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Blood collection in cell-stabilizing tubes does not impact germline DNA quality for pediatric patients.

Author(s): Wollison BM, Thai E, Mckinney A, Ward A, Clapp A, Clinton C, Nag A, Thorner AR, Gastier-Foster JM, Crompton BD

Publication: PLoS One, 2017, Vol. 12, Page e0188835

PubMed ID: 29206863 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared whole exome sequencing (WES) and microarray data obtained from DNA isolated from blood collected in matched EDTA tubes and Streck tubes. Blood was obtained from three pediatric patients at Dana Farber Cancer Institute. The patients were selected based on the expectation of normal or near normal WBC counts and age >11 years, but diagnoses were not provided. Blood was drawn through standard venipuncture into a 5 mL EDTA tube and a 10 mL Streck Cell-Free DNA BCT. Blood specimens were centrifuged at 1000 x g for 10 min at 4˚C. Plasma was removed and DNA was extracted from the cells using the Gentra Puregene Blood Kit. DNA was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. DNA was ultrasonicated to produce an average size of 250 bp and sequencing libraries were constructed using the KAPA HTP Library Preparation Kit, purified using Agencourt AMPure XP beads, evaluated using a bioanalyer, and quantified using an Illumina MiSeq. Capture was performed using the SureSelect XT Hybrid Capture Kit with the Exon v5 bait set and then sequencing was performed on an Illumina Hiseq 2500 in rapid run mode with an expected 80X coverage. Variant calls were performed using MuTect and through comparison to DNA from the CEPH1408 cell line and indels were called using the Somatic Indel Detector Tool. Copy number analysis was also performed using custom 4x180K CGH plus SNP microarrays.

   Summary of Findings:

   For two of three patients, more DNA was obtained from the Streck tube than the EDTA tube which is easily attributable to the higher tube volume; however, sufficient DNA for analysis was obtained from all tubes. The authors report comparable expected library fragment sizes and WES metrics. Even when read coverage was binned by GC content of the targeted regions, differences in normalized mean coverage were <0.02 X for all categories. The number of unfiltered variants identified was comparable between tube types (21672-25478 for EDTA and 22598-25147 for Streck). After filtering for an allelic fraction of >0.25 and coverage depth of >30 read, a comparable percentage of variants were identified in specimens collected in EDTA (57.12-67.32%) and Streck (63.31-65.42%) tubes.  Less than 1.2% of the variants identified were not identified in the matched specimen in the other tube type. The authors state this is comparable to the expected rate of sequencing error. Importantly, less than 0.05% of variants were unique to a single specimen. Further, the mutation pattern was similar in tubes collected in EDTA and Streck tubes with no apparent bias. When the DNA was used for microarray analysis, the derivative log ratio spread and call rate were comparable for Streck (0.1249-0.1322 and 0.937-0.972, respectively) and EDTA specimens (0.1241-0.1292 and 0.95-0.977, respectively). Using microarray analysis, clinically relevant copy number data was identical in specimens from Streck and EDTA tubes and matched specimens showed identical regions of homozygosity.

    

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • Streck/BCT

   Diagnoses:

   • Not specified
   • Neoplastic - Pediatric

   Platform:

   Analyte	Technology Platform
   DNA	DNA microarray
   DNA	Next generation sequencing
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	EDTA tube Streck cell-free DNA BCT

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