DNA Sequence Data Research Articles

CONIPHER: a computational framework for scalable phylogenetic reconstruction with error correction.

Intratumor heterogeneity provides the fuel for the evolution and selection of subclonal tumor cell populations. However, accurate inference of tumor subclonal architecture and reconstruction of tumor evolutionary histories from bulk DNA sequencing data remains challenging. Frequently, sequencing and alignment artifacts are not fully filtered out from cancer somatic mutations, and errors in the identification of copy number alterations or complex evolutionary events (e.g., mutation losses) affect the estimated cellular prevalence of mutations. Together, such errors propagate into the analysis of mutation clustering and phylogenetic reconstruction. In this Protocol, we present a new computational framework, CONIPHER (COrrecting Noise In PHylogenetic Evaluation and Reconstruction), that accurately infers subclonal structure and phylogenetic relationships from multisample tumor sequencing, accounting for both copy number alterations and mutation errors. CONIPHER has been used to reconstruct subclonal architecture and tumor phylogeny from multisample tumors with high-depth whole-exome sequencing from the TRACERx421 dataset, as well as matched primary-metastatic cases. CONIPHER outperforms similar methods on simulated datasets, and in particular scales to a large number of tumor samples and clones, while completing in under 1.5 h on average. CONIPHER enables automated phylogenetic analysis that can be effectively applied to large sequencing datasets generated with different technologies. CONIPHER can be run with a basic knowledge of bioinformatics and R and bash scripting languages.

A TIME-CALIBRATED PHYLOGENY OF VERBESINA (HELIANTHEAE – ASTERACEAE) BASED ON NUCLEAR RIBOSOMAL ITS AND ETS SEQUENCES

Verbesina L. is a genus of the tribe Heliantheae, subtribe Verbesininae (Asteraceae), with distribution in the Americas, where Mexico and the Andes harbour the richest concentration of species. The approximately 325 species in the genus are shrubs, subshrubs, trees and rarely herbs. Despite its high species diversity and biogeographical importance, the only available phylogenetic hypothesis for Verbesina was based on chloroplast DNA restriction site data. In the present study, nuclear ITS and ETS DNA sequence data were used with an expanded taxon sampling, particularly among the South American Verbesina species, to improve phylogenetic resolution and support, clarify infrageneric relationships, and resolve biogeographical questions in the genus. The results of our new analysis corroborate the monophyly of Verbesina, but its current classification into 12 taxonomic sections, based on morphological characters such as phyllotaxis, head size, corolla colour, and presence of ray flowers, is not congruent with the molecular phylogeny, in which most sections are polyphyletic. We also show that Verbesina diverged in the late Miocene of North America, about 8 Ma. At least two independent Pleistocene dispersals into South America across the Isthmus of Panama and along the Southern Andes are evident, beginning around 3.23 Ma (1.27–3.23 Ma) in the Middle Pliocene, and resulting in an extra-Amazonian distribution of the genus inSouth America. Diversification in South America began around 2.83 Ma with occupation of the Andes. Colonisation of Brazil is estimated to have occurred around 2.15 Ma, from Andean lineages.

Comparing the effect of larval dispersal strategies on morphological versus genetic differentiation in two neotropical fiddler crabs

