Discriminant Canonical Tool for Differential Biometric Characterization of Multivariety Endangered Hen Breeds

This study aimed to develop a tool to perform the morphological characterization of Sureña and Utrerana breeds, two endangered autochthonous breeds ascribed to the Mediterranean trunk of Spanish autochthonous hens and their varieties (n = 608; 473 females and 135 males). Kruskal–Wallis H test reported sex dimorphism pieces of evidence (p < 0.05 at least). Multicollinearity analysis reported (variance inflation factor (VIF) >5 variables were discarded) white nails, ocular ratio, and back length (Wilks’ lambda values of 0.191, 0.357, and 0.429, respectively) to have the highest discriminant power in female morphological characterization. For males, ocular ratio and black/corneous and white beak colors (Wilks’ lambda values of 0.180, 0.210, and 0.349, respectively) displayed the greatest discriminant potential. The first two functions explained around 90% intergroup variability. A stepwise discriminant canonical analysis (DCA) was used to determine genotype clustering patterns. Interbreed and varieties proximity was evaluated through Mahalanobis distances. Despite the adaptability capacity to alternative production systems ascribed to both avian breeds, Sureña and Utrerana morphologically differ. Breed dimorphism may evidence differential adaptability mechanisms linked to their aptitude (dual purpose/egg production). The present tool may serve as a model for the first stages of breed protection to be applicable in other endangered avian breeds worldwide.

Computational Biological Modeling Identifies PD-(L)1 Immunotherapy Sensitivity Among Molecular Subgroups of KRAS-Mutated Non–Small-Cell Lung Cancer

PURPOSE KRAS-mutated ( KRASMUT) non–small-cell lung cancer (NSCLC) is emerging as a heterogeneous disease defined by comutations, which may confer differential benefit to PD-(L)1 immunotherapy. In this study, we leveraged computational biological modeling (CBM) of tumor genomic data to identify PD-(L)1 immunotherapy sensitivity among KRASMUT NSCLC molecular subgroups. MATERIALS AND METHODS In this multicohort retrospective analysis, the genotype clustering frequency ranked method was used for molecular clustering of tumor genomic data from 776 patients with KRASMUT NSCLC. These genomic data were input into the CBM, in which customized protein networks were characterized for each tumor. The CBM evaluated sensitivity to PD-(L)1 immunotherapy using three metrics: programmed death-ligand 1 expression, dendritic cell infiltration index (nine chemokine markers), and immunosuppressive biomarker expression index (14 markers). RESULTS Genotype clustering identified eight molecular subgroups and the CBM characterized their shared cancer pathway characteristics: KRAS MUT/ TP53 MUT, KRAS MUT/ CDKN2A/ B/ C MUT, KRAS MUT/ STK11 MUT, KRAS MUT/ KEAP1 MUT, KRAS MUT/ STK11 MUT/ KEAP1 MUT, KRAS MUT/ PIK3CA MUT, KRAS MUT/ ATM MUT, and KRAS MUT without comutation. CBM identified PD-(L)1 immunotherapy sensitivity in the KRAS MUT/ TP53 MUT, KRAS MUT/ PIK3CA MUT, and KRAS MUT alone subgroups and resistance in the KEAP1 MUT containing subgroups. There was insufficient genomic information to elucidate PD-(L)1 immunotherapy sensitivity by the CBM in the KRAS MUT/ CDKN2A/ B/ C MUT, KRAS MUT/ STK11 MUT, and KRAS MUT/ ATM MUT subgroups. In an exploratory clinical cohort of 34 patients with advanced KRASMUT NSCLC treated with PD-(L)1 immunotherapy, the CBM-assessed overall survival correlated well with actual overall survival ( r = 0.80, P < .001). CONCLUSION CBM identified distinct PD-(L)1 immunotherapy sensitivity among molecular subgroups of KRASMUT NSCLC, in line with previous literature. These data provide proof-of-concept that computational modeling of tumor genomics could be used to expand on hypotheses from clinical observations of patients receiving PD-(L)1 immunotherapy and suggest mechanisms that underlie PD-(L)1 immunotherapy sensitivity.

