16th June 2021
- Required settings in MinKNOW in the 'Sequencing and data analysis' section has been updated include instructions to toggle 'Override mid barcoding score' and to increase minimum mid barcoding score from 40 to 50.
16th June 2021
- Required settings in MinKNOW in the 'Sequencing and data analysis' section has been updated include instructions to toggle 'Override mid barcoding score' and to increase minimum mid barcoding score from 40 to 50.
29th April
- Primer pool concentration in the change note table of 'Overview of the protocol' updated to the correct 100 µM for the Eco protocol overview.
29th April
- Primer pool concentration in the change note table of 'Overview of the protocol' updated to the correct 100 µM for the Eco protocol overview.
28th April 2021
- Added screenshots of MinKNOW for barcoding recommendations in 'Data acquisition and basecalling' section
- Updated 'PCR and clean-up' section to include step to dilute primer stocks to 10 µM
28th April 2021
- Added screenshots of MinKNOW for barcoding recommendations in 'Data acquisition and basecalling' section
- Updated 'PCR and clean-up' section to include step to dilute primer stocks to 10 µM
21st April 2021
Inclusion of the optional Adapter Mix II Expansion (EXP-AMII001) kit for instances when a user is running a subset of barcodes in one experiment and needs extra Adapter Mix II for more reactions:
- EXP-AMII001 is included in the "Equipment and consumables" section
- Explanation of when to use EXP-AMII001 in the "Adapter ligation and clean-up" step
21st April 2021
Inclusion of the optional Adapter Mix II Expansion (EXP-AMII001) kit for instances when a user is running a subset of barcodes in one experiment and needs extra Adapter Mix II for more reactions:
- EXP-AMII001 is included in the "Equipment and consumables" section
- Explanation of when to use EXP-AMII001 in the "Adapter ligation and clean-up" step
25th March 2021
- Updated 'Overview of the protocol' to include changelog to clearly show the differences between our PCR tiling of SARS-CoV-2 protocols.
- Updated 'Equipment and consumables' to include Native Barcoding Expansion 96 (EXP-NBD196) and their barcodes.
- Updated 'PCR and clean-up' to include volume per sample and corrected volume totals.
- Updated 'End-prep' to include volume per sample for the end-prep master mix.
25th March 2021
- Updated 'Overview of the protocol' to include changelog to clearly show the differences between our PCR tiling of SARS-CoV-2 protocols.
- Updated 'Equipment and consumables' to include Native Barcoding Expansion 96 (EXP-NBD196) and their barcodes.
- Updated 'PCR and clean-up' to include volume per sample and corrected volume totals.
- Updated 'End-prep' to include volume per sample for the end-prep master mix.
17th March 2021
- Updated title to 'Classic PCR tiling of SARS-CoV-2 virus'
- URL updated from 'pcr-tiling-SARS-CoV-2' to 'classic-pcr-SARS-CoV-2'
- Added a table to the 'Overview of the protocol' to highlight the differences between our PCR tiling protocols.
- Updated 'Library preparation' to outline preparation volumes of 24, 48 and 96 samples in a plate format.
- "Downstream analysis and expected results" step: the recommended analysis pipeline and workflow packaging information has been updated. We have removed the recommendation to use RAMPART and have included the EPI2ME Labs Jupyter notebook tutorial and EPI2ME workflow
17th March 2021
- Updated title to 'Classic PCR tiling of SARS-CoV-2 virus'
- URL updated from 'pcr-tiling-SARS-CoV-2' to 'classic-pcr-SARS-CoV-2'
- Added a table to the 'Overview of the protocol' to highlight the differences between our PCR tiling protocols.
- Updated 'Library preparation' to outline preparation volumes of 24, 48 and 96 samples in a plate format.
- "Downstream analysis and expected results" step: the recommended analysis pipeline and workflow packaging information has been updated. We have removed the recommendation to use RAMPART and have included the EPI2ME Labs Jupyter notebook tutorial and EPI2ME workflow
2nd Sept 2020
The Reverse Transcription step uses LunaScript RT SuperMix Kit to simplify and reduce the workflow time.
The Native Barcode Ligation step uses NEB Blunt/TA Ligase Master Mix.
The protocol has been re-written for processing multiple samples in parallel.
2nd Sept 2020
The Reverse Transcription step uses LunaScript RT SuperMix Kit to simplify and reduce the workflow time.
The Native Barcode Ligation step uses NEB Blunt/TA Ligase Master Mix.
The protocol has been re-written for processing multiple samples in parallel.
26th March 2020
- Link to V3 of the ARTIC primer sequences
- Launch of the SFB Expansion kit (EXP-SBF001) to support the 'one-pot' library preparation which requires more Short Fragment Buffer (SFB)
26th March 2020
- Link to V3 of the ARTIC primer sequences
- Launch of the SFB Expansion kit (EXP-SBF001) to support the 'one-pot' library preparation which requires more Short Fragment Buffer (SFB)
12th March 2020
Update to the library preparation instructions:
- Primer elution volume after the PCR step is reduced from 30 μl to 15 μl
- End-prep total reaction volume is reduced to 15 μl, and the AMPure bead clean-up after end-prep is no longer necessary
- Native barcode ligation requires the NEBNext Ultra II Ligation Module instead of the Blunt/tA Ligation Master Mix
- After native barcode ligation, all samples are pooled and cleaned up with AMPure beads
- Adapter ligation volume is reduced from 100 μl to 50 μl
12th March 2020
Update to the library preparation instructions:
- Primer elution volume after the PCR step is reduced from 30 μl to 15 μl
- End-prep total reaction volume is reduced to 15 μl, and the AMPure bead clean-up after end-prep is no longer necessary
- Native barcode ligation requires the NEBNext Ultra II Ligation Module instead of the Blunt/tA Ligation Master Mix
- After native barcode ligation, all samples are pooled and cleaned up with AMPure beads
- Adapter ligation volume is reduced from 100 μl to 50 μl
12th March 2020
Update to the library preparation instructions:
- DNA elution volume after the PCR step is reduced from 30 μl to 15 μl
- End-prep total reaction volume is reduced to 15 μl, and the AMPure bead clean-up after end-prep is no longer necessary
- Native barcode ligation requires the NEBNext Ultra II Ligation Module instead of the Blunt/tA Ligation Master Mix
- After native barcode ligation, all samples are pooled and cleaned up with AMPure beads
- Adapter ligation volume is reduced from 100 μl to 50 μl
12th March 2020
Update to the library preparation instructions:
- DNA elution volume after the PCR step is reduced from 30 μl to 15 μl
- End-prep total reaction volume is reduced to 15 μl, and the AMPure bead clean-up after end-prep is no longer necessary
- Native barcode ligation requires the NEBNext Ultra II Ligation Module instead of the Blunt/tA Ligation Master Mix
- After native barcode ligation, all samples are pooled and cleaned up with AMPure beads
- Adapter ligation volume is reduced from 100 μl to 50 μl
21st February 2020
- Update to the __Downstream analysis and expected results__ section of the protocol: inclusion of the Medaka software for single-nucleotide variant calling
- In line with World Health Organization recommendations, the nCoV-2019 virus is named COVID-19
- Added 'Library storage recommendations' snippet before flow cell loading step