High-throughput sample tracking using 96 spike-in DNA controls

Sample barcoding using indexed adapters allows for high-throughput multiplexing of large numbers of highly similar samples, such as DNA from patients for clinical screening. Maintaining sample identity, which often relies... [ view full abstract ]

Sample barcoding using indexed adapters allows for high-throughput multiplexing of large numbers of highly similar samples, such as DNA from patients for clinical screening. Maintaining sample identity, which often relies solely on the indexed adapters, is critical for accurate analysis and diagnosis. Successful sample identification requires minimal index cross-talk and sample cross-contamination.

We have developed a method to accurately and sensitively track sample identity and detect cross-contamination using high molecular weight (HMW) DNA spike-in controls (SICs). The SICs comprise repeating units of a core sequence unique to each SIC, flanked by universal sequences. The HMW nature of the SICs mimics the sample DNA throughout NGS library preparation and allows for the SICs to be indelibly linked to the sample from as early as sample collection. Furthermore, introduction of the SICs to crude samples prior to extraction and library preparation further reduces the probability of cross-contamination. The random and synthetic design of the SIC sequences, along with their repetitive structure, allows for unambiguous SIC-derived sequence identification. The large edit distance between SICs enables clear identification of sample cross-contamination. The synthetic design of the SICs is compatible with high-throughput sequencing, where the number of SICs can be scaled to meet requirements.

We have generated a panel of 96 unique SICs that were spiked into three bacterial gDNA samples with differing GC-content, with minimal cross-talk. This demonstrates the feasibility of accurate sample tracking for high-throughput workflows, regardless of genomic complexity. The repetitive structure of the SICs allows for their use in targeted sequencing applications such as hybrid capture and amplicon-based sequencing. This requires minimal probe design and optimization due to the universal flanking regions present in each SIC. The SICs were easily introduced into a standard targeted capture workflow without affecting the efficiency of the capture.