The generation of NGS libraries from single cells is growing in demand as the use of single-cell analysis broadens. The analysis and comparison of an individual cell against a population and against other individual cells allows researchers to implement different sequencing techniques in the fields of cancer, metagenomics, stem cells, developmental biology, immunology, neurobiology, drug discovery and almost any other area in biology. The detection of DNA mutations, CNVs, DNA–protein binding, RNA splicing and the measurement of mRNA expression gives researchers new insight into what is happening at a particular moment in an individual cell.

For the analysis of individual cells, the first step is to isolate them from suspension or tissues. This can be achieved by a variety of methods, such as FACS, serial dilutions, laser capture microdisection and microfluidics. The approach taken depends on the final objective and the resources available. Once the cells are isolated, sample preparation using the kits begins. Most of the kits’ protocols consist of cell lysis, nucleic acid denaturation, neutralization, amplification, fragmentation, end-repair and platform-specific adaptors ligation. The amplification step, of critical importance, is typically done by PCR, but some brands have introduced PCR-free amplification based on methods such as multiple displacement amplification. If the target molecule is RNA, then reverse transcription is needed as an additional step at the beginning.

Due to the novelty of single-cell library preparation techniques, there are only a handful of vendors that focus on single-cell library preparation kits. Among the kits available are QIAGEN’s QIAseq FX Single Cell DNA and RNA Library Kits, Rubicon Genomics’ PicoPLEX WGA Kit, Takara’s SMART-Seq v4 Ultra Low Input RNA Kit, 10x Genomics’ Single Cell 3’ Reagent Kit, and the recently launched SureCell WTA 3ʹ Library Prep Kit from the partnership between Illumina and Bio-Rad Technologies. The two latter kits are substantially different from the other mentioned kits, as single-cell RNA libraries are barcoded as part of the protocol, which allows differentiation of unique reads for more ac­curate downstream analysis. The different kits have their advantages and disadvantages that need to be weighed according to the purpose of the experiment.

There are two fundamental technological challenges on which single-cell library preparation kit suppliers are focused: 1) the ability to work with the highest possible cell-capture capacity, and 2) the amplification of genetic material to yield sufficient material for downstream analyses while minimizing the introduction of artifacts, such as amplification bias, genome loss, mutations and chimeras.

The pharmaceutical and biotechnology sector is the most rapidly growing market for single-cell library preparation kits due to the exponential rise of NGS. Demand from this sector is expected to continue growing due to strong interest in companion diagnostics, biomarker discovery and cell-based applications.

Academic and government research facilities also account for a big portion of the market, as the decreasing cost of NGS instruments and the cost per analysis, along with the emerging flexible and comprehensive NGS systems entering the market, have paved the way for the use of systems in dedicated facilities for rapid single-cell DNA sequencing for identifying disease-causing mutation sequences. Single-cell library preparation is a rapidly growing market that is expected to see advances in clinical research settings in a few years.

NGS Single-Cell Library Preparation Kits at a Glance:

Leading Suppliers:

• QIAGEN
• Rubicon Genomics

Major markets:

• Academia
• Government Research
• Pharmaceutical and Biotech

Kit Cost (96 Reactions):

• $4,000­–$6,000