Exploring the evolution of archaic humans through sedimentary ancient DNA.

Recent success in the retrieval of nuclear DNA of ancient humans and animals from cave sediments paves the way for genome-wide studies of past populations directly from sediments. In three studies, nuclear genomes of different species were obtained from the sediments of multiple archeological caves and their genetic histories were revealed, including an unknown population replacement of Neanderthals from Estatuas cave in Spain, which was recovered using a new DNA capture approach. By extending sediments as a source of DNA beyond fossils, this breakthrough is of particular significance to the field of ancient human genomics, which brings about more possibilities for exploring the history of past population migration, evolution and adaptation within larger time-scales and geographical areas where no fossil remains exist. Here, we mainly review the significance of the technical advances in retrieving ancient nuclear DNA from sediments and present new insights into the genetic history of Neanderthals revealed by this technique. By combining ancient genomes retrieved from fossils and additional mitochondrial DNA extracted from sediments of archaeological sites, we may begin investigating diverse archaic populations and examine their genetic relationships, movements and replacements in detail.

Development of an interspecies interaction model: An experiment on Clostridium cadaveris and Clostridium sporogenes under anaerobic condition.

The specific primer and probe sets for quantifying Clostridium cadaveris and Clostridium sporogenes using a quantitative real-time PCR were designed. Each primer and probe set detected only the target species very specifically. The two species were cultivated in pure and mixed culture in batch mode with glucose as the only carbon source. The designed QPCR sets were used successfully to estimate the biokinetic parameters of each species in pure culture: i.e., maximum specific growth rate µmax, half saturation concentration Ks, growth yield Y, and decay coefficient Kd. of C. cadaveris and C. sporogenes were 0.311 ±â€¯0.020 and 0.360 ±â€¯0.019 h-1, 4.241 ±â€¯1.653 and 5.171 ±â€¯1.097 g/L, 0.301 ±â€¯0.065 and 0.199 ±â€¯0.037 1011 copies/g, 0.005 ±â€¯0.043 and 0.009 ±â€¯0.025 h-1, respectively. The effect of interspecific interaction of on substrate consumption rate and microbial growth was evaluated using mixed culture; curve fitting and comparison of coefficients detected increase in substrate consumption rate but decrease in microbial growth rate; these results imply interspecific interaction effect. A new model was of the interspecific interaction was developed, with focus on accuracy, realism, simplicity and biological significance. This interspecific interaction model may be extended to more-complex bioprocesses such as biological wastewater treatment systems and anaerobic digestion.

Framework for reanalysis of publicly available Affymetrix® GeneChip® data sets based on functional regions of interest.

BACKGROUND: Since the introduction of microarrays in 1995, researchers world-wide have used both commercial and custom-designed microarrays for understanding differential expression of transcribed genes. Public databases such as ArrayExpress and the Gene Expression Omnibus (GEO) have made millions of samples readily available. One main drawback to microarray data analysis involves the selection of probes to represent a specific transcript of interest, particularly in light of the fact that transcript-specific knowledge (notably alternative splicing) is dynamic in nature. RESULTS: We therefore developed a framework for reannotating and reassigning probe groups for Affymetrix® GeneChip® technology based on functional regions of interest. This framework addresses three issues of Affymetrix® GeneChip® data analyses: removing nonspecific probes, updating probe target mapping based on the latest genome knowledge and grouping probes into gene, transcript and region-based (UTR, individual exon, CDS) probe sets. Updated gene and transcript probe sets provide more specific analysis results based on current genomic and transcriptomic knowledge. The framework selects unique probes, aligns them to gene annotations and generates a custom Chip Description File (CDF). The analysis reveals only 87% of the Affymetrix® GeneChip® HG-U133 Plus 2 probes uniquely align to the current hg38 human assembly without mismatches. We also tested new mappings on the publicly available data series using rat and human data from GSE48611 and GSE72551 obtained from GEO, and illustrate that functional grouping allows for the subtle detection of regions of interest likely to have phenotypical consequences. CONCLUSION: Through reanalysis of the publicly available data series GSE48611 and GSE72551, we profiled the contribution of UTR and CDS regions to the gene expression levels globally. The comparison between region and gene based results indicated that the detected expressed genes by gene-based and region-based CDFs show high consistency and regions based results allows us to detection of changes in transcript formation.

