Multiple Nucleotide Polymorphisms Research Articles

Establishment and application of a Multiple nucleotide polymorphism molecular identification system for grape cultivars

Grapes (Vitis L.) are one of the most important and popular fruits in China. However, efficient identification of grapes is important in the protection of domestic grapes and breeders’ rights. Here, we developed a new method called multiple nucleotide polymorphisms (MNP), which combines the advantages of multiple PCR amplification and high-throughput sequencing to improve the efficiency of grape variety identification. We identified 582 universal MNP markers and screened them based on the whole genome sequencing data of 31 representative cultivars. Subsequently, MNP libraries of 60 common cultivars were constructed using multiple PCR amplification and sequencing. The pairing comparison of 60 grape materials revealed a high identification rate (>99 %) of MNP markers. Furthermore, MNP accurately distinguishes diploid and polyploid, which can assist in molecular identification of grapes. Overall, MNP molecular identification technology provides a new way for grape variety identification. Compared with SNP, MNP reduces the cost of identification because it does not need to make expensive gene chips. In addition, MNP can perform the amplification of thousands of primers in a single tube and uses sequencing instead of polyacrylamide gel electrophoresis, which greatly saves experimental consumables and labor compared with SSR. Collectively, the developed technique has the advantages of low cost and high efficiency, which is more competitive than other identification methods at present. The developed technology can assist in resource research and promote plant breeding.

Comparative genomics and phenotypic studies to determine site-specificity of Escherichia coli in the lower gastrointestinal tract of humans

ABSTRACT Escherichia coli (E. coli) is an important commensal in the human gut; however, it is unknown whether strains show site-specificity in the lower gut. To investigate this, we assessed genotypic and phenotypic differences in 37 clone pairs (two strains with very similar multiple locus variable-number-tandem-repeat analysis [MLVA] profiles) of E. coli isolated from mucosal biopsies of two different gut locations (terminal ileum and rectum). The clone pairs varied at the genomic level; single nucleotide polymorphisms (SNPs) were common, multiple nucleotide polymorphisms (MNPs) were observed but less common, and few indels (insertions and deletions) were detected. The variation was higher in clone pairs that are associated with non-human-associated sequence types (ST) compared to human-associated STs, such as ST95, ST131, and ST73. No gene(s) with non-synonymous mutations were found to be commonly associated with either the terminal ileum or the rectal strains. At the phenotypic level, we identified the metabolic signatures for some STs. Rectum strains of some STs showed consistently higher metabolic activity with particular carbon sources. Clone pairs belonging to specific STs showed distinct growth patterns under different pH conditions. Overall, this study showed that E. coli may exhibit genomic and phenotypic variability at different locations in the gut. Although genomics did not reveal significant information suggesting the site-specificity of strains, some phenotypic studies have suggested that strains may display site-specificity in the lower gut. These results provide insights into the nature and adaptation of E. coli in the lower gut of humans. To the best of our knowledge, no study has investigated or demonstrated the site-specificity of commensal E. coli in the human gut.

Development of Multiple Nucleotide Polymorphism Molecular Markers for Enoki Mushroom (Flammulina filiformis) Cultivars Identification.

The enoki mushroom (Flammulina filiformis) is one of the most important and popular edible mushrooms commercially in China. However, traditional mushroom cultivar identification is challenging due to poor accuracy, heavy workloads, and low reproducibility. To overcome this challenge, we developed a method for identifying F. filiformis strains using multiple nucleotide polymorphism sequencing (MNP-seq). This involved screening 179 universal MNP markers based on whole-genome sequencing data, constructing an MNP sequence library, and performing multiplex PCR amplification and high-sequencing. We further screened 69 core MNP markers and used them to build a neighbor-joining (NJ) phylogenetic tree of 232 cultivated and wild strains. Our analysis showed that all cultivars could be accurately separated by computing genetic similarity values and that the cultivars could be separated into 22 distinct evolutionary pedigrees. The specific value of genetic similarity can be used as the standard to distinguish F. filiformis cultivars, however, it needs to be comprehensively defined by the additional phenotype and biological characteristics of those strains in the future work.

