The Drosophila melanogaster Genetic Reference Panel.

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype-phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype-phenotype mapping using the power of Drosophila genetics.

Comparative validation of the D. melanogaster modENCODE transcriptome annotation.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community.

Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines.

The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed toward larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting that their nonrandom distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia, and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome-wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes, and analysis and visualization tools are publicly available.

A Comparison of Seegene Technologies Novaplex SARS-CoV-2 Variants I, II, and IV Assays with Spike Gene Sequencing for Detection of Known Severe Acute Respiratory Syndrome Coronavirus 2 Variants.

Tracking new and emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has become increasingly important for public health responses, primarily because of variant-dependent transmission, disease severity, and treatment decisions. This evaluation compared Seegene Technologies Novaplex SARS-CoV-2 Variants I, II, and IV (I,II&IV) assays to detect known SARS-CoV-2 variants using traditional spike gene Sanger sequencing results as the gold standard reference. Both RNA extraction and extraction-free protocols were assessed. A total of 156 samples were included in this study. There was 100% (109/109) overall agreement (95% CI, 96.7%-100%) between the spike gene sequencing and the I,II&IV results using extracted RNA for the variants included in the Novaplex assay menus. The RNA extraction-free method was 91.7% (143/156) as sensitive (95% CI, 86.2%-95.5%) as the traditional RNA extraction method. Using the extraction-free method on samples with higher cycle threshold values (>30) resulted in some mutations not being detected, presumably due to lower nucleic acid concentrations in the original samples. In conclusion, the I,II&IV assays provide an accurate, rapid, and less labor-intensive method for detecting SARS-CoV-2 and identifying known variants of interest and concern. The RNA extraction-free method for samples with cycle threshold of <30 could be cost-effective for surveillance purposes. However, spike gene sequencing retains the advantage of detecting more and new variants.

Vaccination Decreases the Infectious Viral Load of Delta Variant SARS-CoV-2 in Asymptomatic Patients.

The Delta variant of SARS-CoV-2 has caused many breakthrough infections in fully vaccinated individuals. While vaccine status did not generally impact the number of viral RNA genome copies in nasopharyngeal swabs of breakthrough patients, as measured by Ct values, it has been previously found to decrease the infectious viral load in symptomatic patients. We quantified the viral RNA, infectious virus, and anti-spike IgA in nasopharyngeal swabs collected from individuals asymptomatically infected with the Delta variant of SARS-CoV-2. Vaccination decreased the infectious viral load, but not the amount of viral RNA. Furthermore, vaccinees with asymptomatic infections had significantly higher levels of anti-spike IgA in their nasal secretions compared to unvaccinated individuals with asymptomatic infections. Thus, vaccination may decrease the transmission risk of Delta, and perhaps other variants, despite not affecting the amount of viral RNA measured in nasopharyngeal swabs.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

Vascular-specific increase in exon 1B-encoded CAV1.2 channels in spontaneously hypertensive rats.

BACKGROUND: The vasculature of the adult spontaneously hypertensive rat (SHR) is known to express more functional L-type Ca channels than the vasculature of normotensive Wistar Kyoto (WKY) rats, but it is not known which Ca(V)1.2 channel isoform is upregulated. METHODS: Western blots and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were used to compare the expression levels of Ca(V)1.2 channel protein and message in selected tissues of adult SHR and WKY rats. RESULTS: The results indicate overexpression in SHR vasculature specifically of the short exon 1b-encoded amino terminus Ca(V)1.2 isoform. Brain and visceral smooth muscle expressing the same isoform were not similarly affected. Differences in message levels are insufficient to account for the differences in isoform-specific protein levels. CONCLUSIONS: We conclude that SHR vasculature must regulate the channel postranscriptionally. Further experiments will be required to determine whether this involves translation of protein from exon 1b-specific transcripts more efficiently, posttranslational chaperoning to the surface membrane more efficiently, or selective degradation of the short amino terminus form of the protein more slowly than in WKY vasculature.

Expression of multiple CaV1.2 transcripts in rat tissues mediated by different promoters.

