RT Journal
A1 Tilgner, Hagen
A1 Jahanbani, Fereshteh
A1 Gupta, Ishaan
A1 Collier, Paul
A1 Wei, Eric
A1 Rasmussen, Morten
A1 Snyder, Michael
T1 Microfluidic isoform sequencing shows widespread splicing coordination in the human transcriptome
JF Genome Research 
JO Genome Research 
YR 2018 
FD February 01 
VO 28 
IS 2 
SP 231 
OP 242 
DO 10.1101/gr.230516.117 
UL http://genome.cshlp.org/content/28/2/231.abstract 
AB Understanding transcriptome complexity is crucial for understanding human biology and disease. Technologies such as Synthetic long-read RNA sequencing (SLR-RNA-seq) delivered 5 million isoforms and allowed assessing splicing coordination. Pacific Biosciences and Oxford Nanopore increase throughput also but require high input amounts or amplification. Our new droplet-based method, sparse isoform sequencing (spISO-seq), sequences 100k–200k partitions of 10–200 molecules at a time, enabling analysis of 10–100 million RNA molecules. SpISO-seq requires less than 1 ng of input cDNA, limiting or removing the need for prior amplification with its associated biases. Adjusting the number of reads devoted to each molecule reduces sequencing lanes and cost, with little loss in detection power. The increased number of molecules expands our understanding of isoform complexity. In addition to confirming our previously published cases of splicing coordination (e.g., BIN1), the greater depth reveals many new cases, such as MAPT. Coordination of internal exons is found to be extensive among protein coding genes: 23.5%–59.3% (95% confidence interval) of highly expressed genes with distant alternative exons exhibit coordination, showcasing the need for long-read transcriptomics. However, coordination is less frequent for noncoding sequences, suggesting a larger role of splicing coordination in shaping proteins. Groups of genes with coordination are involved in protein–protein interactions with each other, raising the possibility that coordination facilitates complex formation and/or function. We also find new splicing coordination types, involving initial and terminal exons. Our results provide a more comprehensive understanding of the human transcriptome and a general, cost-effective method to analyze it.