Strategy Optimization and Clinical Analyze of Multiple Nucleotide Polymorphism Analysis in the Chimerism Detection after Allogeneic Hematopoietic Stem Cell Transplantation

The chimerism samples from November 2018 to June 2020 were collected, and Pearson correlation coefficient (r) was used to analyze the consistency of bone marrow and peripheral blood results detected by MNPseq; according to the different information integrity before transplantation, the calibration model was constructed to analyze the correction value of the micro chimerism results in each model; the clinical results were retrospectively analyzed to verify the reliability and practicability of chimerism results correction and the clinical value of MNPseq method. The results of bone marrow and peripheral blood chimerism detected by MNPseq method were consistent with each other and showed significant correlation (r=0.985, P<0.01). The three groups of calibration models were constructed according to different pre-transplant information. For the no donor and pre-transplant patients information group, the correction value was 1%; while for the group with pre-transplant patients and without donor information, 0.61% of the chimerism rate and 13 heterotopic points were used as the correction value; 0.26% of the chimerism rate and 21.57% of the heterotopic points were used as the correction value for the group with pre-transplantation patients and donor information. After correction, the number of the patients with incomplete chimerism decreased from 276 (74.19%) to 141 (37.91%) (P<0.01). Among 18 (18/141, 12.77%) patients with incomplete chimerism, the results of MNPseq in the patients were 25-39 days earlier than those in STR and flow MRD, and the result showed statistical significance. MNPseq method can be used to monitor chimerism with peripheral blood instead of bone marrow samples, and the results can be corrected to detect the changes of graft status in vivo in a more timely manner.

Assessment of the Role of NuclearENDOGGene and mtDNA Variations on Paternal Mitochondrial Elimination (PME) in Infertile Men: An Experimental Study.

In humans and most animals, maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) is considered as an universal assumption. Recently, several lines of evidence suggest that different species seem to employ distinct mechanisms to prevent the inheritance of paternal mtDNA. There are few studies in the literature on the molecular basis of sperm mtDNA elimination in mammals and paternal mtDNA transmission in humans. Endonuclease G (ENDOG) is a mitochondrial nuclease encoded by nuclear ENDOG gene. The critical importance of ENDOG gene on paternal mitochondrial elimination (PME) has been previously demonstrated in model organisms such as C. elegans and D. melanogaster. However, its mechanism in human is still unclear. Therefore, we aimed to evaluate whether nuclear ENDOG gene copy number could be a potential marker of paternal mtDNA transmission or not.Male factor infertility patients diagnosed with different infertility subgroups such as azoospermia, oligoteratozoospermia, astheno-teratozoospermia were included in this study: 13 infertile men and 25 healthy men as control group. Quantitative real-time polymerase chain reaction (qPCR) analysis and dual-color Fluorescence in situ hybridization (FISH) method were used to compare the groups. FISH method was applied to verify qPCR results and two signals were observed in nearly all patients. ENDOG gene copy number data were evaluated by comparing them with entire human mtDNA next-generation sequencing (NGS) analysis results obtained through bioinformatics and proteomics tools. Mitochondrial whole genome sequencing (WGS) data allowed determination of novel and reported variations such as single nucleotide polymorphisms (SNPs), multiple nucleotide polymorphism (MNP), insertion/deletion (INDEL). Missense variants causing amino acid substitution were filtered out from patients' mtDNA WGS data.Relative copy number of target ENDOG gene in male infertility patients [0.49 (0.31 - 0.77)] was lower than healthy controls [1.00 (0.66 - 1.51)], and statistical results showed significant differences between the groups (p < 0.01). A total of 38 missense variants were detected in the genes encoding the proteins involved in the respiratory chain complex. Moreover, we detected paternal mtDNA transmissions in the children of these patients who applied to assisted reproductive techniques.In conclusion, this study reveals that ENDOG gene may be an important factor for the PME mechanism in humans. To the best of our knowledge, this is the first study in humans about this topic and assessment of ENDOG gene sequencing and gene expression studies in a larger sample size including patients with male factor infertility would be our future project.

Phenotypic and Genetic Variability of Isolates of ZIKV-2016 in Brazil.

