DR-Seq

DR-Seq

gDNA-mRNA sequencing (DR-Seq) studies the genomic and transcriptomic relationship of single-cells via sequencing. Nucleic acid amplification prior to physical separation reduces sample loss and risk of contamination. DR-Seq involves multiple amplification steps, including a quasilinear amplification technique similar to MALBAC.

First, mRNA are reverse-transcribed from lysed single-cells using poly-dT primers with Ad-1x adaptors, producing single-stranded cDNA (sscDNA). Ad-1x adaptor sequence contain cell-identifying barcodes, 5' Illumina adaptors, and a T7 promoter. Then, both gDNA and sscDNA are simultaneously amplified via quasilinear whole-genome amplification with Ad-2 primers. These primers are similar to MALBAC adaptors, containing 8 random nucleotide sequence for random priming followed by a constant 27 nucleotide tag at the 5' end. Products of this amplification step are split in 2. One half is prepared for genome sequencing, in which gDNA are PCR amplified and liberated of their Ad-2 adaptors before DNA library prep and sequencing. The other half is used for transcriptome sequencing, where cDNAs are synthesized and amplified by in-vitro transcription. Resulting RNA products are produced only from cDNA fragments flanked with Ad-1x and Ad-2, suppressing amplification of the gDNA fragments. 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.

Pros:

• 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

Cons:

• Manual single-cell isolation prevents high-throughput adaptation
• Quasilinear amplification is temperature-sensitive
• RNA reads are 3' end biased¹

• DR-Seq: Dey S. S., Kester L., Spanjaard B., Bienko M. and van Oudenaarden A. (2015) Integrated genome and transcriptome sequencing of the same cell. Nat Biotechnol
• 1. Macaulay I. C., Haerty W., Kumar P., Li Y. I., Hu T. X., et al. (2015) G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nat Methods

1. Dey S. S., Kester L., Spanjaard B., Bienko M. and van Oudenaarden A. (2015) Integrated genome and transcriptome sequencing of the same cell. Nat Biotechnol