Sequencing to saturation is simply not feasible, Bock said, and his lab recommends aiming for at least 10,000 reads per cell. It also uses a lower level of sequencing due to the scale of experiments. Thus, it uses the 10x ATAC-seq (assay for transposase-accessible chromatin by sequencing) kit for library prep, rather than the 3' gene expression kit, which also adds a specific primer binding site. The method fixes cells to permeabilize the nuclei and reverse transcribes to cDNA prior to encapsulation. "If we were loading more, we could push that more," he said. That led to about five to 10 nuclei per droplet, with as many as 15. "That's mainly because we didn't need more," Bock said. They settled on running experiments at around 750,000 nuclei per channel, about 50 times what's recommended. His team tried to create a hybrid approach and came up with the combination of one round of indexing on a 384-well plate paired with overloading. "You lose a lot of material and the results we were getting from those processes were not that great," he said. "It turned out to be a hassle," he said, and tedious, since it required three rounds of barcoding to provide a benefit over Chromium's base throughput. Researchers led by Jay Shendure and Cole Trapnell of the University of Washington developed sci-Plex, a plate-based method for high-throughput screening that also uses nuclear hashing, publishing it in Science in 2019.īock said his team was spurred to create scifi-RNA-seq after some frustration with multi-round plate-based combinatorial indexing methods. Other groups have also come up with different combinatorial indexing approaches to improve throughput, though not necessarily on droplet-based single-cell platforms. Another cell-hashing method, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), has enabled multiplex readouts from single-cell experiments. 10x is working on its own ways to increase throughput, including the forthcoming Chromium X instrument, which it says will be able to process 1 million cells at a time, and its recently released CellPlex kits, which uses oligo-conjugated lipids to label cells upstream from droplet encapsulation.īut CellPlex is a cell-hashing strategy that flags and discards droplets containing more than one cell, Bock said, providing about a twofold increase. The method offers another way to increase throughput in single-cell sequencing, which could help lower sample preparation costs, a major consideration with these studies. It is an academic method that still requires more investigation and work to commercialize it." "Conceptually, the approach is interesting, but we have yet to see how it will have broader adoption, as it is still in the academic realm. "Nothing is a bad idea in life sciences if you get closer to mastering biology," a 10x Genomics spokesperson said in an email. But Bock's team found that not only can indexing on 384-well plates provide the mathematical feasibility to run more cells, but also that Chromium can physically handle up to 1.5 million nuclei per channel without clogging. With eight channels on its cartridges, that yields about 80,000 transcriptomes per run. Normally, getting more than one cell in a 10x droplet - a doublet - leads to unusable results, so the firm recommends loading up to about 16,000 cells or nuclei per microfluidic channel to capture up to 10,000 cells or nuclei. He's also senior author of a paper published this week in Nature Methods describing and benchmarking the method. "With 10x's standard protocol, most of the emulsion droplets remain empty, and the reagents are used very inefficiently," said Christoph Bock, a researcher at CeMM whose lab came up with the method. That indexing allows massive overloading beyond 10x's recommendations, filling droplets with as many as 15 nuclei, the transcriptomes of which can be picked apart in downstream analyses. Single-cell combinatorial fluidic indexing RNA sequencing (scifi-RNA-seq) employs a single round of barcoding prior to droplet encapsulation, barcoding the transcriptome molecules inside each cell or nucleus prior to "isolation," either on Chromium or another platform. NEW YORK – Researchers from the Austrian Academy of Sciences' Research Center for Molecular Medicine (CeMM) have devised a new single-cell sample preparation method that raises throughput as much as fifteenfold on droplet-based platforms, such as 10x Genomics' Chromium. Advances in Clinical Genomics Profiling.
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