The possibility of a Zika virus epidemic resurgence requires studies to understand its mechanisms of pathogenicity. Here, we describe the isolation of the Zika virus from breast milk (Rio-BM1) and compare its genetic and virological properties with two other isolates (Rio-U1 and Rio-S1) obtained during the same epidemic period. Complete genomic analysis of these three viral isolates showed that they carry characteristics of the American isolates and belong to the Asian genotype. Furthermore, we detected eight non-synonymous single nucleotide variants and multiple nucleotide polymorphisms that reflect phenotypic changes. The new isolate, Rio-BM1, showed the lowest replication rates in mammalian cells, induced lower cell death rates, was more susceptible to treatment with type I IFN, and was less pathogenic than Rio-U1 in a murine model. In conclusion, the present study shows evidence that the isolate Rio-BM1 is more attenuated than Rio-U1, probably due to the impact of genetic alterations in the modulation of virulence. The results obtained in our in vitro model were consistent with the pathogenicity observed in the animal model, indicating that this method can be used to assess the virulence level of other isolates or to predict the pathogenicity of reverse genetic constructs containing other polymorphisms.

Association of ADIPOQ (rs 2241766) Gene Polymorphism with Type 2 Diabetes Mellitus Patients A Case-Control Study

The adiponectin gene (ADIPOQ) is the main genetic determinant of plasma adiponectin amounts from (30 - 70%) of the inherited genetic components. Multiple nucleotide polymorphisms (SNPs) rs 2241766 for the ADIPOQ gene correlate with metabolic syndromes such as insulin resistance, abdominal obesity, impaired glucose tolerance, hyperlipidemia, hypertension, rapid glucose excess, and plasma adiponectin level. A polymorphism study was conducted based on PCR 2241766 in Al-Diwaniyah city in Iraq, and this study included (300) people aged (30-45) years who were distributed into two groups, the first group (G1) included (150) control, and the second group (G2) included (150) type 2 diabetes mellitus patients. The purpose of the current study was to reveal the ADIPOQ polymorphism and its role in the pathophysiology of type 2 diabetes mellitus. The overall genotype of the ADIPOQ rs2241766 gene was significantly different between the type 2 diabetes mellitus patients (G2) and control (G1) for the genotype GG (χ2 = 13.45, p-value = 0.002), G allele (χ2 = 10.324, p-value = 0.001), and TG &amp; TT compared to the GG genotype (χ2 = 13.266, p-value = 0.0001). In conclusion, the results of the genotype and allele distribution of ADIPOQ rs2241766 gene in type 2 diabetes patients group showed that there is an association between ADIPOQ rs2241766 gene and type 2 diabetes mellitus.

Application of multiple nucleotide polymorphism analysis in chimerism detection after allogeneic hematopoietic stem cell transplantation

目的建立一种利用多核苷酸多态性高通量测序（MNPseq）分析异基因造血干细胞移植后嵌合状态的新方法，并探讨其可行性及优越性。方法筛选100个MNP片段，采用高通量测序技术，通过模拟嵌合样本和临床移植后样本，与STR法、融合基因定量检测和流式细胞术微小残留病检测进行对比，验证方法的准确性和敏感性。结果MNPseq的准确性和敏感性均优于STR法，其中敏感性为0.01%，较STR法敏感约100倍；MNPseq可以进一步区分STR完全嵌合的42份样本，且经Cutoff值校正后，与融合基因定量检测结果相关；MNPseq可以纠正因为影子峰所造成的STR法的假阳性，并且可以用于检测缺乏供者和（或）患者移植前信息的嵌合体标本。结论基于高通量测序的MNPseq分析是一种更加准确和敏感的嵌合体检测方法，而且解决了缺乏移植前信息无法检测嵌合体的问题，具有极高的临床应用价值。

Hg19KIndel: ethnicity normalized human reference genome

BackgroundThe most widely used human genome reference assembly hg19 harbors minor alleles at 2.18 million positions as revealed by 1000 Genome Phase 3 dataset. Although this is less than 2% of the 89 million variants reported, it has been shown that the minor alleles can result in 30% false positives in individual genomes, thus misleading and burdening downstream interpretation. More alarming is the fact that, significant percentage of variants that are homozygous recessive for these minor alleles, with potential disease implications, are masked from reporting.ResultsWe have demonstrated that the false positives (FP) and false negatives (FN) can be corrected for by simply replacing nucleotides at the minor allele positions in hg19 with corresponding major allele. Here, we have effectively replaced 2.18 million minor alleles Single Nucleotide Polymorphism (SNPs), Insertion and Deletions (INDELs), Multiple Nucleotide Polymorphism (MNPs) in hg19 with the corresponding major alleles to create an ethnically normalized reference genome called hg19KIndel. In doing so, hg19KIndel has both corrected for sequencing errors acknowledged to be present in hg19 and has improved read alignment near the minor alleles in hg19.ConclusionWe have created and made available a new version human reference genome called hg19KIndel. It has been shown that variant calling using hg19KIndel, significantly reduces false positives calls, which in-turn reduces the burden from downstream analysis and validation. It also improved false negative variants call, which means that the variants which were getting missed due to the presence of minor alleles in hg19, will now be called using hg19KIndel. Using hg19KIndel, one even gets a better mapping percentage when compared to currently available human reference genome. hg19KIndel reference genome and its auxiliary datasets are available at https://doi.org/10.5281/zenodo.2638113

