scholarly journals Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments

2017 ◽  
Author(s):  
Maria A. Nieves-Colón ◽  
Andrew T. Ozga ◽  
William J. Pestle ◽  
Andrea Cucina ◽  
Vera Tiesler ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Ancient Dna ◽  
Extraction Methods ◽  
Optimal Choice ◽  
Skeletal Remains ◽  
Tissue Type ◽  
Dna Fragments ◽  
Illumina Hiseq ◽  
Tropical Environments ◽  
Significant Difference

ABSTRACTObjectivesThe tropics harbor a large part of the world’s biodiversity and have a long history of human habitation. However, paleogenomics research in these climates has been constrained so far by poor ancient DNA yields. Here we compare the performance of two DNA extraction methods on ancient samples of teeth and petrous portions excavated from tropical and semitropical sites in Tanzania, Mexico, and Puerto Rico (N=12).Materials and MethodsAll samples were extracted twice, built into double-stranded sequencing libraries, and shotgun sequenced on the Illumina HiSeq 2500. The first extraction protocol, Method D, was previously designed for recovery of ultrashort DNA fragments from skeletal remains. The second, Method H, modifies the first by adding an initial EDTA wash and an extended digestion and decalcification step.ResultsNo significant difference was found in overall ancient DNA yields or post-mortem damage patterns recovered from samples extracted with either method, irrespective of tissue type. However, Method H samples had higher endogenous content and more mapped reads after quality-filtering, but also higher clonality. In contrast, samples extracted with Method D had shorter average DNA fragments.DiscussionBoth methods successfully recovered endogenous ancient DNA. But, since surviving DNA in ancient or historic remains from tropical contexts is extremely fragmented, our results suggest that Method D is the optimal choice for working with samples from warm and humid environments. Additional optimization of extraction conditions and further testing of Method H with different types of samples may allow for improvement of this protocol in the future.

scholarly journals Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 146
Author(s):  
Catarina Xavier ◽  
Mayra Eduardoff ◽  
Barbara Bertoglio ◽  
Christina Amory ◽  
Cordula Berger ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Ancient Dna ◽  
Extraction Method ◽  
Fragment Size ◽  
Molecular Genetic ◽  
Extraction Methods ◽  
Degraded Dna ◽  
Size Analysis ◽  
Dna Fragments ◽  
Dna Amount

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

scholarly journals Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments

2018 ◽  
Vol 166 (4) ◽  
pp. 824-836 ◽  
Author(s):  
Maria A. Nieves-Colón ◽  
Andrew T. Ozga ◽  
William J. Pestle ◽  
Andrea Cucina ◽  
Vera Tiesler ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Ancient Dna ◽  
Skeletal Remains ◽  
Tropical Environments

scholarly journals Comparason of extraction methods for PCR detection of Burkholderia cepacia complex (BCC) from cystic fibrosis patients

Open Medicine ◽  
2008 ◽  
Vol 3 (2) ◽  
pp. 157-162
Author(s):  
Koray Ergunay ◽  
Pinar Yurdakul ◽  
Burcin Sener ◽  
Ugur Ozcelik ◽  
Erdem Karabulut ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Burkholderia Cepacia ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Extraction Methods ◽  
Rapid Identification ◽  
Burkholderia Cepacia Complex ◽  
Assay Sensitivity ◽  
Spin Column ◽  
Significant Difference

AbstractDirect detection of Burkholderia cepacia complex (BCC) and its genomovars from sputum by molecular tests emerges as a method for rapid identification. In this study, four DNA extraction methods were evaluated for the identification for BCC from sputum of CF patients. Sputa from 28 CF patients were aliquoted and spiked with BCC reference strain. Boiling, phenol-chloroform, CTAB methods and a commercial spin column kit was used for DNA extraction. Total DNA yields were determined by spectrophotometry and single-round recA PCR was used for detection of BCC. No significant difference was observed in DNA yields from different extraction methods. Lower limit of detection for recA PCR was determined as 106 cfu/ml. Amplification was observed in 7/16 (43.7%) of sputa for boiling, 8/16 (50%) of sputa for CTAB and 13/16 (81.2%) of sputa for phenol-chloroform method and spin column kit in the assay sensitivity range determined in the study. Phenol-chloroform and commercial spin column kit were found to be better suited for DNA purification from sputum of CF patients for BCC identification. Diagnostic impact of single-round recA PCR directly from sputum was limited to chronically-infected patients.

