Two Methods for High-Throughput NGS Template Preparation for Small and Degraded Clinical Samples Without Automation.

Clinical samples are difficult to prepare for NGS, because of the small amounts or degraded states of formalin-fixed tissue, plasma, urine, and single-cell DNA. Conventional whole genome amplification methods are too biased for NGS applications, and the existing NGS preparation kits require intermediate purifications and excessive time to prepare hundreds of samples in a day without expensive automation. We have tested two 96-well manual methods to make NGS templates from FFPE tissue, plasma, immunoprecipitates and single cells. Both enable a single person to prepare 200 – 300 indexed samples/day from very small and degraded samples. Data were produced by early adopter labs and companies. ThruPLEX-FD Prep kit is a 1-tube, 2-hour, 3-step ligation-based process for preparing 50 pg – 20 ng amounts of fragmented DNA for NGS using only one AMPure purification before clustering for the MiSeq or HiSeq. Sequence representation was excellent from 15% to 85% GC. NGS from 20 pg to 20 ng FFPE, plasma and ChIP DNA yielded mean quality scores >35, 95% intact PE reads. PCR duplicates were 1, 6, and 60% for 20, 2 and 0.2 ng FFPE DNA; and 3% for 0.2 ng plasma DNA. ThruPLEX enabled high-performance targeted sequencing from ChIP DNA using 10 – 100 X smaller starting amounts of DNA or cells than possible with conventional NGS preparations. PicoPLEX-SC Prep kit is a 1-tube, 3-hour, 4-step process to easily prepare hundreds of single cell or sorted chromosome NGS samples per day. Greater than 95% of sorted cancer cells or blastomeres were successfully prepared for sequencing. Typically 99% of reads were high quality, 98.5% mapped to human, and <0.6% did not match hg19. Indexed NGS was able to reproducibly detect human single copy number changes less than 20 kb in size.