AbstractThe dispersal mode of estuarine invertebrates has a strong effect on genetic variability and phenotypic expression, potentially maintaining gene flow among widely separated populations or limiting connectivity among close ones. Understanding the dispersal strategies effects can support adequate strategies for environment management and understand the evolutionary history of the studied species. A comparative study on the effect of larval dispersal strategies (export vs retention) along the overall geographic distribution ranges of adult populations of two co‐distributed fiddler crab species was performed. Geometric morphometric techniques were used to evaluate morphological differentiation in the shape of the carapace and major cheliped propodus in 10 populations of Minuca mordax and five populations of Leptuca leptodactyla. Potential genetic differentiation was assessed using DNA sequence data from up to 170 fiddler crab specimens, corresponding to the mitochondrial gene cytochrome c oxidase subunit 1 (COX1). Patterns of genetic and morphological characteristics diverged among populations of both species. In terms of morphology, populations of both species showed intraspecific variation in the shape of the carapace and, to a lesser extent, in the major cheliped propodus. This observed intraspecific variation followed a clear geographic pattern of morphometric differentiation, in which the geographic origin is associated with a phenotypic response to the environmental diversity along the extended Brazilian coast. In contrast, there are shared haplotypes in populations separated by more than 4000 km, but with significant ΦST values among most populations of L. leptodactyla and M. mordax, suggesting a moderate to high level of gene flow. Therefore, it can be concluded that the split of the Central South Equatorial Current has not acted as an efficient long‐term barrier to the dispersal of larvae in both species. In M. mordax, a slight limitation of genetic exchange can be observed between the southernmost populations and the remaining ones. This first evidence of genetic structuring within this widely distributed species is consistent with its documented strategy to retain larvae in the estuarine environments. However, occasional transport of larvae to more open coastal waters seems to maintain stepwise gene flow between neighboring populations. Overall, our results still provide evidence of more pronounced genetic homogeneity in a species with a larval export strategy (L. leptodactyla) compared to a species with a larval retention strategy (M. mordax). This suggests that larval dispersal strategies, coupled with the microhabitat in which the respective species occur, have a noticeable effect on the degree of gene flow. Most likely, these biological aspects are further influenced by the estuarine and coastline geomorphology.

CHARR efficiently estimates contamination from DNA sequencing data

DNA sample contamination is a major issue in clinical and research applications of whole-genome and -exome sequencing. Even modest levels of contamination can substantially affect the overall quality of variant calls and lead to widespread genotyping errors. Currently, popular tools for estimating the contamination level use short-read data (BAM/CRAM files), which are expensive to store and manipulate and often not retained or shared widely. We propose a metric to estimate DNA sample contamination from variant-level whole-genome and -exome sequence data called CHARR, contamination from homozygous alternate reference reads, which leverages the infiltration ofreference reads within homozygous alternate variant calls. CHARR uses a small proportion of variant-level genotype information andthus can be computed from single-sample gVCFs or callsets in VCF or BCF formats, as well as efficiently stored variant calls in HailVariantDataset format. Our results demonstrate that CHARR accurately recapitulates results from existing tools with substantially reduced costs, improving the accuracy and efficiency of downstream analyses of ultra-large whole-genome and exome sequencing datasets.

Development of SSR markers identification system for Carex L. based on RAD sequencing

In this study, we designed and evaluated an efficient set of simple sequence repeat (SSR) markers from restriction site-associated DNA (RAD) sequencing data of four widely utilized Carex L. accession across Northern China, especially in Beijing. Based on their genomic sequencing data, we developed 400 SSR markers and evaluated their amplification specificities among which a total of 17 SSR markers were identified as a core set of molecular markers for efficient assessment of diverse Carex L. accessions. Using this molecular identification system, we classified 26 Carex L. accessions into 3 genetically distinct groups. The establishment of a molecular identification system based on a core set of 17 SSR markers provides an essential basis for evaluating genetic relationships among Carex L. accessions along with a wealth of genetic resources for marker-assisted selection and breeding of Carex L..

First Molecular Phylogenetic Identification and Report of Pseudocercospora cannabina Causing Leaf Spot Disease on Cannabis sativa in Thailand

Cannabis sativa is gaining attention as an agronomically important crop in many countries around the world. The identification and control of leaf diseases in cannabis are very important for cannabis cultivators as leaves are the most economically important part of the cannabis plants. In 2022, several cannabis plants in cultivations showing olive leaf spot symptoms emerged from Chiang Rai province, Thailand. Preliminary studies indicated that the causal organism is Pseudocercospora sp. Species of Pseudocercospora are important plant pathogens that are now identified through morphological studies combined with DNA sequence data of Internal Transcribed Spacer (ITS), Actin (act), Translation Elongation Factor (tef), and RNA Polymerase II second largest subunit (rpb2) gene regions. We aimed to investigate and understand the emergence of olive leaf spot disease in cannabis plants in Chiang Rai province, Thailand, with a specific focus on the combined morpho-molecular identification of the pathogen. In our study, Pseudocercospora cannabina, the causal organism of olive leaf spot disease, was identified as the leaf spot-causing pathogen with both morphological and phylogenetic analyses. Our study is the first to provide molecular data for Ps. cannabina as the typenor Ps. cannabina isolates from previous studies have made molecular data available for this species. A pathogenicity test, re-isolation, and identification steps were performed to fulfill Koch’s postulates. This comprehensive approach enhances our understanding of the olive leaf spot disease and its causative agent in cannabis.

