Nimbus: a design-driven analyses suite for amplicon-based NGS data.

Motivation: PCR-based DNA enrichment followed by massively parallel sequencing is a straightforward and cost effective method to sequence genes up to high depth. The full potential of amplicon-based sequencing assays is currently not achieved as analysis methods do not take into account the source amplicons of the detected variants. Tracking the source amplicons has the potential to identify systematic biases, enhance variant calling and improve the designs of future assays. Results: We present Nimbus, a software suite for the analysis of amplicon-based sequencing data. Nimbus includes tools for data pre-processing, alignment, single nucleotide polymorphism (SNP), insertion and deletion calling, quality control and visualization. Nimbus can detect SNPs in its alignment seeds and reduces alignment issues by the usage of decoy amplicons. Tracking the amplicons throughout analysis allows easy and fast design optimization by amplicon performance comparison. It enables detection of probable false positive variants present in a single amplicon from real variants present in multiple amplicons and provides multiple sample visualization. Nimbus was tested using HaloPlex Exome datasets and outperforms other callers for low-frequency variants. The variants called by Nimbus were highly concordant between twin samples and SNP-arrays. The Nimbus suite provides an end-to-end solution for variant calling, design optimization and visualization of amplicon-derived next-generation sequencing datasets. Availability and implementation: https://github.com/erasmus-center-for-biomics/Nimbus. Supplementary information: Supplementary data are available at Bioinformatics online.

Bidirectional action of the Igf2r imprint control element on upstream and downstream imprinted genes.

Imprinting of the maternally-expressed Igf2r gene is controlled by an intronic imprint control element (ICE) known as Region2 that contains the promoter of the noncoding Air RNA, whose transcript overlaps the silenced paternal Igf2r promoter in an antisense orientation. Two novel imprinted genes, Slc22a2 and Slc22a3 are described here that lie 110 and 155 kb 3' to Igf2r and that are not overlapped by the Air transcript but are regulated by the Igf2r-ICE, as previously shown for Igf2r. These results identify a new cluster of imprinted genes whose repression by the bidirectional action of the Region2-ICE is independent of transcript overlap by the Air RNA.

Investigation of elements sufficient to imprint the mouse Air promoter.

Imprinted maternal-allele-specific expression of the mouse insulin-like growth-factor type 2 receptor (Igf2r) gene depends on a 3.7-kb element named region 2, located in the second intron of the gene. Region 2 carries a maternal-allele-specific methylation imprint and contains an imprinted CpG island promoter (Air) that expresses a noncoding antisense RNA from the paternal inherited allele only. Here, we use transgenes to test the minimal requirements for imprinting of Air and to test if the action of region 2 is restricted to Igf2r. Transgenes up to 9 kb with Air as a single promoter are expressed but not imprinted. When coupled to the Igf2r CpG island promoter on a 44-kb transgene, Air was imprinted in one of three lines. However, Air on a 4.6-kb fragment is also imprinted in 2 of 14 lines when inserted in an intron of an adenine phosphoribosyltransferase (Aprt) transgene, and in one line, the imprinted methylation and expression of Air have been transferred onto the Aprt CpG island promoter. These data suggest that a dual CpG island promoter setting may facilitate Air imprinting as a short transgene and also show that Air can transfer imprinting onto other genes. However, for reliable Air imprinting, elements are necessary that are located outside a 44-kb region spanning the Air-Igf2r promoters.

Allelic IGF2R repression does not correlate with expression of antisense RNA in human extraembryonic tissues.

In the mouse, expression of an antisense Igf2r RNA (Air) is correlated with Igf2r repression on the paternal allele. One of the possible models for Igf2r repression could be through promoter competition or through the action of the Air RNA, in, e.g., transcriptional interference or repressor binding. These models predict the conservation of AIR RNA in human samples with monoallelic IGF2R expression and the production of AIR RNA in first-trimester human tissues. However, by strand-specific RT-PCR and by ribonuclease protection assay we have not detected any AIR RNA in first-trimester placental tissue samples, not even in samples that downregulate IGF2R expression in an allele-specific manner. This indicates that in contrast to the mouse, allelic IGF2R repression in the developing human placenta does not correlate with AIR expression.

The uniqueness of the imprinting mechanism.

An epigenetic imprinting mechanism that is based on a gamete-specific methylation imprint restricts expression of a subset of mammalian genes to one parental chromosome. Recent results suggest that imprints may act only indirectly to induce monoallelic expression of coding genes. Instead, atypical non-coding RNAs appear to be a primary target of the imprints, and their parental-specific repression correlates with parental-specific expression of linked coding genes.

