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| ONT Main Workflows | Details | Recommended inputs |
|---|---|---|
| Ligation | A flexible method of preparing sequencing libraries from dsDNA. DNA ends are repaired and dA-tailed before the sequencing adapters, supplied in the kit, are ligated onto the prepared ends. | 1ug (100-200fmol) gDNA in up to 47ul |
| Rapid | A kit optimised for simplicity and speed to sequence up to 24 libraries using a PCR-free method. The kit uses a transposase to simultaneously cleave template molecules and attach barcoded tags to the cleaved ends. | 100ng of gDNA in 10ul |
| Native Barcoding | gDNA is repaired and dA-tailed, and then a unique dT-tailed barcode adapter is ligated on the dA-tailed template. Barcoded samples are then pooled together. Each barcode adapter also has a cohesive end, and this is used as a hook to ligate to the supplied sequencing adapter. | * 400ng gDNA per sample if using 5 or more barcodes in 12ul *1ug gDNA per sample if using 4 or less barcodes in 12ul |
| Direct RNA | The kit is used to prepare and sequence native RNA without conversion to cDNA. Inputs include poly(A)-tailed RNA or total RNA, such as eukaryotic mRNA and viral RNA. | *300ng enriched RNA (Poly(A) or ribodepleted per sample * or 1ug total RNA per sample in 8ul |
| Ultra-Long | Based on transposase chemistry: the transposase simultaneously cleaves template molecules and attaches tags to the cleaved ends. Rapid sequencing adapters are then added to the tagged ends. The last step is an overnight elution of the DNA library. | 6 million cells in 40ul PBS or 750ul of uHMW gDNA |
| cDNA PCR | A strand switching method to select for full length transcripts, allowing the identification of splice variants, with the incorporation of unique molecular identifiers (UMI) during this step. Taking full-length poly(A)+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. Double-stranded cDNA is then generated by PCR amplification using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters. | *10ng enriched RNA (Poly(A) or ribodepleted per sample *or 500ng total RNA per sample in 10ul |