Design of 240,000 orthogonal 25mer DNA barcode probes

Abstract

DNA barcodes linked to genetic features greatly facilitate screening these features in pooled formats using microarray hybridization, and new tools are needed to design large sets of barcodes to allow construction of large barcoded mammalian libraries such as shRNA libraries. Here we report a framework for designing large sets of orthogonal barcode probes. We demonstrate the utility of this framework by designing 240,000 barcode probes and testing their performance by hybridization. From the test hybridizations, we also discovered new probe design rules that significantly reduce cross-hybridization after their introduction into the framework of the algorithm. These rules should improve the performance of DNA microarray probe designs for many applications.

Fig. 1.

The DeLOB DNA barcode design procedure. (A) Ten million random 25mers were generated and sequentially passed through restriction enzyme (RE) site, T_m, GC composition, and repetitive sequence filters. Candidates passing these filters were searched against themselves by BLAST and a subset of orthogonal sequences was selected on the basis of their BLAST results and the network elimination algorithm. After applying a secondary structure filter to eliminate self-folding-prone candidates, we obtained a final set of 240,000 probes. The 2 filters in the dashed box were based on rules discovered from analyzing hybridization data from first round probe design. (B) The network elimination algorithm. (i) Nonorthogonal candidate pairs were represented by a network graph. Each vertex was a candidate and each edge was a longer than 12-base match between the 2 connected candidates. (ii) One candidate was randomly chosen and placed in the orthogonal group (green). All candidates that were connected to this one were labeled in red and then eliminated from the network together with all edges incident to these red vertices. (iii) The random selection and elimination steps were repeated on the remaining network members. (iv) At the end, only orthogonal candidates were left.

Fig. 2.

A representative 2-color hybridization experiment of a single subpool labeled with Cy3 vs. the entire pool labeled with Cy5. Separation of the “present group” (red, probes that have target sequences in the Cy3-labeled subpool) and “absent group” (green, probes that do not have target sequences in the Cy3-labeled subpool) in the first round design (A) and the second round of design (B). The dashed blue lines represent Cy3/Cy5 ratio of 2 and 0.5, respectively. Only 10,000 randomly sampled probes in each group were plotted for clarity.

Fig. 3.

Analysis of probe composition and activity. (A) Distribution of T_m's in the 4-probe groups. (B) Distribution of CCCC motifs along probe lengths in the 4 groups. In the bright group, CCCCs were highly biased toward the very 5′ end, whereas in other groups, CCCCs were depleted from the very 5′ end of probes. (C) Nucleotide compositions at each of the 25 bases on probes in the 4 groups and the starting set of 10 million candidates 25mers. Dim probes had high A and low C compositions along the probe except for the 2 ends. Bright probes had extremely skewed C composition at the 5′ half of probes. The starting set had equal compositions for the 4 nucleotides at all 25 positions.