Identify gestational diabetes mellitus by deep learning model from cell-free DNA at the early gestation stage.

Gestational diabetes mellitus (GDM) is a common complication of pregnancy, which has significant adverse effects on both the mother and fetus. The incidence of GDM is increasing globally, and early diagnosis is critical for timely treatment and reducing the risk of poor pregnancy outcomes. GDM is usually diagnosed and detected after 24weeks of gestation, while complications due to GDM can occur much earlier. Copy number variations (CNVs) can be a possible biomarker for GDM diagnosis and screening in the early gestation stage. In this study, we proposed a machine-learning method to screen GDM in the early stage of gestation using cell-free DNA (cfDNA) sequencing data from maternal plasma. Five thousand and eighty-five patients from north regions of Mainland China, including 1942 GDM, were recruited. A non-overlapping sliding window method was applied for CNV coverage screening on low-coverage (~0.2×) sequencing data. The CNV coverage was fed to a convolutional neural network with attention architecture for the binary classification. The model achieved a classification accuracy of 88.14%, precision of 84.07%, recall of 93.04%, F1-score of 88.33% and AUC of 96.49%. The model identified 2190 genes associated with GDM, including DEFA1, DEFA3 and DEFB1. The enriched gene ontology (GO) terms and KEGG pathways showed that many identified genes are associated with diabetes-related pathways. Our study demonstrates the feasibility of using cfDNA sequencing data and machine-learning methods for early diagnosis of GDM, which may aid in early intervention and prevention of adverse pregnancy outcomes.

Rediscovery and systematics of Rhinophis fergusonianus Boulenger, 1896 (Serpentes: Uropeltidae) from Indias Western Ghats.

The shieldtail snake Rhinophis fergusonianus Boulenger, 1896 was previously known only from the holotype specimen collected approximately 130 years ago from an imprecise Indian locality (Cardamom Hills). We report the rediscovery of this species from four localities, from low-elevation hills on both sides of the Palghat Gap in the southern part of the Western Ghats of peninsular India. We document new specimens of R. fergusonianus and provide a reassessment of the systematics of the species based on external morphology and DNA-sequence data. We also provide a revised key to the identification of Indian species of Rhinophis.

Morphological, acoustic and genetic identification of a reproducing population of the invasive African clawed frog Xenopuslaevis (Anura, Pipidae) recently discovered in Belgium.

Using external morphology of adults and tadpoles, osteology from high-resolution microcomputed tomography, vocalization analysis, and DNA sequence data, the identity of a reproducing Belgian population of invasive Xenopus at the current northernmost edge of the distribution of the genus in Europe was assessed. All data concur to an identification as Xenopus (Xenopus) laevis (Daudin, 1802). Genetically it is most closely related to populations of the Cape region in South Africa. No studies on the natural history of the Belgian Xenopus population and its impact on the local environment have been made to date.