Genetic diversity and population structure among goat genotypes in Kenya

Population structure and relationship information among goats is critical for genetic improvement, utilization and conservation. This study explored population structure and level of gene intermixing among four goat genotypes in Kenya: Alpine (n = 30), Toggenburg (n = 28), Saanen (n = 24) and Galla (n = 12). The population structuring and relatedness were estimated using principal component analysis utilizing allele frequencies of the SNP markers. Genotype relationships were evaluated based on the calculated Reynolds genetic distances. A phylogenetic tree was constructed to represent genotype clustering using iTOL software. Population structure was investigated using model-based clustering (ADMIXTURE) Genotypes relationships revealed four distinctive clusters: Alpine, Galla, Saanen and Toggenburg. The ADMIXTURE results revealed some level of gene intermixing among Alpine, Toggenburg and Saanen with Galla. Saanen goats were the most admixed genotype with 84%, 7% and 4% of its genome derived from Galla, Alpine and Toggenburg respectively. Alpine and Toggenburg goats shared some associations with the Galla goat; 10% and 1% respectively. The association of Galla with other genotypes was anticipated since Galla goat was used as the founder population for crossbreeding with Saanen, Alpine and Toggenburg breed. The genetic variations among the goat genotypes observed, will provide a good opportunity for sustainable utilization, conservation and future genetic resource improvement programs in goat genotypes in Kenya.

souporcell: Robust clustering of single cell RNAseq by genotype and ambient RNA inference without reference genotypes

Methods to deconvolve single-cell RNA sequencing (scRNAseq) data are necessary for samples containing a natural mixture of genotypes and for scRNAseq experiments that multiplex cells from different donors1. Multiplexing across donors is a popular experimental design with many benefits including avoiding batch effects2, reducing costs, and improving doublet detection. Using variants detected in the RNAseq reads, it is possible to assign cells to the individuals from which they arose. These variants can also be used to identify and remove cross-genotype doublet cells that may have highly similar transcriptional profiles precluding detection by transcriptional profile. More subtle cross-genotype variant contamination can be used to estimate the amount of ambient RNA in the system. Ambient RNA is caused by cell lysis prior to droplet partitioning and is an important confounder of scRNAseq analysis3. Souporcell is a novel method to cluster cells using only the genetic variants detected within the scRNAseq reads. We show that it achieves high accuracy on genotype clustering, doublet detection, and ambient RNA estimation as demonstrated across a wide range of challenging scenarios.

Transmission of tuberculosis and predictors of large clusters within three years in an urban setting in Tokyo, Japan: a population-based molecular epidemiological study

ObjectiveMolecular epidemiology is a promising tool for understanding tuberculosis transmission dynamics but has not been sufficiently utilised in Asian countries including Japan. The aim of this study was to estimate the proportion of TB cases attributable to recent transmission and to identify risk factors of genotype clustering and the development of large clusters within 3 years in an urban setting in Japan.Design and settingLong-term cross-sectional observational study combining the characteristics of patients with culture-positive TB notified in Shinjuku City, Tokyo (2002–2013), with genotype data ofMycobacterium tuberculosis.Primary outcome measureGenotype clustering rate and association between genotype clustering status and explanatory variables.ResultsAmong 1025 cases, 515 were localised within 113 genotype clusters. The overall clustering rate was 39.2%. Significantly higher rates were found in patients aged <40 years (adjusted odds ratio (aOR)=1.73, 95% CI 1.23 to 2.44), native Japanese individuals (aOR=3.90, 95% CI 2.27 to 6.72), full-time workers (aOR=1.63, 95% CI 1.17 to 2.27), part-time/daily workers (aOR=2.20, 95% CI 1.35 to 3.58), individuals receiving public assistance (aOR=1.81, 95% CI 1.15 to 2.84) and homeless people (aOR=1.63, 95% CI 1.02 to 2.62). A significant predictor of large genotype clusters within 3 years was a registration interval ≤2 months between the first two cases in a cluster.ConclusionOur results indicated that a large proportion of patients with culture-positive TB were involved in the recent TB transmission chain. Foreign-born persons still have a limited impact on transmission in the Japanese urban setting. Intensified public health interventions, including the active case finding, need to focus on individuals with socioeconomic risk factors that are significantly associated with tuberculosis transmission and clusters with shorter registration intervals between the first two cases.

