TITLE: Barcode Composition by Overlap-Extension PCR
AUTHORS: Mathew Chu

[DESCRIPTION]
Traditionally, DNA barcodes are synthesised as random oligonucleotides. However, this leads to uncertainty regarding the ground truth of barcode sequences in the experimental setting. Without reference sequences, it is impossible to determine with absolute confidence whether or not the observed diversity of barcodes is due to errors arising from sequencing itself or various cellular and molecular processes. 

Here, we propose a modular way to assemble high-diversity libraries of DNA barcodes from units whose individual sequences are known a priori. Barcode units are amplified and then combinatorically spliced in vitro using overlap-extension PCR, yielding products that can be assembled into vectors for downstream applications.



[STEPS]
SECTION: Single Stranded DNA Pools for Combinatorial Assembly
1. ssDNA oligos for combinatorial assembly can be ordered as a pool (oPool). For a final barcode of n units, each with m diversity, order a set of m different barcode sequences for each unit:
 
where unit i (1 ≤ i ≤ n) consists of m barcodes flanked by left (L) and right (R) homology sequences.

Homology sequences 1 to n should be 20 nt long and serve as both primer binding sites as well as junctions for the linear assembly of the barcode units. A set of orthogonal sequences for this purpose has been described and experimentally validated by Subramanian, Russ & Ranganathan (2018).

oPools should be divided into sets of 3-4 units to enable assembly of 3-4 oligo sets (barcode units) per reaction.
SECTION: Single Stranded DNA Pools for Combinatorial Assembly
2. For each set of units, order primers for the left and rightmost homology sequences.

If the assembly will be performed in sets of 3, order primers for L1, R3, L3, R6, …
SECTION: Stage 1 Assembly of Barcode Units
3. For each oPool, assemble 3-4 barcode units by performing 15 rounds of PCR using Q5 DNA polymerase (NEB). If relying on homology sequences from the literature cited above, use the following PCR parameters:

Ta = 65 C
extension time 10 s/kb (for the total length of the first stage assembly)
reaction volume 100 uL
0.5 uM template

SECTION: Stage 1 Assembly of Barcode Units
4. Remove the PCR reaction from the thermocycler and place immediately on ice.
SECTION: Stage 1 Assembly of Barcode Units
5. For each assembly, add primers for the left and rightmost homology sequences at 0.5 uM directly to the reaction. Perform an additional 15 cycles of PCR.
SECTION: Stage 1 Assembly of Barcode Units
6. Perform gel separation and extract the assembly product.
SECTION: Hierarchical Assembly of Barcodes
7. Continue assembling barcodes in a hierarchical manner by combining 0.5 uM of products from the previous stage of assembly into a reaction with the above PCR parameters, adjusting the extension time at each stage.
SECTION: Hierarchical Assembly of Barcodes
8. In each stage, repeat the steps from the initial stage of assembly (15 initial rounds of PCR with template only, followed by 15 rounds of extension PCR) until n barcode units are assembled.
SECTION: Sequencing and Cloning Barcode Libraries
9. Using overhang primers for the left and rightmost homology in the completed assembly (L1 and Rn), perform a final PCR with the same parameters to add adaptor sequences for next-generation sequencing. If deferring sequencing until after cloning is completed, skip this step.
SECTION: Sequencing and Cloning Barcode Libraries
10. To clone the assembled barcodes into a plasmid, design overhang primers with at least 20 nt homology to the linearized backbone at the desired cloning site. Using HiFi assembly (NEB), insert the barcodes as a single piece into the backbone. The resulting plasmid library can be sequenced on the Oxford Nanopore platform. 

The theoretical diversity of the final barcode library is mn.