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Genetic profiling of tumours using both circulating free DNA and circulating tumour cells isolated from the same preserved whole blood sample.

Rothwell DG, Smith N, Morris D, Leong HS, Li Y, Hollebecque A, Ayub M, Carter L, Antonello J, Franklin L, Miller C, Blackhall F, Dive C, Brady G - Mol Oncol (2015)

Bottom Line: However, accurate assessment of both CTCs and ctDNA requires all blood cells to be maintained intact until samples are processed.We demonstrate that yields of circulating free DNA (cfDNA) obtained from whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 h of blood draw.Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples.

View Article: PubMed Central - PubMed

Affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK. Electronic address: dominic.rothwell@cruk.mancehster.ac.uk.

No MeSH data available.


Related in: MedlinePlus

A. Number of single nucleotide variations identified in a pool of HNV cfDNA prepared from either EDTA processed up to 4 h post blood draw and CellSave processed 96 h post blood draw. There was no significant difference in SNPs per million bases for the EDTA and CellSave cfDNA samples (paired t-test p > 0.05). B. Repertoire of mutations detected in each collection with equal frequencies of transitions and transversions seen in both EDTA and CellSave samples. C. Summary of overall quality of NGS data generated from EDTA and CellSave derived cfDNA showing comparable levels of mapping, read alignment and duplication.
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fig2: A. Number of single nucleotide variations identified in a pool of HNV cfDNA prepared from either EDTA processed up to 4 h post blood draw and CellSave processed 96 h post blood draw. There was no significant difference in SNPs per million bases for the EDTA and CellSave cfDNA samples (paired t-test p > 0.05). B. Repertoire of mutations detected in each collection with equal frequencies of transitions and transversions seen in both EDTA and CellSave samples. C. Summary of overall quality of NGS data generated from EDTA and CellSave derived cfDNA showing comparable levels of mapping, read alignment and duplication.

Mentions: Although the CellSave preservative significantly reduced the level of WBC lysis, thereby maintaining the ctDNA fraction within samples, it is possible that the components of the CellSave tube could act as a DNA damaging agent and effectively increase background sequencing errors. To test this, standard EDTA and 96 h CellSave cfDNA samples from the 20 HNV were subjected to WGS. To estimate the overall mutation burden low pass WGS Illumina MiSeq sequencing data were generated from three technical replicates of each sample set, with pooled cfDNA of each sample set being used to obtain the 5 ng cfDNA input. Over 1.0 × 108 bases were sequenced for each library with approximately 9.5 × 103 single nucleotide variants (SNVs) identified per sample when analysed against the Hg19 genome. No significant difference was found between the overall quality of the NGS data in terms of overall coverage, mapability, duplicates and total reads, and mutation rates of the CellSave (60.4 SNV per million bases) compared to the EDTA samples (58.9 SNV per million bases) indicating CellSave cfDNA is compatible with extended NGS strategies (Figure 2A and 2C). Analysis of the types of SNV detected within the cfDNA in each collection tubes was also performed, with similar frequencies of transitions and transversions seen in both sample type suggesting no effect of CellSave preservative on cfDNA integrity (Figure 2B).


Genetic profiling of tumours using both circulating free DNA and circulating tumour cells isolated from the same preserved whole blood sample.

Rothwell DG, Smith N, Morris D, Leong HS, Li Y, Hollebecque A, Ayub M, Carter L, Antonello J, Franklin L, Miller C, Blackhall F, Dive C, Brady G - Mol Oncol (2015)

A. Number of single nucleotide variations identified in a pool of HNV cfDNA prepared from either EDTA processed up to 4 h post blood draw and CellSave processed 96 h post blood draw. There was no significant difference in SNPs per million bases for the EDTA and CellSave cfDNA samples (paired t-test p > 0.05). B. Repertoire of mutations detected in each collection with equal frequencies of transitions and transversions seen in both EDTA and CellSave samples. C. Summary of overall quality of NGS data generated from EDTA and CellSave derived cfDNA showing comparable levels of mapping, read alignment and duplication.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834815&req=5

fig2: A. Number of single nucleotide variations identified in a pool of HNV cfDNA prepared from either EDTA processed up to 4 h post blood draw and CellSave processed 96 h post blood draw. There was no significant difference in SNPs per million bases for the EDTA and CellSave cfDNA samples (paired t-test p > 0.05). B. Repertoire of mutations detected in each collection with equal frequencies of transitions and transversions seen in both EDTA and CellSave samples. C. Summary of overall quality of NGS data generated from EDTA and CellSave derived cfDNA showing comparable levels of mapping, read alignment and duplication.
Mentions: Although the CellSave preservative significantly reduced the level of WBC lysis, thereby maintaining the ctDNA fraction within samples, it is possible that the components of the CellSave tube could act as a DNA damaging agent and effectively increase background sequencing errors. To test this, standard EDTA and 96 h CellSave cfDNA samples from the 20 HNV were subjected to WGS. To estimate the overall mutation burden low pass WGS Illumina MiSeq sequencing data were generated from three technical replicates of each sample set, with pooled cfDNA of each sample set being used to obtain the 5 ng cfDNA input. Over 1.0 × 108 bases were sequenced for each library with approximately 9.5 × 103 single nucleotide variants (SNVs) identified per sample when analysed against the Hg19 genome. No significant difference was found between the overall quality of the NGS data in terms of overall coverage, mapability, duplicates and total reads, and mutation rates of the CellSave (60.4 SNV per million bases) compared to the EDTA samples (58.9 SNV per million bases) indicating CellSave cfDNA is compatible with extended NGS strategies (Figure 2A and 2C). Analysis of the types of SNV detected within the cfDNA in each collection tubes was also performed, with similar frequencies of transitions and transversions seen in both sample type suggesting no effect of CellSave preservative on cfDNA integrity (Figure 2B).

Bottom Line: However, accurate assessment of both CTCs and ctDNA requires all blood cells to be maintained intact until samples are processed.We demonstrate that yields of circulating free DNA (cfDNA) obtained from whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 h of blood draw.Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples.

View Article: PubMed Central - PubMed

Affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK. Electronic address: dominic.rothwell@cruk.mancehster.ac.uk.

No MeSH data available.


Related in: MedlinePlus