Fig. 3: Scalable, programmable oligo subset selection within a hierarchically structured complex oligo library.
From: Oligonucleotide subsets selection by single nucleotide resolution barcode identification
a The complex oligo library is designed to accommodate the hierarchical organization of barcodes. The library encodes data from four classical music pieces totaling 96.88 kB, spread across 12,000 oligos. Each oligo is 200 bp long, with 4 nt for the barcode. Barcodes are hierarchical; the first two nucleotides (N1, N2) differentiate pieces and instruments, while the next two (N3, N4) address specific sections. b Two nt and (c) 4 nt hierarchical barcode-based selection were demonstrated and analyzed using NGS. Read counts for each designed oligo were normalized and plotted in bar graphs. The left graph shows the distribution before selection; the right shows the results after. The red dashed line indicates the normalized read count, averaged at one. Pie charts show the proportion of total read counts, with blue for target barcodes before selection, green for target barcodes after, and gray for non-targets. The 2 nt barcode reveals the library’s hierarchical structure. The barcode ‘TG’ targets the trombone part of Mozart’s Requiem in D minor. Selecting a 4 nt barcode decodes specific musical segments. The barcode ‘ATAC’ targets a segment of Pachelbel’s Canon in D Major Viola part. The sheet music is the result of converting the original MIDI file of the music into an SVG file (https://musescore.com/user/94946395/scores/23202934?share=copy_link) using the MuseScore app (https://musescore.org/ko). The MIDI file is licensed under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 license. d Multiple selection scenarios within the oligo library, with graphs representing selection to 1, 2, and 3 nt barcodes from the left, and the lower graph for a 4 nt barcode. Two barcodes at each length were selected simultaneously, marked in cyan on the tree above each graph.