Recalcitrant deep and shallow nodes in Aristolochia (Aristolochiaceae) illuminated using anchored hybrid enrichment.

Recalcitrant relationships are characterized by very short internodes that can be found among shallow and deep phylogenetic scales all over the tree of life. Adding large amounts of presumably informative sequences, while decreasing systematic error, has been suggested as a possible approach to increase phylogenetic resolution. The development of enrichment strategies, coupled with next generation sequencing, resulted in a cost-effective way to facilitate the reconstruction of recalcitrant relationships. By applying the anchored hybrid enrichment (AHE) genome partitioning strategy to Aristolochia using an universal angiosperm probe set, we obtained 231-233 out of 517 single or low copy nuclear loci originally contained in the enrichment kit, resulting in a total alignment length of 154,756bp to 160,150bp. Since Aristolochia (Piperales; magnoliids) is distantly related to any angiosperm species whose genome has been used for the plant AHE probe design (Amborella trichopoda being the closest), it serves as a proof of universality for this probe set. Aristolochia comprises approximately 500 species grouped in several clades (OTUs), whose relationships to each other are partially unknown. Previous phylogenetic studies have shown that these lineages branched deep in time and in quick succession, seen as short-deep internodes. Short-shallow internodes are also characteristic of some Aristolochia lineages such as Aristolochia subsection Pentandrae, a clade of presumably recent diversification. This subsection is here included to test the performance of AHE at species level. Filtering and subsampling loci using the phylogenetic informativeness method resolves several recalcitrant phylogenetic relationships within Aristolochia. By assuming different ploidy levels during bioinformatics processing of raw data, first hints are obtained that polyploidization contributed to the evolution of Aristolochia. Phylogenetic results are discussed in the light of current systematics and morphology.

Developing Asparagaceae1726: An Asparagaceae-specific probe set targeting 1726 loci for Hyb-Seq and phylogenomics in the family.

Premise: Target sequence capture (Hyb-Seq) is a cost-effective sequencing strategy that employs RNA probes to enrich for specific genomic sequences. By targeting conserved low-copy orthologs, Hyb-Seq enables efficient phylogenomic investigations. Here, we present Asparagaceae1726-a Hyb-Seq probe set targeting 1726 low-copy nuclear genes for phylogenomics in the angiosperm family Asparagaceae-which will aid the often-challenging delineation and resolution of evolutionary relationships within Asparagaceae. Methods: Here we describe and validate the Asparagaceae1726 probe set (https://github.com/bentzpc/Asparagaceae1726) in six of the seven subfamilies of Asparagaceae. We perform phylogenomic analyses with these 1726 loci and evaluate how inclusion of paralogs and bycatch plastome sequences can enhance phylogenomic inference with target-enriched data sets. Results: We recovered at least 82% of target orthologs from all sampled taxa, and phylogenomic analyses resulted in strong support for all subfamilial relationships. Additionally, topology and branch support were congruent between analyses with and without inclusion of target paralogs, suggesting that paralogs had limited effect on phylogenomic inference. Discussion: Asparagaceae1726 is effective across the family and enables the generation of robust data sets for phylogenomics of any Asparagaceae taxon. Asparagaceae1726 establishes a standardized set of loci for phylogenomic analysis in Asparagaceae, which we hope will be widely used for extensible and reproducible investigations of diversification in the family.

A target enrichment probe set for resolving phylogenetic relationships in the coffee family, Rubiaceae.

