DR-Seq studies the genomic and transcriptomic relationship in single cells (Dey et al., 2015). Nucleic acid amplification prior to physical separation reduces sample loss and the risk of contamination. DR-Seq involves multiple amplification steps, including a quasilinear amplification technique similar to MALBAC.
First, mRNA from lysed single-cells is reverse-transcribed using poly(dT) primers with Ad-1x adapters, producing single-stranded cDNA. The Ad-1x adapter sequence contains cell-identifying barcodes, 5ê Illumina adaptors, and a T7 promoter. Next, both gDNA and sscDNA are simultaneously amplified via quasilinear WGA with Ad-2 primers. These primers are similar to MALBAC adapters, containing an 8 nt random sequence for random priming followed by a constant 27 nt tag at the 5ê end. The products of this amplification step are split into 2 batches. One half is prepared for genome sequencing, in which gDNA is PCR-amplified and liberated from the Ad-2 adapters before DNA library preparation and sequencing. The other half is used for transcriptome sequencing, where double-stranded cDNA is synthesized and RNA is amplified by in vitro transcription. The resulting amplified RNA is produced only from cDNA fragments flanked with Ad-1x and Ad-2, suppressing amplification of the gDNA fragments. The RNA library is prepared for sequencing following the Illumina small-RNA protocol. Sequencing gDNA and mRNA from the same cell preserves information between the genome and its expression levels.
- Interrogates genomic and transcriptomic behavior from a single cell
- Amplification prior to separation reduces sample loss and contamination
- Length-based identifier used to remove duplicate reads
- Quasilinear amplification reduces PCR bias
- Manual single-cell isolation prevents high-throughput adaptation
- Quasilinear amplification is temperature-sensitive
- RNA reads are 3ê-end-biased (Macaulay et al., 2015)
Illumina Library prep and Array Kit Selector
Zhang X., Marjani S. L., Hu Z., Weissman S. M., Pan X., et al. Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects. Cancer Res. 2016;76:1305-1312
Saadatpour A., Lai S., Guo G. and Yuan G. C. Single-Cell Analysis in Cancer Genomics. Trends Genet. 2015;31:576-586
Dey S. S., Kester L., Spanjaard B., Bienko M. and van Oudenaarden A. Integrated genome and transcriptome sequencing of the same cell. Nat Biotechnol. 2015;33:285-289