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      Methodology for Y Chromosome Capture: A complete genome sequence of  Y chromosome using flow cytometry, laser microdissection and magnetic streptavidin-beads

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          Abstract

          This study is a comparison of the efficiency of three technologies used for Y chromosome capture and the next-generation sequencing (NGS) technologies applied for determining its whole sequence. Our main findings disclose that streptavidin–biotin magnetic particle-based capture methodology offers better and a deeper sequence coverage for Y chromosome capture, compared to chromosome sorting and microdissection procedures. Moreover, this methodology is less time consuming and the most selective for capturing only Y chromosomal material, in contrast with other methodologies that result in considerable background material from other, non-targeted chromosomes. NGS results compared between two platforms, NextSeq 500 and SOLID 5500xl, produce the same coverage results. This is the first study to explore a methodological comparison of Y chromosome capture and genetic analysis. Our results indicate an improved strategy for Y chromosome research with applications in several scientific fields where this chromosome plays an important role, such as forensics, medical sciences, molecular anthropology and cancer sciences.

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          Temporal Patterns of Nucleotide Misincorporations and DNA Fragmentation in Ancient DNA

          DNA that survives in museum specimens, bones and other tissues recovered by archaeologists is invariably fragmented and chemically modified. The extent to which such modifications accumulate over time is largely unknown but could potentially be used to differentiate between endogenous old DNA and present-day DNA contaminating specimens and experiments. Here we examine mitochondrial DNA sequences from tissue remains that vary in age between 18 and 60,000 years with respect to three molecular features: fragment length, base composition at strand breaks, and apparent C to T substitutions. We find that fragment length does not decrease consistently over time and that strand breaks occur preferentially before purine residues by what may be at least two different molecular mechanisms that are not yet understood. In contrast, the frequency of apparent C to T substitutions towards the 5′-ends of molecules tends to increase over time. These nucleotide misincorporations are thus a useful tool to distinguish recent from ancient DNA sources in specimens that have not been subjected to unusual or harsh treatments.
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            Strict evolutionary conservation followed rapid gene loss on human and rhesus Y chromosomes

            The human X and Y chromosomes evolved from an ordinary pair of autosomes during the past 200–300 million years 1–3 . Due to genetic decay, the human MSY (male-specific region of Y chromosome) retains only three percent of the ancestral autosomes’ genes 4,5 . This evolutionary decay was driven by a series of five “stratification” events. Each event suppressed X-Y crossing over within a chromosome segment or “stratum”, incorporated that segment into the MSY, and subjected its genes to the erosive forces that attend the absence of crossing over 2,6 . The last of these events occurred 30 million years ago (mya), or 5 million years before the human and Old World monkey (OWM) lineages diverged. Although speculation abounds regarding ongoing decay and looming extinction of the human Y chromosome 7–10 , remarkably little is known about how many MSY genes were lost in the human lineage in the 25 million years that have followed its separation from the OWM lineage. To explore this question, we sequenced the MSY of the rhesus macaque, an OWM, and compared it to the human MSY. We discovered that, during the last 25 million years, MSY gene loss in the human lineage was limited to the youngest stratum (stratum 5), which comprises three percent of the human MSY. Within the older strata, which collectively comprise the bulk of the human MSY, gene loss evidently ceased more than 25 mya. Likewise, the rhesus MSY has not lost any older genes (from strata 1–4) during the past 25 million years, despite major structural differences from the human MSY. The rhesus MSY is simpler, with few amplified gene families or palindromes that might enable intrachromosomal recombination and repair. We present an empirical reconstruction of human MSY evolution in which each stratum transitioned from rapid, exponential loss of ancestral genes to strict conservation through purifying selection.
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              Centromere reference models for human chromosomes X and Y satellite arrays

              The human genome sequence remains incomplete, with multimegabase-sized gaps representing the endogenous centromeres and other heterochromatic regions. Available sequence-based studies within these sites in the genome have demonstrated a role in centromere function and chromosome pairing, necessary to ensure proper chromosome segregation during cell division. A common genomic feature of these regions is the enrichment of long arrays of near-identical tandem repeats, known as satellite DNAs, which offer a limited number of variant sites to differentiate individual repeat copies across millions of bases. This substantial sequence homogeneity challenges available assembly strategies and, as a result, centromeric regions are omitted from ongoing genomic studies. To address this problem, we utilize monomer sequence and ordering information obtained from whole-genome shotgun reads to model two haploid human satellite arrays on chromosomes X and Y, resulting in an initial characterization of 3.83 Mb of centromeric DNA within an individual genome. To further expand the utility of each centromeric reference sequence model, we evaluate sites within the arrays for short-read mappability and chromosome specificity. Because satellite DNAs evolve in a concerted manner, we use these centromeric assemblies to assess the extent of sequence variation among 366 individuals from distinct human populations. We thus identify two satellite array variants in both X and Y centromeres, as determined by array length and sequence composition. This study provides an initial sequence characterization of a regional centromere and establishes a foundation to extend genomic characterization to these sites as well as to other repeat-rich regions within complex genomes.
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                Author and article information

                Contributors
                mjesusac@ugr.es
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                21 June 2018
                21 June 2018
                2018
                : 8
                : 9436
                Affiliations
                [1 ]ISNI 0000000121678994, GRID grid.4489.1, GENYO (Pfizer-University of Granada-Andalusian Government Centre for Genomics and Oncological Research), Av. Ilustracion, ; 114 – PTS – 18016 Granada, Spain
                [2 ]ISNI 0000 0001 2300 0941, GRID grid.6530.0, Centro di Antropologia Molecolare per lo Studio del DNA Antico, Dipartimento di Biologia, , Università degli Studi di Roma“Tor Vergata”, via della Ricerca Scientifica n. 1, ; 00173 Rome, Italy
                [3 ]ISNI 0000000121678994, GRID grid.4489.1, University of Granada. Dept. of Biochemistry & Molecular Biology III - Faculty of Medicine - PTS, ; 18016 Granada, Spain
                [4 ]ISNI 0000000121678994, GRID grid.4489.1, University of Granada. Laboratory of Genetic Identification, Legal Medicine and Toxicology Department, Faculty of Medicine -PTS, ; 18016 Granada, Spain
                Author information
                http://orcid.org/0000-0002-5492-9355
                Article
                27819
                10.1038/s41598-018-27819-x
                6013464
                29930304
                14e71f92-a640-4e63-8b9c-fb7116c27cbe
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 2 June 2017
                : 1 June 2018
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