A multiplexed, three-dimensional pooling and next-generation sequencing strategy for creating barcoded mutant arrays: construction of a Schizosaccharomyces pombe transposon insertion library.

Arrayed libraries of defined mutants have been used to elucidate gene function in the post-genomic era. Yeast haploid gene deletion libraries have pioneered this effort, but are costly to construct, do not reveal phenotypes that may occur with partial gene function and lack essential genes required for growth. We therefore devised an efficient method to construct a library of barcoded insertion mutants with a wider range of phenotypes that can be generalized to other organisms or collections of DNA samples. We developed a novel but simple three-dimensional pooling and multiplexed sequencing approach that leveraged sequence information to reduce the number of required sequencing reactions by orders of magnitude, and were able to identify the barcode sequences and DNA insertion sites of 4391 Schizosaccharomyces pombe insertion mutations with only 40 sequencing preparations. The insertion mutations are in the genes and untranslated regions of nonessential, essential and noncoding RNA genes, and produced a wider range of phenotypes compared to the cognate deletion mutants, including novel phenotypes. This mutant library represents both a proof of principle for an efficient method to produce novel mutant libraries and a valuable resource for the S. pombe research community.

Novel recurrently mutated genes in African American colon cancers.

We used whole-exome and targeted sequencing to characterize somatic mutations in 103 colorectal cancers (CRC) from African Americans, identifying 20 new genes as significantly mutated in CRC. Resequencing 129 Caucasian derived CRCs confirmed a 15-gene set as a preferential target for mutations in African American CRCs. Two predominant genes, ephrin type A receptor 6 (EPHA6) and folliculin (FLCN), with mutations exclusive to African American CRCs, are by genetic and biological criteria highly likely African American CRC driver genes. These previously unsuspected differences in the mutational landscapes of CRCs arising among individuals of different ethnicities have potential to impact on broader disparities in cancer behaviors.

Genomewide analysis of divergence of antibiotic resistance determinants in closely related isolates of Acinetobacter baumannii.

Multidrug resistance has emerged as a significant concern with infections caused by Acinetobacter baumannii. Ample evidence supports the involvement of mobile genetic elements in the transfer of antibiotic resistance genes, but the extent of variability and the rate of genetic change associated with the acquisition of antibiotic resistance have not been studied in detail. Whole-genome sequence analysis of six closely related clinical isolates of A. baumannii, including four from the same hospital, revealed extensive divergence of the resistance genotype that correlated with observed differences in antimicrobial susceptibility. Resistance genes associated with insertion sequences, plasmids, and a chromosomal resistance gene island all showed variability. The highly dynamic resistance gene repertoire suggests rapid evolution of drug resistance.

Comparative genome sequence analysis of multidrug-resistant Acinetobacter baumannii.

The recent emergence of multidrug resistance (MDR) in Acinetobacter baumannii has raised concern in health care settings worldwide. In order to understand the repertoire of resistance determinants and their organization and origins, we compared the genome sequences of three MDR and three drug-susceptible A. baumannii isolates. The entire MDR phenotype can be explained by the acquisition of discrete resistance determinants distributed throughout the genome. A comparison of closely related MDR and drug-susceptible isolates suggests that drug efflux may be a less significant contributor to resistance to certain classes of antibiotics than inactivation enzymes are. A resistance island with a variable composition of resistance determinants interspersed with transposons, integrons, and other mobile genetic elements is a significant but not universal contributor to the MDR phenotype. Four hundred seventy-five genes are shared among all six clinical isolates but absent from the related environmental species Acinetobacter baylyi ADP1. These genes are enriched for transcription factors and transporters and suggest physiological features of A. baumannii that are related to adaptation for growth in association with humans.

Transcriptomic analysis of changes in gene expression of immune proteins of gill tissue in response to low environmental temperature in fathead minnows (Pimephales promelas).

