- PgmNr 2574/W: PCR-free whole genome sequencing using isolates from dried blood spot.
Y. Ding; J. Le; S. Batalov; S. Chowdhury; L. Van Der Kraan; C. Yamada; Z. Bezares-Orin; N. Veeraraghavan; M. Niedzwiecki; D. Dimmock; C. Hobbs; S. Nahas; S. Kingsmore
Affiliation: Rady Children's Institute for Genomic Medicine, San Diego, California.
Background: Polymerase Chain Reactions-free rapid Whole Genome Sequencing (PCR-free rWGS) using Illumina sequencing platforms has emerged as a powerful diagnostic tool within hospital intensive care units for children who are acutely ill. Current PCR-free WGS library construction kits utilize genomic DNA (gDNA) isolated from whole blood. Although dried blood spot (DBS) isolates can be used for WGS, whole genome amplification (WGA) or post-ligation PCR is required to ensure sufficient library yield for next generation sequencing. Recent publications have shown that WGA and PCR-based libraries result in downstream sequencing bias such as increased GC content. To demonstrate the application of DBS isolates to generate WGA free and PCR-free WGS, we report feasibility studies and initial results using the DBS isolates and KAPA HyperPlus kits. Method: DBS specimens were collected with Whatman FTA paper or Protein saver cards 903 (40 ul EDTA whole blood). Ten disks of 3x3 mm punches were lysed using proteinase K and lysis buffer. After 60 minutes incubation at 56°C, magnet KAPA Pure Beads were applied for purification purposes. DBS isolates were quantified with Picogreen assay and Nanodrop followed by 0.8% agarose electrophoresis to ensure the integrate of the DNA. KAPA HyperPlus PCR-free library construction kits (pair-end) were utilized following the manufacturer’s protocol. Final libraries were measured using real time PCR assay and fragment analysis. Lastly five PCR-free libraries were pooled and sequenced on Illmnina Novaseq6000 S2 flow cell with read length 101x2 format. Results: Around 100 ng to 600 ng gDNA were extracted from DBS without degradation from both Whatman FTA and Protein saver cards. Approximately 3 to 11 nM of PCR free WGS libraries were generated with peak insert size around 450 bp. Greater than 95% of OMIM genes had >10x coverage for all coding bases. All genome build metrics were equivalent to the data from the previously validated clinical workflow. Discussion and conclusion: DBS are routinely collected for newborn screening. Our approach allows researchers and clinical laboratories to utilize DBS specimens for PCR-free WGS. Additional validation will be performed to further improve sequencing outcomes at Rady Children’s Institute for Genomic Medicine.
PgmNr 2574: PCR-free whole genome sequencing using isolates from dried blood spot.
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