October 15, 2015 (Vol. 35, No. 18)

Glenn Fu Ph.D. Founder and Senior Scientist Cellular Research

Technique Enables Single-Cell Gene Analysis, Eliminating Amplification Bias

Gene expression analysis tools have evolved from hybridization array-based technologies to quantitative reverse transcription (RT-PCR or qPCR), and most recently, RNA sequencing (RNA-Seq). Though RNA-Seq has become the gold standard and can be used to determine relative transcript abundance, it is costly, time intensive, comparatively inaccurate, and requires considerable expertise for assay design, performance, and data analysis.

Generating libraries for RNA-Seq is a laborious process with loss of precious sample at every step. The presence of high-abundance RNAs (rRNA, etc.) has required the inclusion of methods to reduce background RNA and/or enrich for poly-A mRNAs. Although these methods improve data quality, they contribute to the labor and time required, as well as to loss of original mRNA sample.

Low efficiency has led to the requirement for restrictively high amounts of initial sample and/or many rounds of PCR amplification, which have been shown to distort abundance measurements due to differential amplification efficiencies between transcripts. Taken together, these limitations challenge the use of standard RNA-Seq library prep methods for precise, accurate, and reliable mRNA quantification. 

Advantages of Molecular Indexing and Precise Assays

To address the limitations of RNA-Seq for targeted gene expression experiments, Cellular Research has developed the Molecular Indexing™ technology. This novel method eliminates amplification biases and enables direct quantification of molecules by tagging individual copies of DNA molecules with barcodes or molecular indices (Figure 1).

Once encoded, the DNA sample can be amplified as desired without losing the ability to track and distinguish the original templates from clonal replicates. The method allows for absolute quantification and sequencing error correction, and greatly reduces the problem of amplification bias.

Based on Molecular Indexing technology, Precise Assays allow the examination of hundreds to thousands of genes in a high-throughput manner using less than 100 pg of total RNA. The assays provide absolute quantification of target transcripts or the whole transcriptome in an easy-to-follow workflow, and offer unprecedented accuracy and sensitivity for low-expression targets in rare and limited samples. By combining molecular and sample indexing in 96-sample and 384-sample formats, users can sequence up to 4,608 samples at once without new equipment or extensive training.


Figure 1. Molecular Indexing enables direct quantification of molecules by tagging individual copies of DNA molecules with barcodes, or molecular indices. Once encoded, the DNA sample can be amplified as desired without losing the ability to track and distinguish the original templates from clonal replicates.

Assay Design

Precise Assays utilize a pool of 6,000–65,000 unique molecular indexing barcodes to label all poly-A mRNAs in a sample prior to the RT step. This pre-RT labeling step allows individual RNA molecules to be tracked and counted independently of any bias introduced by PCR amplification.

The assays are designed for quantifying targets in purified total RNA, and can be optimized to analyze cell extracts, including single-cell extracts, without the need for nucleic acid purification, poly-A enrichment, or rRNA depletion. The poly-T tails of the molecular indexing primers are annealed to samples in a 96-well plate (or a 384-well plate) immediately prior to RT. These primers include a universal PCR sequence on the 5´ end to provide a template for subsequent PCR amplification steps as well as sample indexing barcodes. Additionally, plate barcodes can be added to further increase sample multiplexing and processing throughput.

This level of barcoding allows all samples to be combined into a single tube for subsequent steps, adding ease of handling and reduced reagent costs. After pooling samples into a single tube, the resulting single-stranded cDNAs undergo second-strand synthesis with a single amplification step using gene-specific primers. A global transcriptome amplification protocol is also available when gene targeting is not ideal. 

Resulting PCR products are size-selected and bead-purified with SPRI beads, and serve as the template for a second nested PCR step with a multiplex set of nested PCR primers suitable for Illumina® sequencers. In many cases, only a fraction of the original pooled cDNA is required for the specific amplification; the remaining cDNA is effectively archived. Since the stored sample contains all of the transcribed mRNA material, the Precise assay provides the opportunity for results validation or for testing additional genes or targets with no need to return to the original samples. After a final size selection with beads, the amplicons are ready for sequencing. Precise assays are designed for simple analysis with the cloud-based Seven Bridges Genomics analysis pipeline.

Advantages for Limited Samples and Rare Transcripts

While many methods have been described for RNA-Seq from limited sample quantities, they all require substantial pre-amplification steps prior to library construction that limit their sensitivity and reproducibility and further exacerbate amplification bias. Because of the count-based nature of RNA-Seq data, sufficient sequencing reads must be collected from each transcript to accurately measure its abundance in the library.

To accurately measure rare transcripts, many users resort to deeper sequencing to increase the number of reads in hope of finding and sequencing a rare molecule, which presents challenges to data analysis, reduces sample throughput, and quickly becomes cost prohibitive.

For applications involving rare transcripts, Precise Assays offer a more streamlined workflow with less chance of sample loss. The Molecular Indexing step further benefits applications involving rare transcripts by providing accurate quantification and drastically reducing PCR bias that could otherwise overshadow signal from rare transcripts. Most importantly, the assay offers a means of assessing when deeper sequencing is useful and when it becomes a matter of diminishing returns. 

Single-Cell Analysis

Single-cell mRNA sequencing is a powerful method for defining cell-to-cell variation, either within a cell population or among different cell populations. Scientists from BD Biosciences and Cellular Research used Molecular Indexing with BD’s fluorescence activated cell sorting (FACS) instruments to build a cost-effective workflow for high-throughput, single-cell mRNA sequencing.

To demonstrate, peripheral blood cells were stained with cell surface markers to identify rare regulatory T cell (Treg) subpopulations, including naïve, effector, and nonsuppressive regulatory T cells. Cells sampled at various time points were individually sorted into 96-well Precise Encoding plates using a BD FACSJazz cell sorter after sample pre-enrichment with BD IMag magnetic separation to increase sort speed and quality. Each cell was lysed and the mRNA content was barcoded with sample and Molecular Indexing during the reverse transcription step. Expression levels were measured for approximately 100 genes.

After sequencing on an Illumina MiSeq instrument, the molecular counts of each gene in each cell were used to build a single cell profile that unambiguously identified the cell types (Figure 2). The assay was sensitive enough to distinguish between cells of the same subtype sorted at four different time points (Figure 3). With this capability, the efficiency of identifying and isolating single cells greatly increases the throughput of cells available for transcriptome analysis, creating the potential to analyze gene expression targets in many thousands of individual cells.


Figure 2. Scientists from BD Biosciences and Cellular Research used Molecular Indexing technology with BD’s fluorescence-activated cell sorting (FACS) technology to identify rare regulatory T cell (Treg) subpopulations, including naïve, effector, and nonsuppressive regulatory T cells. With Molecular Indexing, molecular counts of each gene in each cell could be determined, and single-cell profiles that unambiguously identified the cell types could be assembled.

Conclusion

As technologies improve and scientists continue to gain a greater understanding of the underlying biology affecting cellular systems, more researchers are appreciating the value of higher precision biology. The Precise Assays, based on Cellular Research’s patented Molecular Indexing technology, provide a number of benefits for performing high-quality gene expression studies. These include simplicity in use, high-quality data, elimination of amplification biases, and absolute quantification of targets in limited samples.


Figure 3. Cells sampled at various time points were individually sorted. Each cell was lysed, and the mRNA content was barcoded with a unique sample and Molecular Index during the RT step. Expression levels were measured for approximately 100 genes. The assay was sensitive enough to distinguish between cells of the same subtype sorted at four different time points.

Glenn Fu, Ph.D. ([email protected]), is a founder and senior scientist at Cellular Research.

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