Capture MRE-seq for methylation analysis of highly degraded DNA samples
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA656241 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12421200_1.fastq.gz | Fastqsanger.gz | |||
| SRR12421200_2.fastq.gz | Fastqsanger.gz | |||
| SRR12421176.fastq.gz | Fastqsanger.gz | |||
| SRR12421177.fastq.gz | Fastqsanger.gz | |||
| SRR12421178.fastq.gz | Fastqsanger.gz |
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Project description:Understanding the impact of DNA methylation within different disease contexts often requires accurate assessment of these modifications in a genome-wide fashion. Frequently, patient-derived tissue stored in long-term hospital tissue banks have been preserved using formalin-fixation paraffin-embedding (FFPE). While these samples can comprise valuable resources for studying disease, the fixation process ultimately compromises the DNA’s integrity and leads to degradation. Degraded DNA can complicate CpG methylome profiling using traditional techniques, particularly when performing methylation sensitive restriction enzyme sequencing (MRE-seq), yielding high backgrounds and resulting in lowered library complexity. Here, we provide results using our new MRE-seq protocol (Capture MRE-seq), tailored to preserving unmethylated CpG information when using samples with highly degraded DNA. The results using Capture MRE-seq correlate well (0.92) with traditional MRE-seq calls when profiling non-degraded samples, and can recover unmethylated regions in highly degraded samples when traditional MRE-seq fails, which we validate using bisulfite sequencing-based data (WGBS) as well as methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq).
2023-03-01 | GSE155968 | GEO
Project description:Background: Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3’ mRNA-seq method sequences the 3’ region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data free from PCR amplification bias. In this study, we evaluated the performance of the 3’ mRNA-Seq using Lexogene QuantSeq 3’ mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq stranded mRNA-Seq and RNA Exome Capture. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13% to >70% of DV200 values; input amounts ranged from 1ng to 100ng for validation. Results: The total mapped reads of the 3’ mRNA-Seq to the reference genome ranged from 99% to74% across all the samples. After PCR bias correction, total mapped reads ranged from 3% to 56%. The 3’ mRNA-Seq data showed highly reproducible data in the UHR (R>0.94) at different input amounts from 1ng to 100ng, and FF and FFPE paired samples (R=0.93) at 10 ng. Severely degraded FFPE RNA with 13% to 30% of DV200 value showed good concordance (R>0.90) with 100ng input. A moderate correlation was observed when directly comparing 3’ mRNA-Seq data with TruSeq stranded mRNA-Seq (R=0.78) and RNA Exome Capture data (R>0.67). Conclusion: In this study, 3’ mRNA-Seq with PCR bias correction using UMI is a suitable method for gene quantification in both FF and FFPE RNAs. The 3’ mRNA-Seq may be applied to severely degraded RNA from FFPE tissues and generate high-quality sequencing data.
2021-10-28 | GSE173506 | GEO
Project description:This SuperSeries is composed of the following subset Series: GSE39013: Stability of miRNA in FFPE tumour samples exhibiting degraded mRNA [Cervix samples series 1] GSE39014: Stability of miRNA in FFPE tumour samples exhibiting degraded mRNA [Cervix samples series 2] GSE39015: Stability of miRNA in FFPE tumour samples exhibiting degraded mRNA [Cervix samples miRNA] GSE39016: Stability of miRNA in FFPE tumour samples exhibiting degraded mRNA [Bladder samples] GSE39066: Stability of miRNA in FFPE tumour samples exhibiting degraded mRNA [cell lines] Refer to individual Series
2012-07-03 | E-GEOD-39067 | biostudies-arrayexpress
Project description:Assay performance comparison between intact and artificially degraded RNA samples
2010-01-13 | GSE17564 | GEO
Project description:We compare the performance of two library preparation protocols (poly(A) and exome capture) in in vitro degraded RNA samples
2015-08-03 | GSE64113 | GEO
Project description:soil samples from degraded grassland and mango cultivation (Raw sequence reads)
| PRJNA689669 | ENA