Simple and highly effective DNA extraction methods from old skeletal remains using silica columns

2010 ◽  
Vol 4 (5) ◽  
pp. 275-280 ◽  
Author(s):  
Hwan Young Lee ◽  
Myung Jin Park ◽  
Na Young Kim ◽  
Jeong Eun Sim ◽  
Woo Ick Yang ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Methods ◽  
Skeletal Remains ◽  
Highly Effective ◽  
Dna Extraction Methods

scholarly journals Comparative Analysis of Sample Extraction and Library Construction for Shotgun Metagenomics

2020 ◽  
Vol 14 ◽  
pp. 117793222091545
Author(s):  
Zonghui Peng ◽  
Xiaolong Zhu ◽  
Zhijiao Wang ◽  
Xianting Yan ◽  
Guangbiao Wang ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Methods ◽  
Shannon Index ◽  
Library Preparation ◽  
Shotgun Metagenomics ◽  
Library Construction ◽  
Freeze Thaw ◽  
Significant Difference ◽  
Dna Extraction Methods ◽  
Library Preparation Protocol

Human fecal specimens, serve as important materials, are widely used in the field of microbiome research, in which inconsistent results have been a pressing issue. The possible attribute factors have been proposed including the specimen status after preservation, extracted DNA quality, library preparation protocol, and sample DNA input. In this study, quality comparisons for shotgun metagenomics sequencing were performed between 2 DNA extraction methods for fresh and freeze-thaw samples, 2 library preparation protocols, and various sample inputs. The results indicate that Mag-Bind® Universal Metagenomics Kit (OM) outperformed DNeasy PowerSoil Kit (QP) with a higher DNA quantity. Controlling on library preparation protocol, OM detected on-average more genes than QP. For library construction comparison by controlling on the same DNA sample, KAPA Hyper Prep Kit (KH) outperformed the TruePrep DNA Library Prep Kit V2 (TP) with the higher number of detected genes number and Shannon index. No significant differences were found in taxonomy between 2 library preparation protocols using the fresh, freeze-thaw and mock community samples. No significant difference was observed between 250 and 50 ng DNA inputs for library preparation on both fresh and freeze-thaw samples. Through the preliminary study, a combined protocol is recommended for performing metagenomics studies, by using OM method plus KH protocol as well as suitable DNA quantity on either fresh or freeze-thaw samples. Our findings provide clues for potential variations from various DNA extraction methods, library protocols, and sample DNA inputs, which are critical for consistent and comprehensive profiling of the human gut microbiome.

Comparing the performance of three ancient DNA extraction methods for high-throughput sequencing

2015 ◽  
Vol 16 (2) ◽  
pp. 459-469 ◽  
Author(s):  
Cristina Gamba ◽  
Kristian Hanghøj ◽  
Charleen Gaunitz ◽  
Ahmed H. Alfarhan ◽  
Saleh A. Alquraishi ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Ancient Dna ◽  
High Throughput Sequencing ◽  
Extraction Methods ◽  
Dna Extraction Methods

scholarly journals Ancient mitochondrial genomes recovered from small vertebrate bones through minimally destructive DNA extraction: phylogeography of the New Zealand gecko genus Hoplodactylus.

2021 ◽  
Author(s):  
Lachie Scarsbrook ◽  
Alexander Verry ◽  
Kerry Walton ◽  
Rodney Hitchmough ◽  
Nic Rawlence
Keyword(s):  
New Zealand ◽  
Dna Extraction ◽  
Ancient Dna ◽  
Phylogenetic Analyses ◽  
Relative Size ◽  
Distinct Species ◽  
Glacial Refugia ◽  
Extraction Methods ◽  
Mitochondrial Genomes ◽  
Small Vertebrate

Methodological and technological improvements are continually revolutionizing the field of ancient DNA. Most ancient DNA extraction methods require the partial (or complete) destruction of finite museum specimens, which disproportionately impacts small or fragmentary subfossil remains, and future analyses. We present a minimally destructive ancient DNA extraction method optimized for small vertebrate remains. We applied these methods to detect lost mainland genetic diversity in the large New Zealand diplodactylid gecko genus Hoplodactylus, which is presently restricted to predator-free island sanctuaries. We present the first mitochondrial genomes for New Zealand diplodactylid geckos, recovered from 19 modern, six historic/archival (1898 to 2011) and 16 Holocene Hoplodactylus duvaucelii sensu latu specimens, and one modern Woodworthia sp. specimen. No obvious damage was observed in post-extraction micro-CT reconstructions. All ‘large gecko’ specimens examined from extinct populations were found to be conspecific with extant Hoplodactylus species, suggesting their large relative size evolved only once in the New Zealand diplodactylid radiation. Phylogenetic analyses of Hoplodactylus samples recovered two genetically (and morphologically) distinct North and South Island clades, probably corresponding to distinct species. Finer phylogeographic structuring within Hoplodactylus spp. highlighted the impacts of Late-Cenozoic biogeographic barriers, including the opening and closure of Pliocene marine straits, fluctuations in size and suitability of glacial refugia, and eustatic sea-level change. Recent mainland extinction obscured these signals from the modern tissue derived data. These results highlight the utility of minimally destructive DNA extraction in genomic analyses of less well studied small vertebrate taxa, and the conservation of natural history collections.

