A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44 000 year-old rodent remains from Northern Africa.

We present a cost-effective metabarcoding approach, aMPlex Torrent, which relies on an improved multiplex PCR adapted to highly degraded DNA, combining barcoding and next-generation sequencing to simultaneously analyse many heterogeneous samples. We demonstrate the strength of these improvements by generating a phylochronology through the genotyping of ancient rodent remains from a Moroccan cave whose stratigraphy covers the last 120 000 years. Rodents are important for epidemiology, agronomy and ecological investigations and can act as bioindicators for human- and/or climate-induced environmental changes. Efficient and reliable genotyping of ancient rodent remains has the potential to deliver valuable phylogenetic and paleoecological information. The analysis of multiple ancient skeletal remains of very small size with poor DNA preservation, however, requires a sensitive high-throughput method to generate sufficient data. We show this approach to be particularly adapted at accessing this otherwise difficult taxonomic and genetic resource. As a highly scalable, lower cost and less labour-intensive alternative to targeted sequence capture approaches, we propose the aMPlex Torrent strategy to be a useful tool for the genetic analysis of multiple degraded samples in studies involving ecology, archaeology, conservation and evolutionary biology.

Phylogeography, genetic structure and population divergence time of cheetahs in Africa and Asia: evidence for long-term geographic isolates.

The cheetah (Acinonyx jubatus) has been described as a species with low levels of genetic variation. This has been suggested to be the consequence of a demographic bottleneck 10 000-12 000 years ago (ya) and also led to the assumption that only small genetic differences exist between the described subspecies. However, analysing mitochondrial DNA and microsatellites in cheetah samples from most of the historic range of the species we found relatively deep phylogeographic breaks between some of the investigated populations, and most of the methods assessed divergence time estimates predating the postulated bottleneck. Mitochondrial DNA monophyly and overall levels of genetic differentiation support the distinctiveness of Northern-East African cheetahs (Acinonyx jubatus soemmeringii). Moreover, combining archaeozoological and contemporary samples, we show that Asiatic cheetahs (Acinonyx jubatus venaticus) are unambiguously separated from African subspecies. Divergence time estimates from mitochondrial and nuclear data place the split between Asiatic and Southern African cheetahs (Acinonyx jubatus jubatus) at 32 000-67 000 ya using an average mammalian microsatellite mutation rate and at 4700-44 000 ya employing human microsatellite mutation rates. Cheetahs are vulnerable to extinction globally and critically endangered in their Asiatic range, where the last 70-110 individuals survive only in Iran. We demonstrate that these extant Iranian cheetahs are an autochthonous monophyletic population and the last representatives of the Asiatic subspecies A. j. venaticus. We advocate that conservation strategies should consider the uncovered independent evolutionary histories of Asiatic and African cheetahs, as well as among some African subspecies. This would facilitate the dual conservation priorities of maintaining locally adapted ecotypes and genetic diversity.

A compositional map of human chromosome band Xq28.

The molar fractions of guanine plus cytosine (GC) in DNA were determined for 36 yeast artificial chromosomes (YACs) which almost completely cover human chromosome band Xq28, a terminal reverse band, corresponding to about 8 Mb of DNA. This allowed the construction of the most complete compositional map to date of a chromosomal band; three regions were observed: (i) a proximal 3.5-Mb region formed by GC-poor L and GC-rich H1 isochores; (ii) a middle 2,2-Mb region essentially formed by a GC-rich H2 isochore and a very GC-rich H3 isochore separated by a GC-poor L isochore, YACs from this region being characterized by a striking compositional heterogeneity and instability; and (iii) a distal 1.3-Mb region exclusively formed by GC-poor L isochores. Gene and CpG island concentrations increased with the GC levels of the isochores, as expected. Xq28 exemplifies a subset of reverse bands which are different from the two other subsets, namely from telomeric bands, which are characterized by specific cytogenetic properties and by the predominance of H2 and H3 isochores, and from the majority of reverse bands, which do not contain H2 and H3 isochores.

A radiation hybrid map of human chromosome 18.

A human x Chinese hamster (CH) somatic cell hybrid subclone deficient in HPRT and containing only human chromosome 18 was irradiated with 7000 rad and fused to a thymidine kinase deficient CH cell line. Radiation-rescued hybrid cell lines, selected in HAT medium, were analyzed for human DNA with human interspersed-repeat sequence primers. Size and number of human chromosome fragments retained in a subset of hybrids were determined by FISH. A panel of 98 radiation hybrids (RH) was selected and analyzed for 90 chromosome 18-specific STSs by PCR, and for the D18Z1 centromeric marker by Southern blotting. STSs were developed from previously mapped RFLP loci and from published sequences. In addition, 32 novel STSs were generated from an 18-specific lambda library and from 18-specific YACs previously localized to chromosome bands by FISH. Marker retention frequency varied from 8-65% with an average of 24%. In selected RH the STS typing data were correlated with the chromosome 18 regions retained using 'reverse FISH' of IRS-PCR products from the RH to normal metaphase chromosomes. The order and intermarker distances of loci were determined using two-point and multipoint maximum likelihood methods. The resulting RH map covers most of chromosome 18 with four groups of tightly linked markers and three regions of loosely linked markers, one around the centromere and two on the long arm. More than a third of the markers are polymorphic and allow integration with the linkage map. This RH map provides a framework for establishing a clone contig of the entire chromosome 18.

