Next generation sequencing

Low-volume dispensing and miniaturization of library preparation for next-generation sequencing

Introduction

Next-generation sequencing (NGS) is a high-throughput method of sequencing that is compatible with a variety of samples, including microbial genomes and transcriptomes. Prior to sequencing, the genomes are fragmented into smaller sizes, and adapters are introduced for flow cell attachment during sequencing. These adapters can contain barcodes or indexes for multiplexing. Thus, the NGS library prep of samples is an important final step before sequencing.

As NGS library prep is a significant cost of the total workflow, miniaturization of library prep enables the generation of high-quality samples while reducing overall costs. Cost also becomes more critical when many libraries are sequenced simultaneously. The I-DOT provides easy reaction miniaturization, while maintaining flexibility to perform various types of NGS library protocols.

Normalization

Indexing

Purification

Sequencing

Immediate Drop-on-demand technology (I-DOT) for low-volume, non-contact dispensing

The I-DOT is a patented approach for optimizing noncontact liquid-handling tasks. The system uses eight individually controlled positive pressure channels to generate droplets from 8 to 50 nanoliters from a small outlet at the bottom of each well. Each channel can generate up to 100 droplets per second, which enables a volume range from 8 nanoliters to 80 microliters. The non-contact dispensing significantly reduces the number pipette tips required during NGS library prep, and the low-volume dispensing (dead volume <1 µL) provides significant cost savings on reagents.

Accurate dispensing and built-in droplet verification

The I-DOT supports multiple liquid classes and can dispense a variety of solutions, including PCR buffer, DMSO and glycerol up to 50%, making it compatible with both commercial and in-house generated reagents. Accurate dispensing is essential for producing high-quality data that shows minimal technical variation between samples. The built-in droplet verification detects and counts every droplet released during a single dispensing run. The I-DOT is the only liquid dispenser that offers built-in droplet quality control.

Quantitation and Normalization of NGS libraries

Typically, input DNA needs to be normalized before NGS library preparation to ensure an even read distribution for all samples. Hundreds of libraries can be quantitated and normalized within minutes using the I-DOT, which allows for highly multiplexed dispensing of up to 96 reagents in parallel. Reagent volumes for dye-based normalization assays can be scaled down orders of magnitude for additional cost savings.

Fast and simplified DNA Indexing or Barcoding

A single I-DOT source well plate can contain up to 96 different barcoded primers, a powerful feature for high-throughput indexing or barcoding of NGS samples. With one 96-well source plate, over 2,000 samples can be uniquely barcoded, making the I-DOT readily compatible with high-throughput processing. With the I-DOT’s intuitive software, complex barcoding protocols can be easily generated. Using a non-contact, low-volume technology can save thousands of pipette tips while eliminating the risk of cross-contamination.

Miniaturization and Automation of a Full-length Single-cell RNA-seq Workflow

An introduction to a miniaturized, highthroughput workflow in which single HEK293FT cells were sorted by the f.sight™ into 384-well PCR plates, then libraries were prepared with the I-DOT.

CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

Author(s): Xiaolu Zhang, Silvano Garnerone, Michele Simonetti, Luuk Harbers, Marcin Nicoś, Reza Mirzazadeh, Tiziana Venesio, Anna Sapino, Johan Hartman, Caterina Marchiò, Magda Bienko & Nicola Crosetto

Karolinska Institutet

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I-DOT

A premier solution for noncontact liquid handling tasks. Optimizing your workflow to bring intuitive automation, precision and speed.