TITLE: One-pot native barcoding of amplicons v3 (LoCost)
AUTHORS: Josh Quick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This one-pot native barcoding protocol was developed in conjunction with Oxford Nanopore Technologies, New England Biolabs and BCCDC.</div></div>

[STEPS]
?. In a new PCR strip-tube/plate set up the following reaction for each sample:  AB1ComponentVolume2PCR dilution from previous step3.3 µL3Ultra II End Prep Reaction Buffer1.2 µL4Ultra II End Prep Enzyme Mix0.5 µL5Nuclease-free water5 µL6Total10 µL
AB1ComponentVolume2PCR dilution from previous step3.3 µL3Ultra II End Prep Reaction Buffer1.2 µL4Ultra II End Prep Enzyme Mix0.5 µL5Nuclease-free water5 µL6Total10 µL
Make a master mix of end-preparation reagents and nuclease-free water and aliquot into strip-tube/plate to improve reproducability.
?. Incubate at room temperature for  Incubate at  for Incubate on ice for
65 °C
?. In a new PCR strip-tube/plate set up the following reaction for each sample:  AB1ComponentVolume2End-preparation reaction mixture0.75 µL3NBXX barcode1.25 µL4Blunt/TA Ligase Master Mix5 µL5Nuclease-free water3 µL6Total10 µL
AB1ComponentVolume2End-preparation reaction mixture0.75 µL3NBXX barcode1.25 µL4Blunt/TA Ligase Master Mix5 µL5Nuclease-free water3 µL6Total10 µL
Use one native barcode from the EXP-NBD104 (1-12), EXP-NBD114 (13-24) or EXP-NBD196 per sample. Use 12 or more barcodes per library or there will be insufficient total material to achieve good yields.
?. Incubate at room temperature for Incubate at  for Incubate on ice for
65 °C
The 65°C incubation is to inactivate the DNA ligase to prevent barcode cross-ligation when reactions are pooled in the next step.
?. In a new Eppendorf tube pool all one-pot barcoding reactions together.
1.5 mL
If processing 12-24 samples pool all   from each native barcoding reaction. if processing 48 samples pool   from each native barcoding reaction. If processing 96 samples pool   from each native barcoding reaction so as not to exceed a pool volume of   which would make the clean-up volume too large.
?. Add 0.4x volume of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add  SPRI beads to  pooled one-pot barcoding reactions.
96 µl
240 µl
0.4x volume of SPRI is sufficient to bind 400 bp amplicons in the presence of ligation buffer, do not use 1x as this will result in an excessive large bead pellet.
?. Mix by vortexing and pulse centrifuge to collect all liquid at the bottom of the tube. Incubate for  at room temperature.
?. Place on magnetic rack and incubate for  or until the beads have pelleted and the supernatant is completely clear. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
?. Add  SFB and resuspend beads completely by pipette mixing. Pulse centrifuge to collect all liquid at the bottom of the tube and place on the magnet. Remove supernatant and discard.
250 µl
SFB will remove excess adapter without damaging the adapter-protein complexes. Do not use 70% ethanol as in early clean-ups.
?. Repeat steps 11.9 to perform a second SFB wash. Pulse centrifuge and remove any residual SFB.
You do not need to allow to air dry with SFB washes.
?. Add  of room-temperature ethanol to bathe the pellet. Carefully remove and discard ethanol, being careful not to touch the bead pellet.
200 µl
Only perform 1x 70% ethanol wash
?. Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
?. With the tube lid open incubate for  or until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).
?. Resuspend pellet in  Tris pH 8.0, mix gently by either flicking or pipetting and incubate for .
30 µl
?. Place on magnet and transfer sample to a clean  Eppendorf tube ensuring no beads are transferred into this tube.
1.5 mL