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Ultra-Low DNA Input into Whole Genome Methylation Assays and Detection of Oncogenic Methylation and Copy Number Variants in Circulating Tumour DNA

Background: Abnormal CpG methylation in cancer is ubiquitous and generally detected in tumour specimens using a variety of techniques at a resolution encompassing single CpG loci to genome wide coverage. Analysis of samples with very low DNA inputs, such as formalin fixed (FFPE) biopsy specimens fro...

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Autores principales: Whalley, Celina, Payne, Karl, Domingo, Enric, Blake, Andrew, Richman, Susan, Brooks, Jill, Batis, Nikolaos, Spruce, Rachel, Mehanna, Hisham, Nankivell, Paul, Beggs, Andrew D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610445/
https://www.ncbi.nlm.nih.gov/pubmed/33777442
http://dx.doi.org/10.3390/epigenomes5010006
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author Whalley, Celina
Payne, Karl
Domingo, Enric
Blake, Andrew
Richman, Susan
Brooks, Jill
Batis, Nikolaos
Spruce, Rachel
Mehanna, Hisham
Nankivell, Paul
Beggs, Andrew D.
author_facet Whalley, Celina
Payne, Karl
Domingo, Enric
Blake, Andrew
Richman, Susan
Brooks, Jill
Batis, Nikolaos
Spruce, Rachel
Mehanna, Hisham
Nankivell, Paul
Beggs, Andrew D.
author_sort Whalley, Celina
collection PubMed
description Background: Abnormal CpG methylation in cancer is ubiquitous and generally detected in tumour specimens using a variety of techniques at a resolution encompassing single CpG loci to genome wide coverage. Analysis of samples with very low DNA inputs, such as formalin fixed (FFPE) biopsy specimens from clinical trials or circulating tumour DNA is challenging at the genome-wide level because of lack of available input. We present the results of low input experiments into the Illumina Infinium HD methylation assay on FFPE specimens and ctDNA samples. Methods: For all experiments, the Infinium HD assay for methylation was used. In total, forty-eight FFPE specimens were used at varying concentrations (lowest input 50 ng); eighteen blood derived specimens (lowest input 10 ng) and six matched ctDNA input (lowest input 10 ng)/fresh tumour specimens (lowest input 250 ng) were processed. Downstream analysis was performed in R/Bioconductor for quality control metrics and differential methylation analysis as well as copy number calls. Results: Correlation coefficients for CpG methylation were high at the probe level averaged R2 = 0.99 for blood derived samples and R2 > 0.96 for the FFPE samples. When matched ctDNA/fresh tumour samples were compared, R2 > 0.91 between the two. Results of differential methylation analysis did not vary significantly by DNA input in either the blood or FFPE groups. There were differences seen in the ctDNA group as compared to their paired tumour sample, possibly because of enrichment for tumour material without contaminating normal. Copy number variants observed in the tumour were generally also seen in the paired ctDNA sample with good concordance via DQ plot. Conclusions: The Illumina Infinium HD methylation assay can robustly detect methylation across a range of sample types, including ctDNA, down to an input of 10 ng. It can also reliably detect oncogenic methylation changes and copy number variants in ctDNA. These findings demonstrate that these samples can now be accessed by methylation array technology, allowing analysis of these important sample types.
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spelling pubmed-76104452021-03-26 Ultra-Low DNA Input into Whole Genome Methylation Assays and Detection of Oncogenic Methylation and Copy Number Variants in Circulating Tumour DNA Whalley, Celina Payne, Karl Domingo, Enric Blake, Andrew Richman, Susan Brooks, Jill Batis, Nikolaos Spruce, Rachel Mehanna, Hisham Nankivell, Paul Beggs, Andrew D. Epigenomes Article Background: Abnormal CpG methylation in cancer is ubiquitous and generally detected in tumour specimens using a variety of techniques at a resolution encompassing single CpG loci to genome wide coverage. Analysis of samples with very low DNA inputs, such as formalin fixed (FFPE) biopsy specimens from clinical trials or circulating tumour DNA is challenging at the genome-wide level because of lack of available input. We present the results of low input experiments into the Illumina Infinium HD methylation assay on FFPE specimens and ctDNA samples. Methods: For all experiments, the Infinium HD assay for methylation was used. In total, forty-eight FFPE specimens were used at varying concentrations (lowest input 50 ng); eighteen blood derived specimens (lowest input 10 ng) and six matched ctDNA input (lowest input 10 ng)/fresh tumour specimens (lowest input 250 ng) were processed. Downstream analysis was performed in R/Bioconductor for quality control metrics and differential methylation analysis as well as copy number calls. Results: Correlation coefficients for CpG methylation were high at the probe level averaged R2 = 0.99 for blood derived samples and R2 > 0.96 for the FFPE samples. When matched ctDNA/fresh tumour samples were compared, R2 > 0.91 between the two. Results of differential methylation analysis did not vary significantly by DNA input in either the blood or FFPE groups. There were differences seen in the ctDNA group as compared to their paired tumour sample, possibly because of enrichment for tumour material without contaminating normal. Copy number variants observed in the tumour were generally also seen in the paired ctDNA sample with good concordance via DQ plot. Conclusions: The Illumina Infinium HD methylation assay can robustly detect methylation across a range of sample types, including ctDNA, down to an input of 10 ng. It can also reliably detect oncogenic methylation changes and copy number variants in ctDNA. These findings demonstrate that these samples can now be accessed by methylation array technology, allowing analysis of these important sample types. MDPI 2021-02-19 /pmc/articles/PMC7610445/ /pubmed/33777442 http://dx.doi.org/10.3390/epigenomes5010006 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ).
spellingShingle Article
Whalley, Celina
Payne, Karl
Domingo, Enric
Blake, Andrew
Richman, Susan
Brooks, Jill
Batis, Nikolaos
Spruce, Rachel
Mehanna, Hisham
Nankivell, Paul
Beggs, Andrew D.
Ultra-Low DNA Input into Whole Genome Methylation Assays and Detection of Oncogenic Methylation and Copy Number Variants in Circulating Tumour DNA
title Ultra-Low DNA Input into Whole Genome Methylation Assays and Detection of Oncogenic Methylation and Copy Number Variants in Circulating Tumour DNA
title_full Ultra-Low DNA Input into Whole Genome Methylation Assays and Detection of Oncogenic Methylation and Copy Number Variants in Circulating Tumour DNA
title_fullStr Ultra-Low DNA Input into Whole Genome Methylation Assays and Detection of Oncogenic Methylation and Copy Number Variants in Circulating Tumour DNA
title_full_unstemmed Ultra-Low DNA Input into Whole Genome Methylation Assays and Detection of Oncogenic Methylation and Copy Number Variants in Circulating Tumour DNA
title_short Ultra-Low DNA Input into Whole Genome Methylation Assays and Detection of Oncogenic Methylation and Copy Number Variants in Circulating Tumour DNA
title_sort ultra-low dna input into whole genome methylation assays and detection of oncogenic methylation and copy number variants in circulating tumour dna
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610445/
https://www.ncbi.nlm.nih.gov/pubmed/33777442
http://dx.doi.org/10.3390/epigenomes5010006
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