Evaluating accuracy of DNA pool construction based on white blood cell counts

Authors: ABRAMS, AMY - South Dakota State University; McDaneld, Tara; Keele, John; Chitko-Mckown, Carol; Kuehn, Larry; GONDA, MICHAEL - South Dakota State University

Submitted to: Frontiers in Livestock Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/14/2021
Publication Date: 2/5/2021
Citation: Abrams, A.N., McDaneld, T.G., Keele, J.W., Chitko-McKown, C.G., Kuehn, L.A., Gonda, M.G. 2021. Evaluating accuracy of DNA pool construction based on white blood cell counts. Frontiers in Livestock Genomics. 12. Article 635846. https://doi.org/10.3389/fgene.2021.635846.
DOI: https://doi.org/10.3389/fgene.2021.635846

Interpretive Summary: Combining individual tissue samples into a pool prior to DNA extraction can be used to mitigate the cost of genotyping; however, representing animals equally in pools can be challenging. Unequal representation will bias allele frequency estimates and could lead to incorrect conclusions. The objective of this study was to determine accuracy of pooling based on equalizing the number of white blood cell counts of blood samples prior to DNA extraction compared equalizing the amount of DNA based on each of two common DNA quantification methods. Ten individual bovine blood samples were collected and then pooled with two different individuals represented in each pool. Pools were constructed with the target of equal representation of the 2 animals based on number of white blood cells in the blood sample prior to DNA extraction and two common DNA quantification methods (spectrophotometer and spectrofluorometer). Pools and individual samples that comprised the pools were genotyped using a commercially available genotyping array. Pooling samples based on white blood cell count of the individual blood sample prior to DNA extraction was more predictive of sample representation compared to pooling based on DNA concentration alone. Therefore, constructing pools using white blood cell counts prior to DNA extraction may reduce cost associated with genotyping and improve representation of individuals in a pool.

Technical Abstract: Pooling individual samples prior to DNA extraction can mitigate the cost of DNA extraction and genotyping; however, these methods need to accurately generate equal representation of individuals within pools. The objective of this study was to determine accuracy of pool construction of blood samples based on white blood cell counts compared to two common DNA quantification methods. Fifty individual bovine blood samples were collected, and then pooled with all individuals represented in each pool. Pools were constructed with the target of equal representation of each individual animal based on number of white blood cells, spectrophotometric readings, spectrofluorometric readings, and whole blood volume with 9 pools per method and a total of 36 pools. Pools and individual samples that comprised the pools were genotyped using a commercially available genotyping array. ASReml was used to estimate variance components for individual animal contribution to pools. The correlation between animal contributions between two pools was estimated using bivariate analysis with starting values set to the result of a univariate analysis. Adonis test on distance matrix from the animal correlation showed clustering with method, and higher correlations between methods than within (P < 1 × 10–6). White blood cell count was predictive of sample representation when compared to pooling based on DNA concentration. Therefore, constructing pools using white blood cell counts prior to DNA extraction may reduce cost associated with DNA extraction and genotyping and improve representation of individuals in a pool.