Premise: Rubiaceae is among the most species-rich plant families, as well as one of the most morphologically and geographically diverse. Currently available phylogenies have mostly relied on few genomic and plastid loci, as opposed to large-scale genomic data. Target enrichment provides the ability to generate sequence data for hundreds to thousands of phylogenetically informative, single-copy loci, which often leads to improved phylogenetic resolution at both shallow and deep taxonomic scales; however, a publicly accessible Rubiaceae-specific probe set that allows for comparable phylogenetic inference across clades is lacking. Methods: Here, we use publicly accessible genomic resources to identify putatively single-copy nuclear loci for target enrichment in two Rubiaceae groups: tribe Hillieae (Cinchonoideae) and tribal complex Palicoureeae+Psychotrieae (Rubioideae). We sequenced 2270 exonic regions corresponding to 1059 loci in our target clades and generated in silico target enrichment sequences for other Rubiaceae taxa using our designed probe set. To test the utility of our probe set for phylogenetic inference across Rubiaceae, we performed a coalescent-aware phylogenetic analysis using a subset of 27 Rubiaceae taxa from 10 different tribes and three subfamilies, and one outgroup in Apocynaceae. Results: We recovered an average of 75% and 84% of targeted exons and loci, respectively, per Rubiaceae sample. Probes designed using genomic resources from a particular subfamily were most efficient at targeting sequences from taxa in that subfamily. The number of paralogs recovered during assembly varied for each clade. Phylogenetic inference of Rubiaceae with our target regions resolves relationships at various scales. Relationships are largely consistent with previous studies of relationships in the family with high support (≥0.98 local posterior probability) at nearly all nodes and evidence of gene tree discordance. Discussion: Our probe set, which we call Rubiaceae2270x, was effective for targeting loci in species across and even outside of Rubiaceae. This probe set will facilitate phylogenomic studies in Rubiaceae and advance systematics and macroevolutionary studies in the family.

New design and optimization of an in-house quantitative TaqMan Real-Time PCR-based assay for the detection and monitoring of occult hepatitis B virus (genotype A-J) infection.

PURPOSE: HBV DNA quantification is used for individuals with uninterpretable serological tests, occult HBV infections, decreasing the window period of the disease, and treatment follow-up. Although there are commercial qPCR assays, they are expensive. In this study, we developed a highly sensitive quantitative TaqMan Real-Time PCR with an exogenous internal control to quantify HBV DNA in serum/plasma. METHODS: A specific primer/probe set was designed for the S conserved region of various HBV genotypes. The primer/probe set was evaluated experimentally and in-silico. An exogenous internal control was included to monitor the effects of inhibitors. The standard plasmid was titrated using three different methods to prepare the seven standards for the assay. The functional characteristics of the in-house assay were evaluated using the standards. Two hundred clinical specimens were also tested. RESULTS: The LOD of the in-house assay was 40 IU/mL, and the assay was linear from 3.26Log10 to 9.26Log10 IU/mL. The analytical and clinical sensitivity of the assay was 100% and 92.15%, respectively. The analytical and clinical specificity of the assay was 100% and 98.97%, respectively. The positive and negative predictive values of the assay were determined to be 98.94% and 92.38%, respectively. The highest coefficient of variation of the inter/intra-assay was 5.1%. The accuracy was close to 100% for all standards, and the correlation between the in-house assay and commercial kit AltoStar® PCR Kits 1.5 was remarkable. The results of the clinical samples using the standards titrated using AcroMetrix™ HBV Panel, Artus® HBV RG PCR Kit, and AltoStar® PCR Kits 1.5 were comparable (r â€‹= â€‹0.942, 0.951, 0.951). CONCLUSIONS: The results indicate that the in-house assay is highly sensitive and specific, reproducible, and cost-benefit. Thus, it can be used to detect and quantify HBV DNA in research and clinical settings.

Steric Hindrance On-Off Mass-Tagged Probe Set Enables Detection of Protein Homodimer in Living Cells.

The major challenge in the detection of protein homodimers is that the identical monomers in a homodimer are indistinguishable using most conventional methods and cannot be sequentially recognized. In this study, a steric hindrance on-off mass-tagged probe set strategy was developed for the quantification of HER2 homodimer in living cells. The probe set contained a hindrance probe and a detection probe. The hindrance probe had a DNA dendrimer as a hindrance group to achieve the steric hindrance on-off function and thus the assignment of monomer identity. The detection probe contained a mass tag released for mass spectrometric quantification. Using the steric hindrance on-off mass-tagged probe set, the level of HER2 homodimer in various breast cancer cell lines was quantified. This is the first report to determine the quantity of protein homodimers, and the steric hindrance on-off probe set developed herein can facilitate the illustration of protein function in cancer.

rePROBE: Workflow for Revised Probe Assignment and Updated Probe-set Annotation in Microarrays.