scholarly journals Ancient DNA Methods Improve Forensic DNA Profiling of Korean War and World War II Unknowns

Genes ◽  
2022 ◽  
Vol 13 (1) ◽  
pp. 129
Author(s):  
Elena I. Zavala ◽  
Jacqueline Tyler Thomas ◽  
Kimberly Sturk-Andreaggi ◽  
Jennifer Daniels-Higginbotham ◽  
Kerriann K. Meyers ◽  
...  
Keyword(s):  
World War Ii ◽  
Dna Extraction ◽  
Ancient Dna ◽  
Korean War ◽  
Skeletal Remains ◽  
Dna Profiling ◽  
Library Preparation ◽  
Forensic Dna ◽  
World War ◽  
Preparation Methods

The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service members. However, highly degraded or chemically treated skeletal remains often fail to provide usable DNA profiles, even with sensitive mitochondrial (mt) DNA capture and MPS methods. In parallel, the ancient DNA field has developed workflows specifically for degraded DNA, resulting in the successful recovery of nuclear DNA and mtDNA from skeletal remains as well as sediment over 100,000 years old. In this study we use a set of disinterred skeletal remains from the Korean War and World War II to test if ancient DNA extraction and library preparation methods improve forensic DNA profiling. We identified an ancient DNA extraction protocol that resulted in the recovery of significantly more human mtDNA fragments than protocols previously used in casework. In addition, utilizing single-stranded rather than double-stranded library preparation resulted in increased attainment of reportable mtDNA profiles. This study emphasizes that the combination of ancient DNA extraction and library preparation methods evaluated here increases the success rate of DNA profiling, and likelihood of identifying historical remains.

scholarly journals Comparing two automated methods of DNA extraction from degraded skeletal remains

2020 ◽  
Author(s):  
Linda Rubinstein
Keyword(s):  
Dna Extraction ◽  
Extraction Methods ◽  
Positive Control ◽  
Skeletal Remains ◽  
Variable Environment ◽  
Dna Profile ◽  
Preparation Step ◽  
Dna Profiles ◽  
Skeletal Samples ◽  
Automated Methods

AbstractDNA extraction from degraded skeletal samples is often particularly challenging. The difficulty derives from the fact that variable environment has significant effect on DNA preservation. During the years 2002-2015 unidentified degraded skeletal remains were accumulated at our institute, National Institute of Forensic Medicine (NIFM), most of them with none or partial DNA profile.As new methods rapidly emerge, we revisited the samples with partial DNA profiles. We have chosen to use these samples to compare two automated methods: Prepfiler Express BTA (Applied Biosystems) and QIAcube (Quiagen), in hope of acquiring a more complete DNA profile and eventually even make new identifications. In both methods a preparation step is required, after which the samples undergo automatic DNA extraction.The two protocols are based on different extraction methods. Fresh or non-problematic bone samples as the positive control gave the same results in both methods. In the degraded skeletal samples, the results were significantly better using the QIAcube method.

scholarly journals The influence of sample quantity and lysis parameters on the success of ancient DNA extraction from skeletal remains

BioTechniques ◽  
2021 ◽  
Author(s):  
Alina Euskirchen ◽  
Larissa Hartmann ◽  
Janine Mazanec ◽  
Patrick Wittmeier ◽  
Susanne Hummel
Keyword(s):  
Mitochondrial Dna ◽  
Sample Preparation ◽  
Dna Extraction ◽  
Ancient Dna ◽  
Skeletal Remains ◽  
Sample Material ◽  
Chromosomal Dna ◽  
High Quality ◽  
Dna Analyses ◽  
Lysis Time

DNA extraction is of utmost importance in archaeobiology, as it determines the success of further DNA analyses. This study concentrates on the success of ancient DNA extraction using silica spin columns and PCR-based analysis from archaeological skeletal material and investigates the influence of sample quantity, lysis time and lysis temperature during sample preparation. The results show that lysis times ranging from 2 to 48 h are suitable, and that lysis should be carried out at a constant temperature of 56°C. Concerning sample quantity, 10 mg for mitochondrial DNA and 50 mg for chromosomal DNA are sufficient for high quality analyses. Thus invaluable sample material can be saved, and time of sample preparation can be reduced considerably.

Sign in / Sign up

Export Citation Format

Share Document