Improving DNA extraction from ancient bones
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB10532 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR988478.fastq.gz | Fastqsanger.gz | |||
| ERR988479.fastq.gz | Fastqsanger.gz | |||
| ERR988480.fastq.gz | Fastqsanger.gz | |||
| ERR988481.fastq.gz | Fastqsanger.gz | |||
| ERR988482.fastq.gz | Fastqsanger.gz |
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Project description:A Minimally Destructive Protocol for DNA Extraction from Ancient Teeth
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Project description:Dental calculus is becoming a crucial material in the study of past populations, with increasing interest in its proteomic and genomic content. Here we suggest further development of protocol for analysis of ancient proteins and a combined approach for subsequent ancient DNA extraction. We tested the protocol on recent teeth. We then applied the optimised protocol to ancient teeth to limit the destruction of calculus, as it is a precious and irreplaceable source of dietary, microbiological, and ecological information in the archaeological context. Finally, the applicability of the protocol was proven on samples of ancient calculus.
2023-09-11 | PXD042668 | Pride
Project description:Poor DNA preservation is the most limiting factor in ancient genomic research. In the majority of ancient bones and teeth, endogenous DNA molecules represent a minor fraction of the whole DNA extract, rendering shot-gun sequencing inefficient for obtaining genomic data. Based on ancient human bone samples from temperate and tropical environments, we show that an EDTA-based enzymatic 'pre-digestion' of powdered bone increases the proportion of endogenous DNA several fold. By performing the pre-digestion step between 30?min and 6?hours on five bones, we observe an asymptotic increase in endogenous DNA content, with a 2.7-fold average increase reached at 1?hour. We repeat the experiment using a brief pre-digestion (15 or 30?mins) on 21 ancient bones and teeth from a variety of archaeological contexts and observe an improvement in 16 of these. We here advocate the implementation of a brief pre-digestion step as a standard procedure in ancient DNA extractions. Finally, we demonstrate on 14 ancient teeth that by targeting the outer layer of the roots we obtain up to 14 times more endogenous DNA than when using the inner dentine. Our presented methods are likely to increase the proportion of ancient samples that are suitable for genome-scale characterization.
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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:We compared 3 small RNA library prep kits (CleanTag, NEXTflex, QIAseq) and two RNA extraction methods (miRNeasy and MagnaZol) on plasma. We report that library preparation has a significant effect upon the miRNA profile detected, with QIAseq libraries exhibiting the least sequencing bias of the three library kits. RNA extraction methods also contribute, to a lesser extent, to the miRNA profile detected, with MagnaZol RNA extraction increasing the percentage of reads mapping to miRNAs and the number of individual miRNAs detected.
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Project description:three protein extraction methods were used for cotton leaves protein extraction, and LC-MS/MS analyses of total proteins obtained from cotton leaves.
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Project description:A comparative study of several methods for extraction of soil microbiome
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Project description:A comparative study of several methods for extraction of soil microbiome
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Project description:A simple and effective modified ethanol precipitation-based protocol is described for the preparation of DNA from ancient human bones. This method is fast and requires neither hazardous chemicals nor special devices. After the powdering and incubating of the bone samples Dextran Blue was added as a carrier for removing the PCR inhibitors with selective ethanol precipitation. This method could eliminate the time-consuming separate decalcification step, dialysis, application of centrifugation-driven microconcentrators and the second consecutive PCR amplification. The efficiency of this procedure was demonstrated on ten 500-1200-year-old human bones from four different Hungarian burial sites. A mitochondrial specific primer pair was used to obtain sequence information from the purified ancient DNA. The PCR amplification, after our DNA extraction protocol, was successful from each of the 10 bone samples investigated. The results demonstrate that extraction of DNA from ancient bone samples with this new approach increases the success rate of PCR amplification.
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Project description:Formalin-fixed paraffin-embedded tissues are suitable for proteomic and phosphoproteomic biomarker studies by data-independent acquisition mass spectrometry. The sample preparation method chosen influences the number, intensity, and reproducibility of identifications. By comparing the performance of four deparaffinisation and rehydration methods, we found that heptane and methanol quantified the most protein groups whilst the most widely used xylene and ethanol quantified the least. We then modified five extraction methods from the literature based on their diverse lysis and digestion conditions before evaluating their performance using rat organs of the kidney, leg muscle, lung, and testis. All methods performed well except for SP3, that struggled with tissue lysis. Heat n’ Beat was the fastest, had the highest digestion efficiency, quantified protein groups, and lowest coefficient of variation across replicates. Strap produced the highest peptide yield and SPEED produced a ultraviolet quantitation interference. TFE produced the highest phosphoprecursor intensity with a 10-15% higher enrichment efficiency. We demonstrate that the choice of extraction method has a larger impact on the phosphoproteome than the proteome. We conclude by providing recommendations for choosing the most appropriate method for different settings.
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