Tonight, I went into the lab to wash flow cells and try to reload the cDNA sequencing run. I may have caused more damaged… so I came back and watched the session “Individual cells matter – single-cell answers through nanopore sequencing.” Rachel Rubinstein, a Technical Product Manager at ONT, facilitated this showcase session. Rubinstein spoke about how long reads can produce novel insights. There are 3′ and 5′ protocols compatible with 10X Genomics products for single-cell sequencing with Oxford Nanopore Technologies. Carly Tyer, an Applications Scientist with ONT, described sample library preparation. They noted that it takes about 3 hours from 10X cDNA to nanopore-ready libraries! Ten nanograms of 10X Genomics cDNA and the Ligation Sequencing Kit are needed. One sample is in one tube, and you need one PromethION library. Tyer’s example was 24 PromethION flow cells producing ~1.9 B cell-barcoded reads. The 10X Chromium system is provided with cells or nuclei. Single cells are captured in droplets and labeled with a unique barcode. Droplets are lysed, and cDNAs are amplified from all cells for sequencing. Cell barcodes in each read identify cDNAs from the same cell. Therefore, at the end of this process, all of the transcripts from one cell have a unique UMI. There are two preparations depending on the 10X protocol used: 3′ cDNA and 5′ cDNA> The 3′ protocol uses biotin primers. Tyer highlighted the 3′ protocol: there is a biotin-strep pulldown followed by SPRI clean-up, end prep of 200 fmols (and cleanup) and then ligation of sequencing adapters. There is a PCR step with PRM primers after the biotin-strep pulldown. After the ligation of sequencing adapters, 50-100 fmol are loaded. Tyer noted that this value is calculated based on the expected read length. In their example, 900 bp of read length would result in loading of 65 ng of the library. For the 5′ protocol, there isn’t a pulldown. The protocol takes about 2 hours from cDNA to sequencing-ready library. The protocol starts with an eight-cycle PCR, end prep, ligation of sequencing adapters, and loading of 50-100 fmols. You can either base call live with Dorado or after the run. Tyer compared the 3′ and 5′ protocols. They shared a biological example with B cells from an MMR challenge. Scaling can be done by starting with the P2 and, if needed, loading a PromethION 24!