The expression of two different transcripts for Ca(V)1.2 in rat tissues mirrors that which has previously been described for human tissue, in that expression of transcripts expressing exon 1a is predominant only in heart, whereas expression of transcripts expressing exon 1b is greater in smooth muscle rich tissues such as aorta and intestine. Transcripts expressing exon 1b also predominate in brain and in diaphragm. Western blots indicate that the N-terminus coded for by exon 1b is present in much of the protein in all these tissues except heart. The promoter just upstream of exon 1b has been cloned, sequenced and utilized to drive expression of luciferase in smooth muscle A7r5 cells, cardiac HL-1 cells, skeletal muscle L6 cells and neuronal PC12 cells. The nucleotide sequence of the promoter exhibits 80% identity with the equivalent promoter previously identified in humans and 94% identity with the sequence of the equivalent region of the mouse genome. Evidence in favor of still another promoter upstream of exon 2 has been uncovered.

Negative transcriptional regulation of human colonic smooth muscle Cav1.2 channels by p50 and p65 subunits of nuclear factor-kappaB.

BACKGROUND & AIMS: The expression of Cav1.2 channels in colonic circular smooth muscle cells and the contractility of these cells are suppressed in inflammation. Our aim was to investigate whether the activation of p50 and p65 nuclear factor-kappaB subunits mediates these effects. METHODS: Primary cultures of human colonic circular smooth muscle cells and muscle strips were used. RESULTS: The messenger RNA and protein expression of the pore-forming alpha1C subunit of Cav1.2 channels decreased time dependently in response to tumor necrosis factor alpha. This effect was blocked by prior transient transfection of the cells with antisense oligonucleotides to p50 or p65. The overexpression of p50 and p65 inhibited the constitutive expression of alpha1C. Three putative kappaB binding motifs were identified on the 5' flanking region of exon 1b of the human L-type calcium channel alpha1C gene. Progressive 5' deletions of the promoter and point mutations of the kappaB binding motifs indicated that the two 5' binding sites, but not the third 3' binding site, were essential for the suppression of alpha1C. Transient transfection of human colonic circular muscle strips with antisense oligonucleotides to p50 and p65 decreased expression of the 2 nuclear factor-kappaB units and reversed the suppression of alpha1C, as well as that of the contractile response to acetylcholine, by 24 hours of treatment with tumor necrosis factor alpha. CONCLUSIONS: The activation of p50 and p65 by tumor necrosis factor alpha suppresses the expression of the alpha1C subunit of Cav1.2 channels in human colonic circular smooth muscle cells and their contractile response to acetylcholine. Nuclear factor-kappaB must bind concurrently to the two 5' kappaB motifs on the promoter of alpha1C to produce this effect.

Smooth muscle uses another promoter to express primarily a form of human Cav1.2 L-type calcium channel different from the principal heart form.

Several different first exons and amino termini have been reported for the cardiac Ca channel known as alpha(1C) or Ca(V)1.2. The aim of this study was to investigate whether the expression of this channel is regulated by different promoters in smooth muscle cells and in heart in humans. Ribonuclease protection assay (RPA) indicates that the longer first exon 1a is found in certain human smooth muscle-containing tissues, notably bladder and fetal aorta, but that it is not expressed to any significant degree in lung or intestine. On the other hand, all four smooth muscle-containing tissues examined strongly express transcripts containing exon 1b, first reported cloned from human fibroblast cells. In addition, primary cultures of human colonic myocytes and coronary artery smooth muscle cells express predominantly transcripts containing exon 1b. The promoter immediately upstream of exon 1b was cloned, and it displays functional promoter activity when luciferase-expressing constructs were transfected into three different cultured smooth muscle cells: primary human coronary artery smooth muscles cells, primary human colonocytes, and the fetal rat aorta-derived A7r5 cell line. These results indicate that expression in smooth muscle is primarily driven by a promoter different from that which drives expression in cardiac myocytes.

A new promoter for alpha1C subunit of human L-type cardiac calcium channel Ca(V)1.2.

The cardiac Ca channel known as alpha1C or Ca(V)1.2 is shown to express a new longer first exon equivalent to that formerly reported in rabbit heart or rat aorta. Ribonuclease protection assay indicates that this exon is found in the majority of Ca(V)1.2 transcripts in human heart RNA. The presence of this exon also suggests that expression of this transcript is driven by a promoter immediately upstream of this exon and its 5' untranslated region. The putative promoter exhibits 69% homology to its rat counterpart and displays functional promoter activity when transfected into heart cells in culture in luciferase-expressing constructs.