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Comparison of three methods of DNA extraction from human bones with different degrees of degradation.

ABSTRACT: There is a necessity for deceased identification as a result of many accidents and sometimes bones are the only accessible source of DNA. So far, a universal method that allows for extraction of DNA from materials at different stages of degradation does not exist. The aims of this study were: the comparison of three methods of DNA extraction from bones with different degree of degradation and an evaluation of the usefulness of these methods in forensic genetics. The efficiency of DNA extraction, the degree of extract contamination by polymerase chain reaction (PCR) inhibitors and the possibility of determining the STR loci profile were especially being compared. Nuclear DNA from bones at different states of degradation was isolated using three methods: classical, organic phenol-chloroform extraction, DNA extraction from crystal aggregates and extraction by total demineralisation. Total demineralisation is the best method for most cases of DNA extraction from bones, although it does not provide pure DNA. DNA extraction from aggregates removes inhibitors much better and is also a good method of choice when identity determination of exhumed remains is necessary. In the case of not buried bones (remains found outside) total demineralisation or phenol-chloroform protocols are more efficient for successful DNA extraction.

SUBMITTER: Jakubowska J

PROVIDER: S-EPMC3254873 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Comparison of three methods of DNA extraction from human bones with different degrees of degradation.

Jakubowska Joanna J Maciejewska Agnieszka A Pawłowski Ryszard R

International journal of legal medicine 20110630 1

There is a necessity for deceased identification as a result of many accidents and sometimes bones are the only accessible source of DNA. So far, a universal method that allows for extraction of DNA from materials at different stages of degradation does not exist. The aims of this study were: the comparison of three methods of DNA extraction from bones with different degree of degradation and an evaluation of the usefulness of these methods in forensic genetics. The efficiency of DNA extraction, ...[more]

PMID: 21717154

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Project description:BackgroundDNA isolation and PCR amplification from molluscan taxa is considered as problematic because polysaccharides in tissue and mucus presumably co-precipitate with the DNA and inhibit the activity of DNA polymerase. In the present study we tested two common extraction methods on specimens from the mollusc collection of the Natural History Museum Vienna (NHMW). We analysed a broad variety of taxa covering a large temporal span (acquisition years 1877 to 1999), which distinguishes our study from previous ones where mostly fresh material was used. We also took other factors into account: effects of sample age, effects of formaldehyde treatment and taxon-specific problems. We used several primer combinations to amplify amplicons of different lengths of two mitochondrial genes: cytochrome c oxidase subunit 1 (COI) and 16S rRNA gene (16S).ResultsOverall PCR success was 43 % in the 576 extractions (including all primer combinations). The smallest amplicon (~240 bp) showed the best results (49 % positive reactions), followed by the 400 bp amplicon (40.5 %). Both short sections yielded significantly better results than the 700 bp long amplicon (27 %). Comparatively, the Gen-ial-First, All-tissue DNA-Kit-extraction method performed significantly better than Promega-Tissue and Hair Extraction Kit. Generally, PCR success is age-dependent. Nonetheless, we were able to obtain the longest amplicon even from 137-year-old material. Importantly, formaldehyde traces did not totally inhibit amplification success, although very high concentrations did.ConclusionsMuseum material has gained importance for DNA analysis in recent years, especially for DNA barcoding projects. In some cases, however, the amplification of the standard barcoding region (partial sequence of the COI) is problematic with old material. Our study clearly shows that the COI barcoding region could be amplified in up to 49 % of PCRs (varying with amplicon length), which is, for museum samples, quite a high percentage. The difference between extraction methods was minimal and we recommend using an established kit for a first attempt because experience and routine in handling might be more important than slight performance differences of the various kits. Finally, we identify fixation, storage, sample conservation and documentation of the specimens' history rather than the DNA extraction method to be the most crucial factors for PCR success.

Project description:When studying the microbiome using next-generation sequencing, the DNA extraction method, sequencing procedures, and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity, and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with a maximum 3-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for observed richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs, and variation induced by DNA extraction methods was lower than intersubject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally, with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we again demonstrate the importance of including negative controls when analyzing low-bacterial-biomass samples.IMPORTANCE Method choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiota studies. Our methods were applied not only to commonly studied samples for microbiota analysis, e.g., feces, but also to more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g., urine and tumor biopsy specimens) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiota studies, especially when low bacterial biomass is expected.

Project description:BackgroundAutomated ICD coding on medical texts via machine learning has been a hot topic. Related studies from medical field heavily relies on conventional bag-of-words (BoW) as the feature extraction method, and do not commonly use more complicated methods, such as word2vec (W2V) and large pretrained models like BERT. This study aimed at uncovering the most effective feature extraction methods for coding models by comparing BoW, W2V and BERT variants.MethodsWe experimented with a Chinese dataset from Fuwai Hospital, which contains 6947 records and 1532 unique ICD codes, and a public Spanish dataset, which contains 1000 records and 2557 unique ICD codes. We designed coding tasks with different code frequency thresholds (denoted as [Formula: see text]), with a lower threshold indicating a more complex task. Using traditional classifiers, we compared BoW, W2V and BERT variants on accomplishing these coding tasks.ResultsWhen [Formula: see text] was equal to or greater than 140 for Fuwai dataset, and 60 for the Spanish dataset, the BERT variants with the whole network fine-tuned was the best method, leading to a Micro-F1 of 93.9% for Fuwai data when [Formula: see text], and a Micro-F1 of 85.41% for the Spanish dataset when [Formula: see text]. When [Formula: see text] fell below 140 for Fuwai dataset, and 60 for the Spanish dataset, BoW turned out to be the best, leading to a Micro-F1 of 83% for Fuwai dataset when [Formula: see text], and a Micro-F1 of 39.1% for the Spanish dataset when [Formula: see text]. Our experiments also showed that both the BERT variants and BoW possessed good interpretability, which is important for medical applications of coding models.ConclusionsThis study shed light on building promising machine learning models for automated ICD coding by revealing the most effective feature extraction methods. Concretely, our results indicated that fine-tuning the whole network of the BERT variants was the optimal method for tasks covering only frequent codes, especially codes that represented unspecified diseases, while BoW was the best for tasks involving both frequent and infrequent codes. The frequency threshold where the best-performing method varied differed between different datasets due to factors like language and codeset.

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Comparison of three methods of DNA extraction from human bones with different degrees of degradation.

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Comparison of three methods of DNA extraction from human bones with different degrees of degradation.

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