PF782 MNPSEQ AS A HIGHLY SENSITIVE METHOD FOR DETECTING CHIMERISM AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

Background:Chimerism testing after allogeneic hematopoietic stem cell transplantation (Allo‐HSCT) can be used to monitor donor engraftment or to diagnose relapse. At present, short tandem repeats (STR) analysis is the most popular method for monitoring chimerism following Allo‐HSCT. However, this method suffers from a large number of technical and quality issues such as low detection sensitivity (3∼5%), stutter interference, and the need for genetic information of donor and recipient. Multiple Nucleotide Polymorphism (MNP) refers to the combination of multiple SNPs within &lt;200 bp DNA, and the polymorphisms of different SNPs are MNP. As MNP contains multiple SNPs, it is a multi‐allele genetic marker, which contains more abundant genetic information.Aims:In this study, we carried out a clinical validation study of using MNPseq to detect chimerism based on next‐generation sequencing that enable us to analyze MNPseq's sensitivity and accuracy, and to assess the possibility of MNPseq used in the detection of chimerism without genetic information of donor and recipient.Methods:A total of 106 peripheral blood or bone marrow samples were collected from donors and patients undergoing Allo‐HSCT in Hematology Department of Henan Cancer Hospital, among which these samples were simultaneously detected fusion gene by real‐time PCR (RT‐PCR) and minimal residual disease (MRD) by flow cytometry (FC). In addition, linearity and detection sensitivity were tested by serially diluting white blood cells from two unrelated normal donors with varying dilution factors by 50, 25, 12.5, 6.25, 3.12, 1, 0.5, 0.1 and 0.01%. MNPseq was detected based on next‐generation sequencing (Ion Torrent PGM), which containing 100 amplicons and 646 SNPs. Mean read depth ranged from 3,427 × to 9,524 × , and the coverage of 96.8% of SNPs was over 2,000 × . Chimerism was calculated according to the polymorphism and frequency of MNP.Results:In a comparison study using serially diluting mixed samples, chimerism measured by MNPseq showed an excellent correlation and detection sensitivity with that measured by STR analysis (correlation: r2 = 0.9992 vs. r2 = 0.9968; sensitivity threshold: 0.01% vs. 1%). Among 57 samples with no recipient chimerism detected by STR, 32 samples were positive (0.04∼2.13%) by MNPseq analysis, all were positive detected by RT‐PCR, and only 2 samples were FC positive. MNPseq, RT‐PCR and FC results were all positive in 9 samples with recipient chimerism ranged from 6% to 71%. And MNPseq analysis was also performed in the case of incomplete donor and recipient genetic information. When separate genetic information was availiable or none, MNPseq could not only be used for chimerism detection, but also for detection sensitivity could reach 0.01% or 1%. Furthermore, MNPseq results were 2.13∼1.21% in 13 of 57 samples with no recipient chimerism when genetic information of donor and recipient was absent.Summary/Conclusion:MNPseq is not only a highly sensitive chimerism detection method, but also suitable for chimerism detection without genetic information of donor and recipient.

Investigation of human paternal mitochondrial DNA transmission in ART babies whose fathers with male infertility