Commercial and customized microarrays are valuable tools for the analysis of holistic expression patterns, but require the integration of the latest genomic information. This study provides a comprehensive workflow implemented in an R package (rePROBE) to assign the entire probes and to annotate the probe sets based on up-to-date genomic and transcriptomic information. The rePROBE package can be applied to available gene expression microarray platforms and addresses both public and custom databases. The revised probe assignment and updated probe-set annotation are applied to commercial microarrays available for different livestock species, i.e., chicken (Gallus gallus; ChiGene-1_0-st: 443,579 probes and 18,530 probe sets), pig (Sus scrofa; PorGene-1_1-st: 592,005 probes and 25,779 probe sets), and cattle (Bos Taurus; BovGene-1_0-st: 530,717 probes and 24,759 probe sets), as well as available for human (Homo sapiens; HuGene-1_0-st) and mouse (Mus musculus; HT_MG-430_PM). Using current species-specific transcriptomic information (RefSeq, Ensembl, and partially non-redundant nucleotide sequences) and genomic information, the applied workflow reveals 297,574 probes (15,689 probe sets) for chicken, 384,715 probes (21,673 probe sets) for pig, 363,077 probes (21,238 probe sets) for cattle, 481,168 probes (23,495 probe sets) for human, and 324,942 probes (32,494 probe sets) for mouse. These are representative of 12,641, 15,758, 18,046, 20,167, and 16,335 unique genes that are both annotated and positioned for chicken, pig, cattle, human, and mouse, respectively. Additionally, the workflow collects information on the number of single nucleotide polymorphisms (SNPs) within respective targeted genomic regions and thus provides a detailed basis for comprehensive analyses such as expression quantitative trait locus (eQTL) studies to identify quantitative and functional traits. The rePROBE R package is freely available at https://github.com/friederhadlich/rePROBE.

Relative performance of customized and universal probe sets in target enrichment: A case study in subtribe Malinae.

PREMISE: Custom probe design for target enrichment in phylogenetics is tedious and often hinders broader phylogenetic synthesis. The universal angiosperm probe set Angiosperms353 may be the solution. Here, we test the relative performance of Angiosperms353 on the Rosaceae subtribe Malinae in comparison with custom probes that we specifically designed for this clade. We then address the impact of bioinformatically altering the performance of Angiosperms353 by replacing the original probe sequences with orthologs extracted from the Malus domestica genome. METHODS: To evaluate the relative performance of these probe sets, we compared the enrichment efficiency, locus recovery, alignment length, proportion of parsimony-informative sites, proportion of potential paralogs, the topology and support of the resulting species trees, and the gene tree discordance. RESULTS: Locus recovery was highest for our custom Malinae probe set, and replacing the original Angiosperms353 sequences with a Malus representative improved the locus recovery relative to Angiosperms353. The proportion of parsimony-informative sites was similar between all probe sets, while the gene tree discordance was lower in the case of the custom probes. DISCUSSION: A custom probe set benefits from data completeness and can be tailored toward the specificities of the project of choice; however, Angiosperms353 was equally as phylogenetically informative as the custom probes. We therefore recommend using both a custom probe set and Angiosperms353 to facilitate large-scale systematic studies, where financially possible.

Direct Conjugation of Streptavidin to Encoded Hydrogel Microparticles for Multiplex Biomolecule Detection with Rapid Probe-Set Modification.