Project description:BackgroundMitochondrial DNA (mtDNA) typing can be a useful aid for identifying people from compromised samples when nuclear DNA is too damaged, degraded or below detection thresholds for routine short tandem repeat (STR)-based analysis. Standard mtDNA typing, focused on PCR amplicon sequencing of the control region (HVS I and HVS II), is limited by the resolving power of this short sequence, which misses up to 70% of the variation present in the mtDNA genome.MethodsWe used in-solution hybridisation-based DNA capture (using DNA capture probes prepared from modern human mtDNA) to recover mtDNA from post-mortem human remains in which the majority of DNA is both highly fragmented (<100 base pairs in length) and chemically damaged. The method 'immortalises' the finite quantities of DNA in valuable extracts as DNA libraries, which is followed by the targeted enrichment of endogenous mtDNA sequences and characterisation by next-generation sequencing (NGS).ResultsWe sequenced whole mitochondrial genomes for human identification from samples where standard nuclear STR typing produced only partial profiles or demonstrably failed and/or where standard mtDNA hypervariable region sequences lacked resolving power. Multiple rounds of enrichment can substantially improve coverage and sequencing depth of mtDNA genomes from highly degraded samples. The application of this method has led to the reliable mitochondrial sequencing of human skeletal remains from unidentified World War Two (WWII) casualties approximately 70 years old and from archaeological remains (up to 2,500 years old).ConclusionsThis approach has potential applications in forensic science, historical human identification cases, archived medical samples, kinship analysis and population studies. In particular the methodology can be applied to any case, involving human or non-human species, where whole mitochondrial genome sequences are required to provide the highest level of maternal lineage discrimination. Multiple rounds of in-solution hybridisation-based DNA capture can retrieve whole mitochondrial genome sequences from even the most challenging samples.
| S-EPMC3879034 | biostudies-literature
Project description:Background: As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits ability to expression profile, we explored factors predicting success for FFPE profiling and investigated an approach overcoming this limitation. Methods: Bladder (n=141, stored 3-8 years) and cervix (n=160, stored 8-23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon1.0ST arrays. Percent detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. Precursor mir-205 was measured by Exon array and mature miR-205 by qRT-PCR. Eight-old and -young cervix samples were compared using Affymetrix miRNA 2.0 arrays. For comparison, the 'cervix_tumour_cs1_113' (previsously submitted GSM677307) was included and re-analyzed with the samples from the current study (total 161 Cervix samples). Results: RNA quality controls (e.g. RNA integrity number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R2=-0.30, p<0.01) and cervix (R2=-0.69, p<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT-PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; p=1.10x10-5). Median miRNA probeset expression of eight-old and eight-young cervix samples correlated well (R2=0.95) overcoming the age-related bias of mRNA probesets, suggesting miR-205 stability generalises across microRNA. Conclusions: microRNA profiling overcomes the limitation of degraded FFPE samples
2012-07-04 | GSE39016 | GEO
Project description:Background: As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits ability to expression profile, we explored factors predicting success for FFPE profiling and investigated an approach overcoming this limitation. Methods: Bladder (n=141, stored 3-8 years) and cervix (n=160, stored 8-23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon1.0ST arrays. Percent detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. Precursor mir-205 was measured by Exon array and mature miR-205 by qRT-PCR. Eight-old and -young cervix samples were compared using Affymetrix miRNA 2.0 arrays. For comparison, the 'cervix_tumour_cs1_113' (previsously submitted GSM677307) was included and re-analyzed with the samples from the current study (total 161 Cervix samples). Results: RNA quality controls (e.g. RNA integrity number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R2=-0.30, p<0.01) and cervix (R2=-0.69, p<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT-PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; p=1.10x10-5). Median miRNA probeset expression of eight-old and eight-young cervix samples correlated well (R2=0.95) overcoming the age-related bias of mRNA probesets, suggesting miR-205 stability generalises across microRNA. Conclusions: microRNA profiling overcomes the limitation of degraded FFPE samples
2012-07-04 | GSE39015 | GEO
Project description:Transcriptome analysis of partially degraded and fragmented RNA samples from PAO1
| PRJNA655510 | ENA