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--- submission:ont_submissions_lps [2024/11/25 15:59] – [Adjacent Workflows] Johanna Barbieri
+++ submission:ont_submissions_lps [2025/02/11 15:41] (current) – [Adjacent Workflows] Johanna Barbieri
@@ Line 4: / Line 4: @@
-^ ONT Main Workflows ^Details ^Recommended inputs^
+^ 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 in up to 47ul|
+|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 of gDNA in 10ul|
+| 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 gDNA per sample if using 5 or more barcodes in 12ul \\ *1ug gDNA per sample if using 4 or less barcodes in 12ul|
+|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 in 8ul|
+|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 in 40ul PBS or 750ul of uHMW gDNA|
+|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 in 10ul|\\
+|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|\\
@@ Line 22: / Line 22: @@
-^ ONT Adjacent Workflows ^Details ^Recommended inputs^
+^ ONT Adjacent Workflows ^Details ^Recommended inputs ^Nucleic acid ^Volume^
-|Ligation Amplicons | Ligation sequencing of amplicons | 100-200 fmol of amplicon DNA in up to 49ul|
+|Ligation Amplicons | Ligation sequencing of amplicons | 100-200 fmol | amplicon DNA | 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 Low-input by PCR| Ligation sequencing of low input gDNA using PCR adaptors| 100-200 fmol | gDNA | 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 Direct cDNA| Sequencing of  cDNA samples using RT and strand-switching | 100 ng Poly(A)+ RNA\\ OR\\ 1 µg of total RNA |RNA| 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 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 | 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 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 | 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|
+|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 |PCR product| 45ul|
-|Multiplex Ligation Sequencing| PCR-free multiplexing of dsDNA samples such as gDNA and amplicons| 1ug gDNA per sample in 12ul|
+|Multiplex Ligation Sequencing| PCR-free multiplexing of dsDNA samples such as gDNA and amplicons| 1ug per sample |gDNA  | 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|
+|Native Barcoding Amplicons| Native barcoding of amplicons|200 fmol (130 ng for 1 kb amplicons) per sample to be barcoded |DNA | 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| Rapid Barcoding of gDNA, quick protocol that allows multiplexing of samples on the same flow cell| 200ng per sample |gDNA  | 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 Plasmids| Rapid Barcoding of plasmids, quick protocol that allows multiplexing of plasmids on the same flow cell| 50ng per sample |HMW plasmid DNA  | 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|
+|Rapid Barcoding Amplicons| Rapid Barcoding of amplicons, quick protocol that allows multiplexing of amplicons on the same flow cell|50ng per sample (500bp - 5kb amplicon size) |amplicon DNA  | 9ul|
-|Direct RNA Sequence Specific|Kit allows sequence-specific sequencing of native RNA|1ug total RNA in 8.5ul|
+|Direct RNA Sequence Specific|Kit allows sequence-specific sequencing of native RNA|1ug |total RNA | 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|
+|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 | RNA| 10ul|
+==== Complete the Pool Information Fields ====
+  * Please submit your samples in 1.5mL tubes, clearly and concisely named.
+  * Complete the submission form:
+     * Complete __Billing Information__. This is essential for correct invoicing: please follow the popup instructions in the field. Only enter a PO Number if required by your department or organisation. __Leave PO Number blank if it does not apply__ (resist the temptation to put "N/A" or "none": it will complicate your department's invoice).
+     * The __RIN__ field is only relevant for RNA submissions.
+     * Leave unused sample rows empty.
+  * Run your completed submission form through this submission application. Be sure to click the "Confirm Submission" button to finish the submission process.
+  * Hand your sample to a member of Genomics.