SOP: Equimolar Pooling of Barcoded ONT PCR-cDNA Libraries

Applies to: Individually barcoded PCR-cDNA libraries prepared with Oxford Nanopore barcoding kits (e.g. SQK-PCB111.24, SQK-PCB114.24) Kit context: This is the pooling step between individual library barcoding and adapter ligation/flow cell loading Location: __________

Document Control

• SOP ID: __________
• Version: 1.0
• Effective Date: __________
• Review Due: __________
• Author: __________
• Approved By: __________

Purpose

To combine individually barcoded PCR-cDNA libraries into a single equimolar pool, ensuring equal representation of each barcode during sequencing. Equimolar pooling prevents over- or under-representation of individual samples caused by differences in library concentration.

Scope

All personnel performing library pooling prior to Oxford Nanopore sequencing.

Principle

Each barcoded library is quantified, its mass concentration (ng/µL) is converted to molar concentration (fmol/µL) using the estimated average fragment size, and volumes are calculated to normalise all libraries to the same molarity. Equal volumes of normalised libraries are then combined into a single pool at the target loading amount.

Materials and Equipment

• Quantified barcoded cDNA libraries (Qubit dsDNA HS or BR assay, or NanoDrop)
• Fragment size estimate from Fragment Analyzer, TapeStation, or Bioanalyzer
• Nuclease-free water
• Low-bind 1.5 mL tubes
• Pipettes (0.5–10 µL range) with filter tips
• Vortex mixer
• Microcentrifuge
• Ice or cold block
• Pooling calculator spreadsheet (see below)

Preparations

1. Determine Target Loading

ONT recommended loading for PCR-cDNA libraries:

2. Determine Average Fragment Size

Use the Fragment Analyzer (or equivalent) main peak size for the library population. If no electrophoresis data is available, use the expected size based on the cDNA protocol:

Procedure

1. Quantify Each Library

1. Measure the concentration of each barcoded library in ng/µL using Qubit dsDNA HS assay (preferred) or NanoDrop A260.
2. Record concentrations for all libraries.

2. Convert to Molar Concentration

Convert ng/µL to fmol/µL using:

fmol/µL = (ng/µL × 10⁶) / (average_fragment_size_bp × 649)

Where 649 Da is the average molecular weight per base pair of dsDNA.

Quick reference (1.5 kb average fragment):

3. Calculate Pooling Volumes

1. Choose target pool molarity:

   target_fmol_per_µL = target_total_fmol / pool_volume
   

   Example: 25 fmol in 11 µL = 2.27 fmol/µL

2. For each library, calculate the volume needed:

   vol_library (µL) = target_fmol_per_µL / library_fmol_per_µL
   

3. Calculate water to bring to a convenient unit volume:

   vol_water (µL) = unit_volume − vol_library
   

4. Scale up if calculated volumes are below 1 µL (difficult to pipette accurately). A 4× scale-up is recommended:

   vol_library_scaled = vol_library × 4
   vol_water_scaled = vol_water × 4
   total_dilution_volume = 4 µL per library
   

5. Calculate the pool volume per library:

   pool_vol_per_library = total_pool_volume / number_of_libraries
   

   Example: 11 µL / 6 libraries = 1.83 µL per library

4. Prepare Normalised Dilutions

1. For each library, combine the calculated volumes of library and nuclease-free water in a fresh low-bind tube.
2. Vortex briefly and spin down.
3. Keep on ice.

5. Pool Libraries

1. Into a fresh low-bind 1.5 mL tube, pipette the calculated pool volume from each normalised dilution.
2. Vortex briefly and spin down.
3. The final pool should be 11 µL (or target volume) at the target molarity.
4. Keep on ice. Proceed to adapter ligation or flow cell loading within 1 hour.

6. Verify Pool (Optional)

1. Measure pool concentration by Qubit.
2. Expected: approximately the target fmol/µL when back-calculated using the same fragment size.
3. Run on Fragment Analyzer to confirm size distribution if sufficient volume remains.

Example Calculation

6 barcoded mouse PCR-cDNA libraries, 1.5 kb average fragment, target 25 fmol in 11 µL:

Pool total: 6 × 1.83 µL = 11 µL containing ~25 fmol

Troubleshooting

Records to Maintain

• Library concentrations (ng/µL) and instrument used
• Fragment size estimate and source (FA, TapeStation, assumption)
• Pooling spreadsheet with all calculated volumes
• Target fmol and actual pool volume
• Date, operator, and any deviations

End of SOP