Aim: Next Generation Sequencing (NGS) workflows can produce libraries that differ in fragment size, index length, clustering efficiency, and more. Pooling different NGS libraries before sequencing maximizes flow cell use, reduces cost per sample, and minimizes turnaround time. With careful consideration, HLA (11 Loci), chimerism, and cfDNA NGS libraries can be pooled and sequenced simultaneously on Illumina instruments. Evaluation of various dilution methods and pooling ratios between different assays enabled adequate distribution of reads and coverage depth for different NGS assays.
Method: Eight to twenty nine QC DNA samples were processed with 11 Loci Multiplex PCR amplification and library preparation using a commercial NGS HLA assay. Two screening and six monitoring samples with known chimerism or donor-derived cell-free DNA (dd-cfDNA) status were amplified and processed for library preparation using chimerism or cfDNA assays up to the point of library dilution. Chimerism and cfDNA library concentrations were adjusted prior to pooling with the HLA library. A sequence coverage calculator was used to determine pooling ratios between the HLA, chimerism, and cfDNA NGS libraries. Pooled libraries were sequenced on 3 Illumina instruments. Data was analyzed with each assay’s respective commercial software.
Results: The distribution of reads between the HLA, chimerism, and cfDNA assays was adequate. All HLA samples surpassed read depth requirements in key exons and typing concordance was 100% at 3-Field for all sequencing runs. Not all screening or monitoring samples for chimerism or cfDNA met the required read depth recommended by vendor. Nevertheless, all screening samples revealed an identical number of informative markers and all monitoring samples showed consistent measurements of recipient or dd-cfDNA with coefficient of determination values of 0.996 to 0.999 across Illumina instruments.
Conclusion: This study demonstrates the feasibility of simultaneous sequencing of NGS libraries pooled from HLA, chimerism and cfDNA NGS assays on Illumina instruments.