Species delimitation in the presence of strong incomplete lineage sorting and hybridization: lessons from Ophioderma (Ophiuroidea: Echinodermata)

Accurate species delimitation is essential to properly assess biodiversity, but also for management and conservation purposes. Yet, it is not always trivial to accurately define species boundaries in closely related species due to incomplete lineage sorting. Additional difficulties may be caused by hybridization, now evidenced as a frequent phenomenon. The brittle star cryptic species complex Ophioderma longicauda encompasses six mitochondrial lineages, including broadcast spawners and internal brooders, yet the actual species boundaries are unknown. Here, we combined three methods to delimit species in the Ophioderma longicauda complex and to infer its divergence history: i) unsupervised species discovery based on multilocus genotypes; ii) divergence time estimation using the multi-species coalescent; iii) divergence scenario testing (including gene flow) using Approximate Bayesian Computation (ABC) methods. 30 sequence markers (transcriptome-based, mitochondrial or non-coding) for 89 O. longicauda and outgroup individuals were used. First, multivariate analyses revealed six genetic clusters, which globally corresponded to the mitochondrial lineages, yet with many exceptions, suggesting ancient hybridization events and challenging traditional mitochondrial barcoding approaches. Second, multi-species coalescent-based analyses confirmed the occurrence of six species and provided divergence time estimates, but the sole use of this method failed to accurately delimit species, highlighting the power of multilocus genotype clustering to delimit recently diverged species. Finally, Approximate Bayesian Computation showed that the most likely scenario involves hybridization between brooders and broadcasters. Our study shows that despite strong incomplete lineage sorting and past hybridization, accurate species delimitation in Ophioderma was possible using a combination of complementary methods. We propose that these methods, especially multilocus genotype clustering, may be useful to resolve other complex speciation histories.HighlightsMultivariate analysis was used for species delimitationSix Ophioderma species were delimited using nuclear and mitochondrial dataOphioderma speciation history is complex and included hybridizationMitochondrial and nuclear histories differed, challenging barcoding approachesWe propose that using multilocus genotypes can resolve complex speciation histories

Snapback Primer Genotyping of the Gilbert Syndrome UGT1A1 (TA)n Promoter Polymorphism by High-Resolution Melting

BACKGROUND Gilbert syndrome, a chronic nonhemolytic unconjugated hyperbilirubinemia, is associated with thymine–adenine (TA) insertions in the UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1) promoter. The UGT1A1 promoter genotype also correlates with toxicity induced by the chemotherapeutic drug irinotecan. Current closed-tube assays for genotyping the UGT1A1 (TA)n promoter polymorphism require multiple labeled probes and/or have difficulty classifying the (TA)5 and (TA)8 alleles. METHODS An unlabeled 5′ extension on one primer that creates a hairpin after asymmetric PCR was used to develop a snapback primer high-resolution melting assay for the (TA)n polymorphism. A new method that plots the local deviation from exponential decay to improve genotype clustering was used to remove background fluorescence and to analyze the data. The snapback assay was compared with small-amplicon melting and fragment length analyses in a blinded study of DNA samples from 100 African Americans. RESULTS Genotyping results obtained by small-amplicon melting and snapback primer melting were 83% and 99% concordant, respectively, with results obtained by fragment analysis. Reanalysis of the single discordant sample in the results of the snapback genotyping assay and the fragment analysis revealed an error in the fragment analysis. High-resolution melting was required for accurate snapback genotyping of the UGT1A1 (TA)n polymorphism. The 100% accuracy obtained with a capillary-based instrument fell to ≤81% with plate-based instruments. CONCLUSIONS In contrast to small-amplicon genotyping, snapback primer genotyping can distinguish all UGT1A1 promoter genotypes. Rapid-cycle PCR combined with snapback primer analysis with only 2 unlabeled PCR primers (one with a 5′ extension) and a saturating DNA dye can genotype loci with several alleles in &lt;30 min.