ObjectiveTo investigate the paternal mitochondrial DNA’s effect on assisted reproductive technology (ART) applications and possible paternal mitochondrial DNA transmission in male factor infertility diagnosed fathers. Study designStudy group was designed according to the families all of which applied to assisted reproductive technologies as a result of male infertility. A total of 16 trios (48 mother-father-child samples) which contain 7 newborns and 9 infants born by in vitro fertilization method (IVF-ICSI) were studied using “Illumina, MiSeq” next-generation sequencing platform (Case-parent trio study). The study has been conducted between February 2017 and May 2018. Result(s)Sequencing analysis results were investigated on the basis of “mother-father-child”, “mother-child” and “father-child” mitochondrial DNA whole genome sequence data, respectively. In 14 “trios” of 16; maternal mitochondrial DNA haplotype were detected for children, the remaining 2 “trios” had different mitochondrial DNA haplotypes when compared to their mother and fathers. Also; “father-child” sharing same genetic variants (SNP (“Single nucleotide polymorphism”) / MNP (“Multiple nucleotide polymorphism”) / INDEL (“Insertion/Deletion”) were found in 8 “trios”. In 5 “trios” of 16; 98–99% paternal mitochondrial DNA genome sequence similarity were obtained by alignment of “father-child” mitochondrial DNA genome. Conclusion(s)This study is the first whole mitochondrial genome investigation for paternal mitochondrial DNA contribution in human IVF / ICSI applied trio cases. Our findings for paternally derived variants could be the result of intermolecular recombination between maternal and paternal mitochondrial DNA.

Unravelling the GSK3β-related genotypic interaction network influencing hippocampal volume in recurrent major depressive disorder.

Glycogen synthase kinase 3β (GSK3β) has been implicated in mood disorders. We previously reported associations between a GSK3β polymorphism and hippocampal volume in major depressive disorder (MDD). We then reported similar associations for a subset of GSK3β-regulated genes. We now investigate an algorithm-derived comprehensive list of genes encoding proteins that directly interact with GSK3β to identify a genotypic network influencing hippocampal volume in MDD. We used discovery (N=141) and replication (N=77) recurrent MDD samples. Our gene list was generated from the NetworKIN database. Hippocampal measures were derived using an optimized Freesurfer protocol. We identified interacting single nucleotide polymorphisms using the machine learning algorithm Random Forest and verified interactions using likelihood ratio tests between nested linear regression models. The discovery sample showed multiple two-single nucleotide polymorphism interactions with hippocampal volume. The replication sample showed a replicable interaction (likelihood ratio test: P=0.0088, replication sample; P=0.017, discovery sample; Stouffer's combined P=0.0007) between genes associated previously with endoplasmic reticulum stress, calcium regulation and histone modifications. Our results provide genetic evidence supporting associations between hippocampal volume and MDD, which may reflect underlying cellular stress responses. Our study provides evidence of biological mechanisms that should be further explored in the search for disease-modifying therapeutic targets for depression.

Genetic Variability of MicroRNA Genes in 15 Animal Species.

MicroRNAs (miRNA) are a class of non-coding RNAs important in posttranscriptional regulation of target genes. Previous studies have proven that genetic variability of miRNA genes (miR-SNP) has an impact on phenotypic variation and disease susceptibility in human, mice and some livestock species. MicroRNA gene polymorphisms could therefore represent biomarkers for phenotypic traits also in other animal species. We upgraded our previously developed tool miRNA SNiPer to the version 4.0 which enables the search of miRNA genetic variability in 15 animal genomes: http://www.integratomics-time.com/miRNA-SNiPer. Genome-wide in silico screening (GWISS) of 15 genomes revealed that based on the current database releases, miRNA genes are most polymorphic in cattle, followed by human, fruitfly, mouse, chicken, pig, horse, and sheep. The difference in the number of miRNA gene polymorphisms between species is most probably not due to a biological reason and lack of genetic variability in some species, but to different stage of sequencing projects and differences in development of genomic resource databases in different species. Genome screening revealed several interesting genomic hotspots. For instance, several multiple nucleotide polymorphisms (MNPs) are present within mature seed region in cattle. Among miR-SNPs 46 are present on commercial whole-genome SNP chips: 16 in cattle, 26 in chicken, two in sheep and two in pig. The update of the miRNA SNiPer tool and the generated catalogs will serve researchers as a starting point in designing projects dealing with the effects of genetic variability of miRNA genes.

Rare allele of HvLox-1 associated with lipoxygenase activity in barley (Hordeum vulgare L.)