Encoded hydrogel microparticles synthesized via flow lithography have drawn attention for multiplex biomarker detection due to their high multiplex capability and solution-like hybridization kinetics. However, the current methods for preparing particles cannot achieve a flexible, rapid probe-set modification, which is necessary for the production of various combinations of target panels in clinical diagnosis. In order to accomplish the unmet needs, streptavidin was incorporated into the encoded hydrogel microparticles to take advantage of the rapid streptavidin-biotin interactions that can be used in probe-set modification. However, the existing methods suffer from low efficiency of streptavidin conjugation, cause undesirable deformation of particles, and impair the assay capability. Here, we present a simple and powerful method to conjugate streptavidin to the encoded hydrogel microparticles for better assay performance and rapid probe-set modification. Streptavidin was directly conjugated to the encoded hydrogel microparticles using the aza-Michael addition click reaction, which can proceed in mild, aqueous condition without catalysts. A highly flexible and sensitive assay was developed to quantify DNA and proteins using streptavidin-conjugated encoded hydrogel microparticles. We also validated the potential applications of our particles conducting multiplex detection of cancer-related miRNAs.

Target Nuclear and Off-Target Plastid Hybrid Enrichment Data Inform a Range of Evolutionary Depths in the Orchid Genus Epidendrum.

Universal angiosperm enrichment probe sets designed to enrich hundreds of putatively orthologous nuclear single-copy loci are increasingly being applied to infer phylogenetic relationships of different lineages of angiosperms at a range of evolutionary depths. Studies applying such probe sets have focused on testing the universality and performance of the target nuclear loci, but they have not taken advantage of off-target data from other genome compartments generated alongside the nuclear loci. Here we do so to infer phylogenetic relationships in the orchid genus Epidendrum and closely related genera of subtribe Laeliinae. Our aims are to: 1) test the technical viability of applying the plant anchored hybrid enrichment (AHE) method (Angiosperm v.1 probe kit) to our focal group, 2) mine plastid protein coding genes from off-target reads; and 3) evaluate the performance of the target nuclear and off-target plastid loci in resolving and supporting phylogenetic relationships along a range of taxonomical depths. Phylogenetic relationships were inferred from the nuclear data set through coalescent summary and site-based methods, whereas plastid loci were analyzed in a concatenated partitioned matrix under maximum likelihood. The usefulness of target and flanking non-target nuclear regions and plastid loci was assessed through the estimation of their phylogenetic informativeness. Our study successfully applied the plant AHE probe kit to Epidendrum, supporting the universality of this kit in angiosperms. Moreover, it demonstrated the feasibility of mining plastome loci from off-target reads generated with the Angiosperm v.1 probe kit to obtain additional, uniparentally inherited sequence data at no extra sequencing cost. Our analyses detected some strongly supported incongruences between nuclear and plastid data sets at shallow divergences, an indication of potential lineage sorting, hybridization, or introgression events in the group. Lastly, we found that the per site phylogenetic informativeness of the ycf1 plastid gene surpasses that of all other plastid genes and several nuclear loci, making it an excellent candidate for assessing phylogenetic relationships at medium to low taxonomic levels in orchids.

Preprocessing Steps for Agilent MicroRNA Arrays: Does the Order Matter?

MOTIVATION/BACKGROUND: Previous publications on microarray preprocessing mostly focused on method development or comparison for an individual preprocessing step. Very few, if any, focused on recommending an effective ordering of the preprocessing steps, in particular, normalization in relationship to log transformation and probe set summarization. In this study, we aim to study how the relative ordering of the preprocessing steps influences differential expression analysis for Agilent microRNA array data. METHODS: A set of 192 untreated primary gynecologic tumor samples (96 endometrial tumors and 96 ovarian tumors) were collected at Memorial Sloan Kettering Cancer Center during the period of 2000-2012. From this same sample set, two datasets were generated: one dataset had no confounding array effects by experimental design and served as the benchmark, and another dataset exhibited array effects and served as the test data. We preprocessed our test dataset using different orderings between the following three steps: quantile normalization, log transformation, and median summarization. Differential expression analysis was performed on each preprocessed test dataset, and the results were compared against the results from the benchmark dataset. True positive rate, false positive rate, and false discovery rate were used to assess the effectiveness of the orderings. RESULTS: The ordering of log transformation, quantile normalization (on probe-level data), and median summarization slightly outperforms the other orderings. CONCLUSION: Our results ease the anxiety over the uncertain effect that the orderings could have on the analysis of Agilent microRNA array data.