EPCO-09. CHARACTERIZING THE GBM CELLULAR LANDSCAPE BY LARGE-SCALE SINGLE-NUCLEUS RNA-SEQUENCING

Abstract Tumor heterogeneity is a well-known hallmark of glioblastoma (GBM). Single-cell RNA-sequencing (scRNA-seq) technologies significantly extended our understanding of GBM intra-tumor heterogeneity. While the landmark scRNA-seq studies were limited by scale, recent technological advances have enabled analyzing increasing numbers of cells. Here, leveraging a large-scale single-nucleus RNA-sequencing (snRNA-seq) dataset containing 457,442 cells (271,444 malignant and 185,995 non-malignant) from 121 IDH-wildtype GBM tumor samples and matching whole-exome/whole-genome sequencing data, we dissected the GBM malignant and tumor microenvironment (TME) compartments with unprecedented resolution and scale. We identified three novel malignant cellular states - Glial progenitor cell-like (GPC-like), Neuron-like and Cilia-like - in addition to the cellular states previously defined in GBM (NPC-like, OPC-like, AC-like and MES-like). Cross-referencing our dataset with published scRNA-seq datasets suggests that GBM states mirror the developmental hierarchy observed in the developing human brain. Functional enrichment analysis demonstrated the heterogeneous nature of pathway and metabolic activities across the spectrum of cellular states. Analysis of the matched DNA-sequencing data revealed novel associations between the occurrence of certain genetic events and the abundance of specific cellular states. Similarly, we dissected the TME and robustly defined cellular states for different TME cell types, underscoring the heterogeneity found also within the TME compartment. Leveraging the large number of samples in our dataset and controlling for intra-tumor cell state frequency exposed three baseline gene expression programs - neuronal activity, glial development and extra-cellular matrix remodeling - termed State-Controlled Profiles (SCPs), that reflect the inter-tumor heterogeneity and are not influenced by the intra-tumor cell state distribution. Integrated analysis of the intra- and inter-tumor heterogeneity components revealed striking associations between certain TME cell types and states, malignant cell states and SCPs, suggesting that an interplay between the TME and malignant cells shapes the frequency of malignant states and baseline expression profile in each tumor.

Laurencia mediterranea sp. nov. (Ceramiales, Rhodophyta) from the central Mediterranean Sea

Abstract The identification of macroalgal species within the Laurencia complex is challenging, due to the presence of inconspicuous morphological characters, extensive variation in these traits and a diverse biogeography. Moreover, for a number of these species, no DNA sequence data are available in online databases. For this study, five algal specimens, tentatively assigned to Laurencia, were collected from Malta in the central Mediterranean Sea and studied using an integrative systematics approach. An analysis of the data resulted in the description of a new species, Laurencia mediterranea using combined morphological and molecular criteria, including COI-5P and rbcL. Morphologically L. mediterranea sp. nov. was distinct from other Laurencia spp. in the type of holdfast, the colour and shape of the thallus, the presence of secondary pit connections, lenticular thickenings and ‘corps en cerise’. Moreover, a new genetic species cluster indicated a separate taxon at the species level. Our study demonstrates that genetic sequences having clear links to sample metadata and digital herbaria are indispensable for macroalgal biodiversity research. This approach could offer a solution for algal taxonomy in cases where biogeography is important and limited sequence data exist in online DNA libraries.

Addition to Pseudoplagiostomataceae: Pseudoplagiostoma inthanonense sp. nov. from Doi Inthanon National Park, Northern Thailand

Fungi are dominant microorganisms in plant litter, especially in pristine environments. This study collected leaf litter of broadleaf species within Doi Inthanon National Park, Chiang Mai, Thailand. Phylogenetic analyses utilizing DNA sequence data from ITS, LSU, tef1-α, rpb2, and tub2 regions and morphological characteristics revealed a new species, Pseudoplagiostoma inthanonense (Pseudoplagiostomataceae). The phylogeny and morphology of this novel species are thoroughly described and supported with detailed illustrations. Pseudoplagiostoma inthanonense is characterized by the shape and size of the conidiogenous cell and spore size. The discovery of P. inthanonense confirms that Pseudoplagiostoma consists of pathogenic, endophytic, and saprobic life forms while creating new research opportunities on potential lifestyle changes of Pseudoplagiostoma species.

