Experimental approaches for ancient DNA extraction and sample preparation for next generation sequencing in ultra-clean
- PDF / 1,821,437 Bytes
- 9 Pages / 595.276 x 790.866 pts Page_size
- 74 Downloads / 199 Views
METHODS AND RESOURCES ARTICLE
Experimental approaches for ancient DNA extraction and sample preparation for next generation sequencing in ultra-clean conditions A. D. Matsvay1,2 · I. E. Alborova1 · E. V. Pimkina2 · M. L. Markelov2 · K. Khafizov1,2 · K. K. Mustafin1 Received: 15 June 2017 / Accepted: 23 February 2018 © Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract In this study, we aimed to develop an approach for genetic material extraction and sample preparation of ancient DNA for next generation sequencing that minimizes intra-laboratory contamination. A special module system consisting of four sterile gloveboxes connected by antechambers, which maintain a high purity atmosphere and allow conducting all stages of sample preparation in an isolated clean environment, starting with the processing of archaeological material, was developed. Furthermore, a fast and cost-effective double-stranded library preparation protocol for massive parallel sequencing was developed using non-standard modified sequencing adaptors, which enabled obtaining a library of fragments from extremely low amounts of starting degraded material. Bioinformatics data processing showed that the distribution of substitution frequencies in a subset of reads, mapped to the human reference genome, completely coincides with the pattern of postmortem modifications expected when the double-stranded library preparation method is employed. This is a strong evidence for the endogenous origin of the sequenced fragments of the human genome. Thus, the results of ancient sample and negative control sample sequencing, indicated the absence of visible contamination during sample preparation procedure. Keywords Ancient DNA · Bioinformatics · Human genetics · Next generation sequencing · DNA extraction
Introduction The field of study of ancient DNA (aDNA) extracted from museum specimens and archaeological and paleontological samples began about 30 years ago (Paabo 1989). The main aim of the studies at that time was to determine the nucleotide sequence (so-called sequencing) of short mitochondrial DNA (mtDNA) fragments. The development of new DNA extraction techniques and subsequent bioinformatics analysis has made it possible to reconstruct the complete mitochondrial genomes of ancient organisms (Krause et al. 2007). These techniques have helped researchers to draw conclusions about the organisms that became extinct tens to hundreds of thousands years ago as well as also about the population structure of those species. The development of * K. Khafizov [email protected] 1
Moscow Institute of Physics and Technology, Institutsky pereulok 9, Dolgoprudny, Moscow Region, Russian Federation 141701
Central Research Institute of Epidemiology, Novogireevskaya Str. 3A, Moscow, Russian Federation 111123
2
next generation sequencing (NGS) technologies has made it feasible to determine the nucleotide sequences of whole nuclear genomes, which, in turn, has allowed conclusions to be drawn about the phenotypic traits of organisms. Nowad
Data Loading...
