Ontology highlight
ABSTRACT:
PROVIDER: PRJNA526013 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8695910_1.fastq.gz | Fastqsanger.gz | |||
| SRR8695910_2.fastq.gz | Fastqsanger.gz | |||
| SRR8695920_1.fastq.gz | Fastqsanger.gz | |||
| SRR8695920_2.fastq.gz | Fastqsanger.gz | |||
| SRR8695930_1.fastq.gz | Fastqsanger.gz |
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Project description:This experiment contains the RNA-Seq samples only. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5 % success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80 % of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.
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2016-05-17 | E-ERAD-37 | biostudies-arrayexpress
Project description:In this study, the efficiency of four RNA extraction methods was compared on 23 FFPE cardiac tissue specimens. The Qiagen AllPrep DNA/RNA FFPE kit (Method QP), Qiagen AllPrep DNA/RNA FFPE kit, with protocol modification on the ethanol wash step after deparaffinization (Method QE), CELLDATA RNAstorm 2.0 FFPE RNA Extraction kit (Method BP) and CELLDATA RNAstorm 2.0 FFPE RNA Extraction Kit with protocol modifications on the lysis step (Method BL). In comparing RNA quality metrics across FFPE RNA extract, nucleic acids extracted with Method QE and QP had the highest RNA yield. However, Method QE outperformed Method QP as more extract from Method QE had DV 200 values above 30%. Both method BL and BP produced a similar range of RNA purity and yield but more extract from Method BL had DV 200 values above 30% compared to Method BP. When accessing distribution value, Method BL outperformed Methods BP, QE, and QP as more extracts from Method BL had DV 200 values above 30% (16/23 samples) compared to other methods (PDV200<0.001; Kruskal-Wallis). However, method QE outperformed other methods in terms of RNA yield. The sequencing performance of RNA extracts from Method QE and Method BL was further tested on 8 matching samples with high RNA yield and high DV200 value respectively. RNA extracts from Method QE which yielded the highest RNA quantity among all methods exhibited comparable sequencing performance to extract obtained through Method BL, which yielded extract with high DV200 value. This study suggests that the DV200 and RNA yield are both reliable pre-analytic metrics in determining a suitable method for successful transcriptome sequencing of FFPE samples and have important implications for future studies exploring transcriptome sequencing of FFPE cardiac specimens.
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Project description:Comparative Evaluation of DNA Extraction Methods from Feces of Multiple Host Species for Downstream Next-Generation Sequencing
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Project description:In the study of tumor genetics, formalin-fixed paraffin-embedded (FFPE) tumors are the most readily available tissue samples. While DNA derived from FFPE tissue has been validated for array comparative genomic hybridization (aCGH) application, the suitability of such fragmented DNA for single-nucleotide polymorphism (SNP) array analysis has not been well examined. Furthermore, whole-genome amplification (WGA) has been used in the study of small precursor lesions to produce sufficient amount of DNA for aCGH analysis. It is unclear whether the same approach can be extended to SNP analysis. In this study, we examined the utility and limitations of genotyping platform performed on whole-genome amplified DNA from FFPE tumor samples for both copy number and SNP analyses. We analyzed the results obtained using DNA derived from matched FFPE and frozen tissue samples on Affymetrix 250K Nsp SNP array. Two widely used WGA methods, Qiagen (isothermal protocol) and Sigma (thermocycling protocol), were used to determine how WGA methods affect the results. We found that the use of DNA derived from FFPE tumors (without or with WGA) for high-resolution SNP array application can produce a significant amount of false positive and false negative findings. While some of these misinterpretations appear to cluster in genomic regions with high or low GC contents, the majority appears to occur randomly. Only large-scale chromosome LOH (>10Mb) can be reliably detected from FFPE tumor DNA samples (without or with WGA) but not smaller LOH or copy number alterations. Our findings here indicate a need for caution in SNP array data interpretation when using FFPE tumor-derived DNA, particularly with WGA.
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