Origins of cryophilous lineages and the role of Beringia: evidence from the cosmopolitan angiosperm genus Stellaria L. (Caryophyllaceae)

Abstract The cosmopolitan angiosperm genus Stellaria L. (Caryophyllaceae) occurs across the Arctic and is most diverse in the high southern Asian mountains, including presence at the most extreme latitudinal and elevational limits of vascular plants. Using the primary fossil record and double digest restriction site-associated DNA sequencing data, we estimated divergence times of Stellaria and relatives across the Caryophyllaceae. We then used ancestral area and ancestral state reconstructions to interpret the biogeographical history of the group. We found that Stellaria originated in the Miocene and gave rise to repeated New World lineages, mostly from temperate Old World regions and probably via Bering Land Bridges. Circumboreal lineages were recovered of recent, Pleistocene origin and several might have originated in southerly mountains of both the Old World and the New World before subsequently colonizing the Arctic. Ancestral state reconstruction of inhabitance of cold vs. temperate climates and of wet vs. dry habitats revealed repeated evolutionary transitions across these extremes by members of the genus worldwide. Our study, which samples nearly all species within a diverse and cosmopolitan lineage of flowering plants, recovers a group characterized by niche lability and helps to support prior findings of temperate origins of many cryophilous plant lineages. Pre-adaptation to cold might have been a prerequisite for colonization of the Arctic.

Diversity of colacosome-interacting mycoparasites expands the understanding of the evolution and ecology of Microbotryomycetes.

Mycoparasites in Basidiomycota comprise a diverse group of fungi, both morphologically and phylogenetically. They interact with their hosts through either fusion-interaction or colacosome-interaction. Colacosomes are subcellular structures formed by the mycoparasite at the host-parasite interface, which penetrate the parasite and host cell walls. Previously, these structures were detected in 19 fungal species, usually by means of transmission electron microscopy. Most colacosome-forming species have been assigned to Microbotryomycetes (Pucciniomycotina, Basidiomycota), a highly diverse class, comprising saprobic yeasts, mycoparasites, and phytoparasites. In general, these myco- and phytoparasites are dimorphic organisms, with a parasitic filamentous morph and saprobic yeast morph. We investigated colacosome-forming mycoparasites based on fungarium material, freshly collected specimens, and cultures of yeast morphs. We characterised the micromorphology of filamentous morphs, the physiological characteristics of yeast morphs, and inferred phylogenetic relationships based on DNA sequence data from seven loci. We outline and employ an epifluorescence-based microscopic method to assess the presence and organisation of colacosomes. We describe five new species in the genus Colacogloea, the novel dimorphic mycoparasite Mycogloiocolax gerardii, and provide the first report of a sexual, mycoparasitic morph in Colacogloea philyla and in the genus Slooffia. We detected colacosomes in eight fungal species, which brings the total number of known colacosome-forming fungi to 27. Finally, we revealed three distinct types of colacosome organisation in Microbotryomycetes. Taxonomic novelties and typifications: New family: Mycogloiocolacaeae Schoutteten & Yurkov; New genus: Mycogloiocolax Schoutteten & Rödel; New species: Colacogloea bettinae Schoutteten & Begerow, C. biconidiata Schoutteten, C. fennica Schoutteten & Miettinen, C. microspora Schoutteten, C. universitatis-gandavensis Schoutteten & Verbeken, Mycogloiocolax gerardii Schoutteten & Rödel; New combinations: Slooffia micra (Bourdot & Galzin) Schoutteten, Fellozyma cerberi (A.M. Yurkov et al.) Schoutteten & Yurkov, Fellozyma telluris (A.M. Yurkov et al.) Schoutteten & Yurkov; Epitypifications (basionyms): Achroomyces insignis Hauerslev, Platygloea micra Bourdot & Galzin, Platygloea peniophorae Bourdot & Galzin; Lectotypification (basionym): Platygloea peniophorae Bourdot & Galzin Citation: Schoutteten N, Yurkov A, Leroux O, Haelewaters D, Van Der Straeten D, Miettinen O, Boekhout T, Begerow D, Verbeken A (2023). Diversity of colacosome-interacting mycoparasites expands the understanding of the evolution and ecology of Microbotryomycetes. Studies in Mycology 106: 41-94. doi: 10.3114/sim.2022.106.02.

Genomic divergence and the evolution of ecotypes in bottlenose dolphins (genus Tursiops).