In the face of ongoing climate change, it is imperative to understand better the effects of temperature on immune function in freshwater teleosts. It is unclear whether previously observed changes were caused by temperature per se. We studied changes in the gill transcriptome of fathead minnows (Pimephales promelas) at low temperature to understand better the effects of temperature on immune function. De novo assembly of the transcriptome using Trinity software resulted in 73,378 assembled contigs. Annotation using the Trinotate package yielded 58,952 Blastx hits (accessions). Expression of 194 unique mRNA transcripts changed in gill tissue of fathead minnows acclimatized to 5° compared to controls at 22 °C. At 5 °C mRNAs coding for proteins involved in innate immune responses were up-regulated. Those included proteins that block early-stage viral replication and macrophage activation. Expression of mRNAs coding for pro-inflammatory molecules and mucus secretion were also enhanced. Messenger RNAs coding for proteins associated with adaptive immune responses were down-regulated at 5 °C. Those included antigen-presenting proteins and proteins involved in immunoglobin production. Messenger RNAs coding for proteins that stimulate the cell cycle were also down-regulated at 5 °C. Histological comparison revealed that gills of cold acclimated fish had fewer mucus cells but cells contained larger mucus droplets. We conclude that decreased temperature modifies the immune systems of freshwater teleosts, leading to genome-wide upregulation of innate immunity and down regulation of adaptive immunity. Such acclimation likely evolved as an adaptive strategy against seasonal changes in infectious insults.

The correlation between ancestry and color in two cities of Northeast Brazil with contrasting ethnic compositions.

The degree of admixture in Brazil between historically isolated populations is complex and geographically variable. Studies differ as to what the genetic and phenotypic consequences of this mixing have been. In Northeastern Brazil, we enrolled 522 residents of Salvador and 620 of Fortaleza whose distributions of self-declared color were comparable to those in the national census. Using the program Structure and principal components analysis there was a clear correlation between biogeographic ancestry and categories of skin color. This correlation with African ancestry was stronger in Salvador (r=0.585; P<0.001) than in Fortaleza (r=0.236; P<0.001). In Fortaleza, although self-declared blacks had a greater proportion of European ancestry, they had more African ancestry than the other categories. When the populations were analyzed without pseudoancestors, as in some studies, the relationship of 'race' to genetic ancestry tended to diffuse or disappear. The inclusion of different African populations also influenced ancestry estimates. The percentage of unlinked ancestry informative markers in linkage disequilibrium, a measure of population structure, was 3-5 times higher in both Brazilian populations than expected by chance. We propose that certain methods, ascertainment bias and population history of the specific populations surveyed can result in failure to demonstrate a correlation between skin color and genetic ancestry. Population structure in Brazil has important implications for genetic studies, but genetic ancestry is irrelevant for how individuals are treated in society, their health, their income or their inclusion. These track more closely with perceived skin color than genetic ancestry.

Inactivating mutation in the prostaglandin transporter gene, SLCO2A1, associated with familial digital clubbing, colon neoplasia, and NSAID resistance.

HPGDand SLCO2A1 genes encode components of the prostaglandin catabolic pathway, with HPGD encoding the degradative enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH), and SLCO2A1 encoding the prostaglandin transporter PGT that brings substrate to 15-PGDH. HPGD-null mice show increased prostaglandin E2 (PGE2), marked susceptibility to developing colon tumors, and resistance to colon tumor prevention by nonsteroidal anti-inflammatory drugs (NSAID). But in humans, HPGD and SLCO2A1 mutations have only been associated with familial digital clubbing. We, here, characterize a family with digital clubbing and early-onset colon neoplasia. Whole-exome sequencing identified a heterozygous nonsense mutation (G104X) in the SLCO2A1 gene segregating in 3 males with digital clubbing. Two of these males further demonstrated notably early-onset colon neoplasia, 1 with an early-onset colon cancer and another with an early-onset sessile serrated colon adenoma. Two females also carried the mutation, and both these women developed sessile serrated colon adenomas without any digital clubbing. Males with clubbing also showed marked elevations in the levels of urinary prostaglandin E2 metabolite, PGE-M, whereas, female mutation carriers were in the normal range. Furthermore, in the male proband, urinary PGE-M remained markedly elevated during NSAID treatment with either celecoxib or sulindac. Thus, in this human kindred, a null SLCO2A1 allele mimics the phenotype of the related HPGD-null mouse, with increased prostaglandin levels that cannot be normalized by NSAID therapy, plus with increased colon neoplasia. The development of early-onset colon neoplasia in male and female human SLCO2A1 mutation carriers suggests that disordered prostaglandin catabolism can mediate inherited susceptibility to colon neoplasia in man.