PCR Barcoding Protocol

After FACS sorting and overnight outgrowth, colony PCRs with well-specific barcoded primers encode the plate and well position on each amplicon. PCR barcoding This enables multiplexed amplicon sequencing to determine the sequence identity of each well.

Overview

The barcoding strategy involves:

1. Well-specific primers containing unique DNA barcodes
2. PCR amplification of the variable region from bacterial lysate
3. Pooling of barcoded amplicons across all plates
4. Sequencing of the pooled library
5. Computational demultiplexing to assign reads to wells

Barcode Design

We recommend ordering primers described in Long et al. 2024 which have been optimized for multiplexed, long-read amplicon sequencing. The adaptor region can be designed to target a common promoter-terminator pair to sequence the entire gene on a single amplicon.

Materials

Protocol

1. Prepare plates

1. Spin all plates at 4,000×g for 2 minutes.
2. Glycerol plates: aliquot 10 µL of filtered 50% glycerol per well.
3. PCR plates: prepare a large tub of ice and arrange PCR plates on ice.

2. Barcoding PCR setup (10 µL total reaction)

1. Prepare PCR master mix for plates

2. Gently invert master mix to mix, then aliquot 7 µL across PCR plates (repeat-dispense on Integra pipette).
3. Liquid handler workflow (minimize tips; do not change tips for each 384 or 96-well equivalent to prevent cross-contamination):
   1. Add 2 µL LevSeq barcodes to the PCR plate.
   2. Set tip volume to 10 µL; resuspend cultures in the sort plate by mixing.
   3. Add 10 µL cultures to glycerol plate and mix.
   4. Set tip volume to 1 µL; add 1 µL cultures to the PCR plate.
   5. Set tip volume to 7 µL; mix PCR reactions.

3. Thermocycler program (touchdown)

Note: Adjust extension time based on expected amplicon length. Highlighted cells (20 sec/kb) update automatically when a length is entered above.

4. Pooling + cleanup

1. Aliquot 30 µL of 100 mM EDTA into two 8×200 µL strip tubes per plate.
2. Pipette 5 µL from each well row-wise into the associated strip tube.
3. Pool 75 µL from strip tubes into a single 1.5 mL Eppendorf.
4. Repeat for each plate.
5. Perform bead cleanup with SPRIselect using a >300 bp cutoff (per Beckman Coulter protocol).

Sequencing Submission

Submit the pooled, purified amplicon library for sequencing. The total amount of sequencing data you need depends on how many wells you sorted, the length of your amplicon, and the read depth required to confidently call each well. Use the calculator below to estimate your sequencing need.

Plasmidsaurus (Recommended)

# Submit purified pool (500 ng - 2 µg in 15 µL)
# Turnaround: 3-5 business days
# Cost: ~$15 per sample (volume discounts available)

Oxford Nanopore (In-House)

# Use ligation sequencing kit (SQK-LSK109 or newer)
# Target: 200-500 ng input DNA
# Run time: 12-48 hours
# Cost: ~$500-900 per flow cell

Quality Control

Expected Metrics

• PCR success rate: 95–98% of wells with growth should amplify
• Pooled concentration: 50–200 ng/µL after purification
• Fragment size: Insert + ~40 bp (primers + barcodes)

Troubleshooting

Next Steps

After receiving sequencing data, use the uSort-M CLI to demultiplex and analyze your results. LevSeq barcodes are built into the pipeline — no separate barcode CSV is needed.

usortm demux project/ \
  --fastq sequencing_data.fastq \
  --library-csv variants.csv

See Demultiplexing for detailed instructions.