Climatic changes have caused major environmental restructuring throughout the world's oceans. Marine organisms have responded to novel conditions through various biological systems, including genomic adaptation. Growing accessibility of next-generation DNA sequencing methods to study non-model species has recently allowed genomic changes underlying environmental adaptations to be investigated. This study used double digest restriction-site associated DNA (ddRAD) sequence data to investigate the genomic basis of ecotype formation across currently recognised species and subspecies of bottlenose dolphins (genus Tursiops) in the Southern Hemisphere. Subspecies-level genomic divergence was confirmed between the offshore common bottlenose dolphin (T. truncatus truncatus) and the inshore Lahille's bottlenose dolphin (T. t. gephyreus) from the southwestern Atlantic Ocean (SWAO). Similarly, subspecies-level divergence is suggested between inshore (eastern Australia) Indo-Pacific bottlenose dolphin (T. aduncus) and the proposed Burrunan dolphin (T. australis) from southern Australia. Inshore bottlenose dolphin lineages generally had lower genomic diversity than offshore lineages, a pattern particularly evident for T. t. gephyreus, which showed exceptionally low diversity. Genomic regions associated with cardiovascular, musculoskeletal and energy production systems appear to have undergone repeated adaptive evolution in inshore lineages across the Southern Hemisphere. We hypothesise that comparable selective pressures in the inshore environment drove similar adaptive responses in each lineage, supporting parallel evolution of inshore bottlenose dolphins. With climate change altering marine ecosystems worldwide, it is crucial to gain an understanding on the adaptive capacity of local species and populations. Our study provides insights into key adaptive pathways that may be important for the long-term survival of cetaceans and other organisms in a changing marine environment.

MicroRNA Promoter Identification in Human With a Three-level Prediction Method.

The accurate annotation of miRNA promoters is critical for the mechanistic understanding of miRNA gene regulation. Various computational methods have been developed for the prediction of miRNA promoters solely employing a single classifier. Most of these computational methods extract either sequence features or one-sided signal features, and the accuracy and reliability of predictions need to be improved. To address these issues, we present miPTP, a three-level prediction method that combines SVM, RF, and correlation coefficients. It is capable of identifying miRNA promoters based on both DNA sequence and ChIP-Seq data (RPol II). By sequentially integrating these two types of information sources with the three methods selected, miPTP can identify miRNA promoters with higher accuracy and sensitivity compared to specific existing methods. Finally, the reliability of miPTP is validated by examining the conservation, CpG content, and activating histone marks in the identified miRNA promoters.

Integrating SNVs and CNAs on a phylogenetic tree from single-cell DNA sequencing data.

Single-cell DNA sequencing enables the construction of evolutionary trees that can reveal how tumors gain mutations and grow. Different whole-genome amplification procedures render genomic materials of different characteristics, often suitable for the detection of either single-nucleotide variation or copy number aberration, but not ideally for both. Consequently, this hinders the inference of a comprehensive phylogenetic tree and limits opportunities to investigate the interplay of SNVs and CNAs. Existing methods such as SCARLET and COMPASS require that the SNVs and CNAs are detected from the same sets of cells, which is technically challenging. Here we present a novel computational tool, SCsnvcna, that places SNVs on a tree inferred from CNA signals, whereas the sets of cells rendering the SNVs and CNAs are independent, offering a more practical solution in terms of the technical challenges. SCsnvcna is a Bayesian probabilistic model using both the genotype constraints on the tree and the cellular prevalence to search the optimal solution. Comprehensive simulations and comparison with seven state-of-the-art methods show that SCsnvcna is robust and accurate in a variety of circumstances. Particularly, SCsnvcna most frequently produces the lowest error rates, with ability to scale to a wide range of numerical values for leaf nodes in the tree, SNVs, and SNV cells. The application of SCsnvcna to two published colorectal cancer data sets shows highly consistent placement of SNV cells and SNVs with the original study while also supporting a refined placement of ATP7B, illustrating SCsnvcna's value in analyzing complex multitumor samples.