Concerted action of cytosolic Ca2+ and protein kinase C in receptor-mediated phospholipase D activation in Chinese hamster ovary cells expressing the cholecystokinin-A receptor.

Receptor-mediated activation of phosphatidylcholine phosphatidohydrolase or phospholipase D (PLD) was studied in Chinese hamster ovary (CHO) cells expressing the cholecystokinin-A (CCK-A) receptor. Cells were labelled with [3H]myristic acid for 24 h and PLD-catalysed [3H]phosphatidylethanol formation was measured in the presence of 1% (v/v) ethanol. Cholecystokinin-(26-33)-peptide amide (CCK8) increased PLD activity both time- and dose-dependently. Maximal activation of protein kinase C (PKC) with 1 microM PMA or sustained elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) with 1 microM thapsigargin increased PLD activity to 50% and 70% of the maximal value obtained with CCK8 respectively. The stimulatory effects of CCK8, PMA and thapsigargin were abolished in cells in which PKC was downregulated or inhibited by chelerythrine. PMA/Ca2+-stimulated PLD activity was absent in a homogenate of PKC-downregulated cells but could be restored upon addition of purified rat brain PKC. CCK8-induced PLD activation was inhibited by 90% in the absence of external Ca2+, demonstrating that receptor-mediated activation of PKC in itself does not significantly add to PLD activation but requires a sustained increase in [Ca2+]i. Taken together, the results presented demonstrate that, in CHO-CCK-A cells, receptor-mediated PLD activation is completely dependent on PKC, but that the extent to which PLD becomes activated depends largely, if not entirely, on the magnitude and duration of the agonist-induced increase in [Ca2+]i.

A highly conserved red pigment-concentrating hormone precursor in the blue crab Callinectes sapidus.

A cDNA library was established from the eyestalk ganglia of the blue crab Callinectes sapidus. One clone was isolated (644 bp excluding the poly(A) tail) which encodes the red pigment-concentrating hormone (RPCH)-precursor, consisting of the 25 amino acid residue signal peptide, the RPCH, and a 73 amino acid residue RPCH-precursor related peptide. This clone displays high sequence similarity with a clone isolated from an eyestalk cDNA library of the shore crab Carcinus maenas, in accordance with the close phylogenetic relationship between these species. Northern blot experiments indicated the presence of two different mRNA transcripts which hybridized with a specific RPCH-cDNA probe pointing to the possibility of multiple RPCH isoforms in the blue crab. Although crustacean RPCH and the insect adipokinetic hormones (AKH) are structurally related, their precursors show little similarity.

Molecular cloning of two pigment-dispersing hormone (PDH) precursors in the blue crab Callinectes sapidus reveals a novel member of the PDH neuropeptide family.

A cDNA library was established from the eyestalk ganglia of the blue crab Callinectes sapidus. Screening resulted in the isolation of a clone (497 bp excluding poly(A) tail) which encodes a beta-PDH previously found in several crustacean species. It displays high sequence similarity with a clone isolated from an eyestalk cDNA library of the shore crab Carcinus maenas, indicating the close phylogenetic relationship of both species. A second clone (414 bp exclusive of the poly(A) tail) encodes a novel beta-PDH analog which displays 400-fold less potency in crab bioassays. Both cDNAs encode open reading frames of 234 bp for the prepropeptides, consisting of signal peptides, PDH-precursor-related peptides, and PDH sequences.

Cloning and expression of mRNA encoding prepro-gonad-inhibiting hormone (GIH) in the lobster Homarus americanus.

The gonad-inhibiting hormone (GIH) is produced in the eyestalk X-organ sinus gland complex of male and female lobsters, and plays a prominent role in the regulation of reproduction, e.g. inhibition of vitellogenesis in female animals. To study this neurohormone at the mRNA level, we cloned and sequenced a cDNA which encodes GIH in the lobster Homarus americanus. The structure of preproGIH consists of a signal peptide and the GIH peptide itself. A comparative analysis revealed that lobster GIH, together with crab molt-inhibiting hormone, belongs to a separate group of the crustacean hyperglycemic hormone (CHH) peptide family which seems to be unique for crustaceans. Expression studies showed that GIH mRNA is expressed in the eyestalk, indicating that the neuroendocrine center in this optic structure is the only source of GIH. As this center modulates the other (neuro)endocrine organs in crustaceans, it is postulated that GIH regulates production and release of hormones involved in reproduction/molting processes.