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Table of Contents
Oxford Nanopore Library Prep Submissions
Main Workflows
| ONT Main Workflows | Details | Recommended inputs | Nucleic acid | Volume |
|---|---|---|---|---|
| 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 | 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 | gDNA | 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 per sample if using 5 or more barcodes *1ug per sample if using 4 or less barcodes | gDNA | 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 | RNA | 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 or uHMW gDNA | Cells or gDNA | * 40uL if cell * 750uL if 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 | RNA | 10ul |
Adjacent Workflows
| ONT Adjacent Workflows | Details | Recommended inputs |
|---|---|---|
| Ligation Amplicons | Ligation sequencing of amplicons | 100-200 fmol of amplicon DNA in up to 49ul |
| Ligation Low-input by PCR | Ligation sequencing of low input gDNA using PCR adaptors | 100-200 fmol of gDNA in up to 48ul |
| Ligation Direct cDNA | Sequencing of cDNA samples using RT and strand-switching | 100 ng Poly(A)+ RNA OR 1 µg of total RNA in 7.5ul |
| Ligation gDNA Whole Genome Amp | Whole genome amplification (WGA) of genomic DNA using the Ligation Sequencing Kit (SQK-LSK114) and the QIAGEN REPLI-g Midi kit. | 50 pg high molecular weight genomic DNA in up to 4ul |
| Ligation 10X Genomics Single-cell Transcriptomics | Sequencing of full-length transcripts generated by 10X Genomics 3'/5'/Visium FF PolyA capture using custom oligos to enrich. | 10ng cDNA in up to 21ul |
| Ligation PCR Barcoding | PCR Barcoding of gDNA/ PCR product allowing for multiplexing on same flow cell | 100-200fmol of each sample or each first round PCR product with tailed primers in 45ul |
| Multiplex Ligation Sequencing | PCR-free multiplexing of dsDNA samples such as gDNA and amplicons | 1ug gDNA per sample in 12ul |
| Native Barcoding Amplicons | Native barcoding of amplicons | 200 fmol (130 ng for 1 kb amplicons) DNA per sample to be barcoded in 11.5ul |
| Rapid Barcoding | Rapid Barcoding of gDNA, quick protocol that allows multiplexing of samples on the same flow cell | 200ng gDNA per sample in up to 10ul |
| Rapid Barcoding Plasmids | Rapid Barcoding of plasmids, quick protocol that allows multiplexing of plasmids on the same flow cell | 50ng HMW plasmid DNA per sample in 9ul |
| Rapid Barcoding Amplicons | Rapid Barcoding of amplicons, quick protocol that allows multiplexing of amplicons on the same flow cell | 50ng amplicon DNA per sample (500bp - 5kb amplicon size) in 9ul |
| Direct RNA Sequence Specific | Kit allows sequence-specific sequencing of native RNA | 1ug total RNA in 8.5ul |
| cDNA-PCR Barcoding | Sequencing of multiple cDNA samples using strand-switching and barcoding for multiplex on the same flow cell | 10ng enriched RNA (Poly(A) or ribodepleted per sample or 500ng total RNA per sample in 10ul |