a million cells.”

 PRESENTATION VIDEO

Implementation of Acoustic Dispensing into HT Single Cell Sequencing Workflows

with Dr. Stephan Lorenz

Dr. Stephan Lorenz, Head of the Single Cell Genomics Core Facility at the Wellcome Trust Sanger Institute, UK was filmed speaking at the Labcyte Genomics Symposium - Edinburgh 2016. Dr. Lorenz explains the importance of single cell analysis versus cell population studies for obtaining better discrimination of individual gene expression within a population.

Streamlining RNA Library Construction

Focusing primarily on RNA, Dr. Lorenz describes, in detail, standard library construction steps and how they have streamlined them to increase productivity. After isolating cells into 96- or 384-well plates, reverse transcription of single cell RNA and amplification is carried out. This is followed by clean-up and then library construction, converting cDNA into sequencing ready libraries. A template switching, Smart-seq protocol is used at the core facility that produces amplified sequences with universal handles incorporated at either end. Dr. Lorenz describes how they have incorporated semi-automated liquid handling reverse transcription that is rapid, RNAse free easy-to-use and incorporates cDNA quality control (QC) monitoring.

Automated solid phase reversible immobilization (SPRI) clean-up is followed by sample transfer using Echo liquid handling and Access Workstation for quantification, normalization and library construction. Dr. Lorenz presents data demonstrating how acoustic liquid handling reliably transfers DNA with wide ranging sizes and concentrations. He also describes how the technology can routinely quantify eight 384-well plates, in one run with standards and normalization.

The Nextera Method for DNA Sample Preparation

The Nextera method employed at the facility is described. This method involves DNA fragmentation, with addition of a small overhang using transposases, so PCRs can be run with an adapter and primer against the overhangs, which introduces barcodes and flow cell adapters.

We use Labcyte acoustic dispenser because it allows us to perform complex assays in very high throughput...

Selected for speed, Dr. Lorenz describes how the Nextera protocols have been adapted for the Echo and miniaturized to eliminate issues of high tip consumption and high cost per sample, whilst maintaining the same performance quality. The team are currently extending this to accommodate a 1536-well plate format.

The final stages of library construction prior to sequencing are described, in particular library QC using BioAnalyser qPCR that has been simplified using acoustic liquid handling, to reduce reaction volumes 10-fold, and save time and consumables by further using a simulated serial dilution.