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URL: https://dx.doi.org/DOI:10.17504/protocols.io.bh69j9h6

Authors: Abebe A. Fola , Jack Dorman, Maria Levy, Ilinca Ciubotariu, Giovanna Carpi

Summary: Genomic DNA (gDNA) extracts from human dried blood spots (DBS) are commonly used for infectious diseases surveillance, genomic epidemiology studies of targeted pathogens, and Biobanking. In particular, DBS samples are routinely collected during cross-sectional and large-scale epidemiological studies (i.e. Malaria Indicator Survey) in malaria-endemic countries for malaria surveillance and genomic studies. DBS samples have multiple advantages over venous blood, including less invasive sample blood collection (finger prick) and the recommended short- and long-term storage at room temperature, which facilitates sample transportation (domestically or internationally). Here we present an optimized high throughput gDNA extraction protocol to recover Plasmodium gDNA from human DBS (collected on Whatman filter paper) using field samples from Zambia and stored at room temperature with desiccant pellet (up to 2 years). Using the QIAcube HT instrument and QIAmp DNA 96-well kit, this protocol allows for gDNA extraction from human DBS samples in a manner that is automated, reproducible, and fast when compared to other methods. After gDNA extraction is completed, genomic DNA quantity and integrity is assessed using Qubit 1x dsDNA High Sensitivity Assay and Genomic DNA ScreenTape on the Agilent TapeStation 4150, respectively. In this case, gDNA samples that were obtained from field collected human DBS samples and extracted using this optimized protocol were then used for Plasmodium falciparum whole-genome sequencing by hybrid capture.Using this protocol, we first compared gDNA concentration between 2X6mm and 3X3mm Uni Core full punch (100% blood punch) and found a statistically significant difference in DNA concentration obtained from 2x6mm (ng/µL) vs 3x3mm DBS punches (range =0.44-2.01ng/µL) (P-value= 0.0003) with a median increased of ~4-fold in the gDNA yield when using 2x6mm hole punches. See the attached Comparison of DNA concentration 2X6mm vs 3X3mm Uni Core Punch documents. Furthermore, we tested and demostrated that using this protocol with 2X6mm punches, it is feasible to successfully extract sufficient gDNA for P. falciparum whole genome capture and deep sequencing from DBS samples (n=93) that had been stored at RT for up to 2 years with variable level of parasitemia. While generally concentrations were outside the recommended range for the Genomic DNA ScreenTape for the TapeStation DIN calculation, from those samples DIN was estimated, values ranged from 6.2 - 8.7 with average fragment peaks of 35,325 bp, indicating that DNA integrity is suitable for downstream analysis. Becuase these were field DBS samples, we reported high variation in gDNA concentration (ranges= 0.50-4.88ng/µL) which reflected the variation in blood volume and adsorbance across DBS samples. See the attached gDNA concentration for 93 DBS field samples documents.

Affiliations: The Department of Biological Sciences, Purdue University, The Department of Biological Sciences, Purdue University, The Department of Biological Sciences, Purdue University, The Department of Biological Sciences, Purdue University, The Department of Biological Sciences, Purdue University

Version: 1

Publication Date: 2020

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