Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens.
Author(s): Wood HM, Belvedere O, Conway C, Daly C, Chalkley R, Bickerdike M, McKinley C, Egan P, Ross L, Hayward B, Morgan J, Davidson L, MacLennan K, Ong TK, Papagiannopoulos K, Cook I, Adams DJ, Taylor GR, Rabbitts P
Publication: Nucleic Acids Res, 2010, Vol. 38, Page e151
PubMed ID: 20525786 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of using FFPE rather than snap-frozen specimens, input amount and reaction multiplexing on detection of CNV by NGS. DNA was extracted from snap-frozen tissue using phenol-chloroform and ethanol precipitation and from microdissected FFPE sections with QIAamp.
Summary of Findings:
The copy number karyograms were very similar between matched FFPE and frozen specimens, but in some specimens, the magnitude of the changes appeared larger when FFPE specimens were used, which the authors attribute to higher concentration of tumor cells in those FFPE specimens due to microdissection. While the authors report that fewer reads were produced when less DNA was used, the copy number data generated using 5, 10 and 50 ng of DNA was similar. When DNA from 10 specimens were combined, the smallest copy number variants detected by NGS were larger than when using DNA from a single specimen (0.9-1.5 Mb versus 15 kb).
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform DNA DNA sequencing DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
DNA sequencing Specific Template/input amount 5 ng
10 ng
50 ng
DNA sequencing Specific Technology platform Multiplex (DNA from 10 specimens)
Single