title: DNA storage under high temperature conditions does not affect performance in human leukocyte antigen genotyping via next-generation sequencing (DNA integrity maintained in extreme conditions).
abstract: BACKGROUND
  Stable dry-state storage of DNA is desirable to minimize required storage space and to reduce electrical and shipping costs. DNA purified from various commercially available dry-state stabilization matrices has been used successfully in downstream molecular applications (e.g., quantitative polymerase chain reaction [qPCR], microarray, and sequence-based genotyping). However, standard DNA storage conditions still include freezing of DNA eluted in aqueous buffers or nuclease-free water. Broad implementation of dry-state, long-term DNA storage requires enhancement of such dry-state DNA stabilization products to control for temperature fluctuations at specimen collection, transit, and storage. This study tested the integrity of genomic DNA subjected to long-term storage on GenTegra(™) DNA stabilization matrices (GenTegra LLC, Pleasanton, CA) at extreme conditions, as defined by a 4-year storage period at ambient temperature with an initial incubation for 7 months at 37°C, 56°C, or ambient temperature. Subsequently, purified DNA performance and integrity were measured by qPCR and next-generation sequencing (NGS)-based human leokocyte antigen (HLA) genotyping.
  
  
  RESULTS
  High molecular weight genomic DNA samples were recovered from the GenTegra product matrix and exhibited integrity comparable to a highly characterized commercial standard under assessment by qPCR. Samples were genotyped for classical HLA loci using next generation sequencing-based methodolgy on the Roche 454 GS Junior instrument. Amplification efficiency, sequence coverage, and sequence quality were all comparable with those produced from a cell line DNA sequenced as a control. No significant differences were observed in the mean, median, or mode quality scores between samples and controls (p≥0.4).
  
  
  CONCLUSIONS
  Next generation HLA genotyping was chosen to test the integrity of GenTegra-treated genomic DNA due to the requirment for long sequence reads to genotype the highly polymorphic classical HLA genes. Experimental results demonstrate the efficacy of the GenTegra product as a suitable genomic DNA preservation tool for collection and long-term biobanking of DNA at fluctuating and high temperatures.
authors.affiliation: 1 Children's Hospital Oakland Research Institute , Oakland, California.
authors.forename: Shana L
authors.initials: SL
authors.lastname: McDevitt
authors.forename: Michael E
authors.initials: ME
authors.lastname: Hogan
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Pappas
authors.forename: Lily Y
authors.initials: LY
authors.lastname: Wong
authors.forename: Janelle A
authors.initials: JA
authors.lastname: Noble
mesh_terms: D018070:Biological Specimen Banks; D004247:DNA / Q000737:chemistry*; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D013696:Temperature
chemical_list: D006680:HLA Antigens; D004247:DNA