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.

了解转录组的复杂性对于了解人类生物学和疾病至关重要。合成长读RNA测序（SLR-RNA-seq）等技术可提供500万个同工型，并可以评估剪接的协调性。太平洋生物科学公司和牛津纳米孔公司也提高了通量，但需要大量的投入或放大。我们基于液滴的新方法，稀疏同工型测序（spISO-seq），一次可对10-200个分子的100k-200k分区进行测序，可分析10-100,000,000个RNA分子。SpISO-seq需要少于1 ng的输入cDNA，从而限制或消除了对其相关偏倚进行事先扩增的需要。调整每个分子的读段数可以减少测序通道和成本，而检测能力几乎没有损失。分子数量的增加扩展了我们对同工型复杂性的理解。除了确认我们先前发布的拼接协调案例（例如，BIN1），更大的深度揭示了许多新情况，例如MAPT。内部外显子的协调在蛋白质编码基因中广泛存在：具有远距离替代外显子的高表达基因的23.5％–59.3％（95％置信区间）表现出协调性，这表明需要长期阅读转录组学。但是，非编码序列的协调频率较低，这表明剪接协调在蛋白质形成中的作用更大。具有协调作用的基因组参与蛋白质彼此之间的相互作用，从而增加了协调促进复杂的形成和/或功能的可能性。我们还发现了新的剪接协调类型，涉及初始和末端外显子。我们的结果提供了对人类转录组的更全面的了解，以及一种通用的，经济有效的方法来对其进行分析。