Robust and High-throughput PCR-Free whole genome sequencing pipeline for Illumina HiSeq X and NovaSeq platforms

Large scale whole genome sequencing (WGS) of population based cohorts and for clinical applications is now possible using Illumina’s HiSeq X and NovaSeq systems. Baylor College of Medicine’s Human Genome Sequencing Center... [ view full abstract ]

Large scale whole genome sequencing (WGS) of population based cohorts and for clinical applications is now possible using Illumina’s HiSeq X and NovaSeq systems. Baylor College of Medicine’s Human Genome Sequencing Center (HGSC) is a large-scale sequencing facility which supports NHGRI and NHLBI whole genome sequencing (WGS) projects in the Center for Common Disease Genomics (CCDG) program, Trans-Omics for Precision Medicine (TOPMed) program, Alzheimer’s disease sequencing project (ADSP).

Currently, >2000 PCR-Free libraries/month are constructed for 30X WGS on Illumina’s HiSeq X system and to support the recently installed NovaSeq sequencer. Protocols have been established to generate high quality libraries from a spectrum of DNA quality and quantities. Robust library preparation and quality control (QC) measures have also been implemented to support this pipeline. Libraries prepared using this protocol are sequenced on HiSeq X instruments generating on average about 124GB (unique/lane). The libraries are also currently being tested on S2 flow cell of the recently installed NovaSeq sequencer. PCR-Free library workflow used KAPA Hyper kit with modifications and libraries are prepared in batches of 96 on the Biomek FX^P robots in 7 hours. The workflow has generated 26,054 PCR-Free libraries with <1% fail rate since December 2015. Novel QC steps using Fragment Analyzer and qPCR assay accurately quantifies libraries and screens for adapter dimer contamination. Newly synthesized adapter barcode oligos are screened in-house for cross-contamination using amplicons to detect contamination rates below current HPLC screening levels and guarantees that adapter contamination is <0.5% to allow for multiplexing of libraries. A qPCR assay has also been developed to detect and quantify rDNA of >110 bacterial species is used to screen DNA extracted from saliva. Fluidigm SNP Trace panel assay is used to confirm sample identity and VerifyBamID to detect sample contamination. Quality control metrics related to coverage, sample integrity and variant representation are generated at-scale with HgV, the HGSC analysis pipeline. To date 15,382 samples for the TOPMed project were sequenced using these PCR-Free libraries and generated an average sequence coverage of 40X, with 97% of the bases in the genome having 20X or greater coverage. These PCR-free protocols and workflow is planned for implementation in the HGSC clinical laboratory.