Permafrost microbial communities follow shifts in vegetation, soils, and megafauna extinctions in Late Pleistocene NW North America

AbstractWe analyzed the microbial constituent of sedimentary ancient DNA sequence data recovered from subarctic loessal permafrost sediments dating between 30,000 and 4000 years ago. These data were originally studied for paleo‐ecological shifts in plants and animals associated with the Pleistocene–Holocene transition. Here, we explore whether there were changes in microbial communities paralleling the transition from distinctive cold‐adapted Ice Age megafauna and vegetation communities—the mammoth steppe ecosystem—toward the expansion of woody shrubs, extirpation of grazing megaherbivores, and development of the boreal forest. We observe a clear shift in the relative proportions of prokaryotic taxa after ca. 13,300 years ago associated with the collapse of the mammoth steppe. These data are consistent among study sites and between replicates processed with different methodologies (shotgun sequencing and targeted capture), which highlights that the “off‐target” fraction of metagenomic data used to study macro‐ecosystems can also be used to investigate synchronous changes in microbial communities. Functional analyses were performed with SEED and KEGG databases where we observed a shift in methane metabolism pathways after ~13,100 years ago, which suggests that there was a shift in methanogenesis away from animal gut microflora at the end of the Pleistocene. There does not appear to be a significant shift in the overall diversity of microbial communities despite the observed taxonomic and functional changes.

Revelation of genetic diversity and genomic footprints of adaptation in Indian pig breeds

In the present study, the genetic diversity measures among four Indian domestic breeds of pig namely Agonda Goan, Ghurrah, Ghungroo, and Nicobari, of different agro-climatic regions of country were explored and compared with European commercial breeds, European wild boar and Chinese domestic breeds. The double digest restriction site-associated DNA sequencing (ddRADseq) data of Indian pigs (102) and Landrace (10 animals) were generated and whole genome sequencing data of exotic pigs (60 animals) from public data repository were used in the study. The principal component analysis (PCA), admixture analysis and phylogenetic analysis revealed that Indian breeds were closer in ancestry to Chinese breeds than European breeds. European breeds exhibited highest genetic diversity measures among all the considered breeds. Among Indian breeds, Agonda Goan and Ghurrah were found to be more genetically diverse than Nicobari and Ghungroo. The selection signature regions in Indian pigs were explored using iHS and XP-EHH, and during iHS analysis, it was observed that genes related to growth, reproduction, health, meat quality, sensory perception and behavior were found to be under selection pressure in Indian pig breeds. Strong selection signatures were recorded in 24.25–25.25 Mb region of SSC18, 123.25–124 Mb region of SSC15 and 118.75–119.5 Mb region of SSC2 in most of the Indian breeds upon pairwise comparison with European commercial breeds using XP-EHH. These regions were harboring some important genes such as EPHA4 for thermotolerance, TAS2R16, FEZF1, CADPS2 and PTPRZ1 for adaptability to scavenging system of rearing, TRIM36 and PGGT1B for disease resistance and CCDC112, PIAS1, FEM1B and ITGA11 for reproduction.

Developing the Protocol Infrastructure for DNA Sequencing Natural History Collections.

Intentionally preserved biological material in natural history collections represents a vast repository of biodiversity. Advances in laboratory and sequencing technologies have made these specimens increasingly accessible for genomic analyses, offering a window into the genetic past of species and often permitting access to information that can no longer be sampled in the wild. Due to their age, preparation and storage conditions, DNA retrieved from museum and herbarium specimens is often poor in yield, heavily fragmented and biochemically modified. This not only poses methodological challenges in recovering nucleotide sequences, but also makes such investigations susceptible to environmental and laboratory contamination. In this paper, we review the practical challenges associated with making the recovery of DNA sequence data from museum collections more routine. We first review key operational principles and issues to address, to guide the decision-making process and dialogue between researchers and curators about when and how to sample museum specimens for genomic analyses. We then outline the range of steps that can be taken to reduce the likelihood of contamination including laboratory set-ups, workflows and working practices. We finish by presenting a series of case studies, each focusing on protocol practicalities for the application of different mainstream methodologies to museum specimens including: (i) shotgun sequencing of insect mitogenomes, (ii) whole genome sequencing of insects, (iii) genome skimming to recover plant plastid genomes from herbarium specimens, (iv) target capture of multi-locus nuclear sequences from herbarium specimens, (v) RAD-sequencing of bird specimens and (vi) shotgun sequencing of ancient bovid bone samples.