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.

Materials

Item Supplier Catalog #
LevSeq F+R barcodes (1 µM) IDT Custom order
Q5 Polymerase NEB M0491L
Q5 5X Reaction Buffer NEB B9027S
10 mM dNTPs NEB N0447L
DMSO Sigma D8418
BioRad Hardshell 384-well PCR plates Bio-Rad HSP3801
Foil plate seals Thermo AB0626
Filtered 50% glycerol — —
100 mM EDTA — —
Beckman Coulter SPRIselect beads Beckman Coulter B23317

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.

Protocol

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.

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.
Plates to prepare:
384 wells × 1 plate + 10% excess = 423 rxns
Component Rxn (µL) Total (µL) 423 rxns Added
Q5 Polymerase0.1
Q5 5X Buffer2.0
10 mM dNTPs0.2
DMSO0.4
H₂O4.3
Total 7.0

Thermocycler program (touchdown)

Note: Adjust extension time based on expected amplicon length. Highlighted cells (30 sec/kb) update automatically when a length is entered above.

Note: Touchdown step decreases from 65°C to 55°C over 10 cycles (ΔT = −1°C/cycle). PCR takes ~1 hour. During PCR, measure ODs of sort plates and store glycerol stocks at −80°C.
Enter length to calculate extension times
Cycles Temperature (°C) Time
1 98 2 min
10 98 10 sec
65 → 55* 30 sec
72 30 sec/kb
25 98 10 sec
66 30 sec
72 30 sec/kb
1 72 3 min
4 hold

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 Need Calculator

Wells sorted × amplicon length × read depth per well.

Total sequencing need —

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 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

Troubleshooting

Problem Possible Cause Solution
No amplification Insufficient template, primer issue Increase template to 1 µL, check primer concentration
Multiple bands Non-specific amplification Optimize annealing temperature, redesign primers
Low yield after pooling Many wells failed PCR Check cell viability before PCR, optimize cycling
Unequal representation Variable amplification efficiency Normalize input or use fewer PCR cycles

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)

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.