Genomic distribution and transcription of solitary HERV-K LTRs.

The human genome contains a family of endogenous retroviruses, HERV-K, with sequence homology to the B-type mouse mammary tumor virus. We have now identified a single HERV-K LTR within the C-type-related human retroviral element S71. The HERV-K LTR is located in the antisense direction between the S71 gag and the pol gene, replacing the 5' half of S71 pol. A number of HERV-K LTR-related cDNA clones were detected by screening various human cDNA libraries with an S71 HERV-K LTR probe, indicating abundant transcription of HERV-K-related LTRs in human tissues. Sequence analysis of four cDNA clones revealed LTR sequences with a nucleotide identity of 70 to 90% with HERV-K10 LTR. Some HERV-K-related LTR sequences contain potential short open reading frames. The analyzed cDNA clones do not harbor any retroviral sequences other than those related to HERV-K LTRs. However, most of the solitary LTRs were found to be coexpressed with cellular sequences. Transcription of these LTRs is probably directed by external cellular promoters. We show that HERV-KLTR-like sequences entered the primate genome about 33-40 million years ago. We estimate the human genome to contain about 25,000 copies of HERV-K-related LTRs, which are distributed over most human chromosomes in an irregular manner.

The repair of double-strand breaks and S1 nuclease-sensitive sites can be monitored chromosome-specifically in Saccharomyces cerevisiae using pulse-field gel electrophoresis.

Repair under non-growth conditions of DNA double-stranded breaks (DSBs) and S1 nuclease-sensitive sites (SSSs; e.g. DNA damage which is processed by in vitro treatment with S1 nuclease to DSBs) induced by [60Co]-gamma-rays (200 Gy; anoxic conditions) was monitored in a diploid repair-competent strain of Saccharomyces cerevisiae. We used pulsed-field gel electrophoresis (PFGE), which allows the separation of chromosome-sized yeast DNA molecules, to determine the number of DSBs and SSSs in individual chromosome species of yeast. Our results indicate that SSSs which have been regarded as clusters of base damage in opposite DNA strands are repaired efficiently in a repair-proficient diploid strain of yeast. The time course of SSS repair is comparable to the one of DSB repair, indicating similarities in the molecular mechanism. Both types of repair kinetics are different for different chromosome species.

Repair of gamma ray-induced S1 nuclease hypersensitive sites in yeast depends on homologous mitotic recombination and a RAD18-dependent function.

Repair under non-growth conditions of DNA double-strand breaks (DSB) and chromatin sites sensitive to S1 endonuclease (SSS) induced by 60Cobalt-gamma rays were monitored in repair-competent and deficient strains of Saccharomyces cerevisiae by pulsed field gel-electrophoresis. In stationary-phase cells of a repair-competent RAD diploid, and an excision-deficient rad3-2 diploid, SSS are repaired as efficiently as DSB, whereas in a repair-competent RAD haploid, and a rad 50-1 diploid, neither SSS nor DSB are repaired. The rad18-2 diploid repairs DSB well but is defective in SSS repair. Obviously, SSS repair in yeast chromatin, like DSB repair, depends on recombination, but unlike DSB repair depends additionally on RAD18 function.

Chromosome-specific identification and quantification of S1 nuclease-sensitive sites in yeast chromatin by pulsed-field gel electrophoresis.

Sites that are sensitive to the single-strand-specific endonuclease S1 ('S1-sensitive sites', SSS) occur in native chromatin and, like DNA double-stranded breaks (DSB), they are induced by DNA-damaging agents, such as ionizing radiation. We have developed a method to quantify SSS and DSB in yeast chromatin by using pulsed-field gel electrophoresis (PFGE) to separate the intact chromosomal-length DNA molecules from the lower molecular-weight broken ones. Direct evaluation of the photonegatives of the ethidium bromide-stained gels by laser densitometry enabled us to calculate the numbers of DSB and SSS per DNA molecule. These numbers were determined from the bulk of the non-separated genomic DNA of yeast, corresponding to a single band in the PFGE (pulse time 10 seconds), and in each of the eight largest yeast chromosomes, corresponding to distinct bands in the PFGE gels (pulse time 50 seconds), which were not superimposed by the smear of the broken, low molecular-weight DNA. Furthermore, the induction of DSB and SSS in a specific chromosome (circular chromosome III) was determined by Southern hybridization of the PFGE gels with a suitable centromere probe, followed by densitometry of the autoradiographs. Our method allows the chromosome-specific monitoring of DSB and all those DNA structures that are processed either in vivo or in vitro into DSB and which may not be distributed randomly within the genome.