Key message Identification and allele-specific marker development of a functional SNP of HvLox - 1 which associated with barley lipoxygenase activity. Improving the stability of the flavor of beer is one of the main objectives in breeding barley for malting, and lipoxygenase-1 (LOX-1) is a key enzyme controlling this trait. In this study, a modified LOX activity assay was used for null LOX-1 mutant screening. Four barley landraces with no detected level of LOX-1 activity were screened from 1,083 barley germplasm accessions from China. The genomic sequence diversity of the HvLox-1 gene of the four null LOX-1 Chinese landraces was compared with that of a further 76 accessions. A total of 104 nucleotide polymorphisms were found, which contained 83 single-nucleotide polymorphisms (SNPs), 7 multiple-nucleotide polymorphisms, and 14 insertions and deletions. Most notably, we found a rare C/G mutation (SNP-61) in the second intron which led to null LOX-1 activity through an altered splicing acceptor site. In addition, an allele-specific polymerase chain reaction marker was developed for the genotyping of SNP-61, which could be used in breeding programs for barley to be used for malting. The objective was to improve beer quality.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-014-2362-3) contains supplementary material, which is available to authorized users.

IMP-HRM: an automated pipeline for high throughput SNP marker resource development for molecular breeding in orphan crops

Orphan crops have played a major role in daily diet, health nourishment, economy and livelihood of marginal farmers in under developed and developing world. For various reasons, the majority of these crops including finger millet and tef in cereals; lentil, cowpea and groundnut in legumes; cassava and yam in root crops; have remained neglected and underutilized. With little or no investment these ‘orphan’ crops have lagged far behind in developing genomic resources and are deprived of the application of advanced molecular breeding approaches for their rapid improvement. Single nucleotide polymorphisms, multiple nucleotide polymorphisms, insertions/deletions (indels) and size polymorphisms are important tools for developing rich molecular marker resources. Cataloguing and use of intron based polymorphism have also been demonstrated for applications in molecular breeding. Currently the process of predicting putative introns and designing flanking primers for their amplification requires multiple computer programs and a high level of human intervention. We automated this process by developing intron mapping pipeline (IMP) which allows the generation of large primer sets with minimal human intervention and greater efficiency. Since IMP can take advantage of the highly conserved nature of the introns flanking sequences across a wide range of species, any available model plant genome can be used to predict intron boundaries in related orphan crop species. IMP combined with high resolution melt analysis demonstrated potential for high throughput polymorphism discovery, validation and molecular marker development platform which can generate genomic resources rapidly in orphan species.

Global Genomic Diversity of Oryza sativa Varieties Revealed by Comparative Physical Mapping

Bacterial artificial chromosome (BAC) physical maps embedding a large number of BAC end sequences (BESs) were generated for Oryza sativa ssp. indica varieties Minghui 63 (MH63) and Zhenshan 97 (ZS97) and were compared with the genome sequences of O. sativa spp. japonica cv. Nipponbare and O. sativa ssp. indica cv. 93-11. The comparisons exhibited substantial diversities in terms of large structural variations and small substitutions and indels. Genome-wide BAC-sized and contig-sized structural variations were detected, and the shared variations were analyzed. In the expansion regions of the Nipponbare reference sequence, in comparison to the MH63 and ZS97 physical maps, as well as to the previously constructed 93-11 physical map, the amounts and types of the repeat contents, and the outputs of gene ontology analysis, were significantly different from those of the whole genome. Using the physical maps of four wild Oryza species from OMAP (http://www.omap.org) as a control, we detected many conserved and divergent regions related to the evolution process of O. sativa. Between the BESs of MH63 and ZS97 and the two reference sequences, a total of 1532 polymorphic simple sequence repeats (SSRs), 71,383 SNPs, 1767 multiple nucleotide polymorphisms, 6340 insertions, and 9137 deletions were identified. This study provides independent whole-genome resources for intra- and intersubspecies comparisons and functional genomics studies in O. sativa. Both the comparative physical maps and the GBrowse, which integrated the QTL and molecular markers from GRAMENE (http://www.gramene.org) with our physical maps and analysis results, are open to the public through our Web site (http://gresource.hzau.edu.cn/resource/resource.html).

A High-Density Gene Map of Loblolly Pine (Pinus taedaL.) Based on Exome Sequence Capture Genotyping

Loblolly pine (Pinus taeda L.) is an economically and ecologically important conifer for which a suite of genomic resources is being generated. Despite recent attempts to sequence the large genome of conifers, their assembly and the positioning of genes remains largely incomplete. The interspecific synteny in pines suggests that a gene-based map would be useful to support genome assemblies and analysis of conifers. To establish a reference gene-based genetic map, we performed exome sequencing of 14729 genes on a mapping population of 72 haploid samples, generating a resource of 7434 sequence variants segregating for 3787 genes. Most markers are single-nucleotide polymorphisms, although short insertions/deletions and multiple nucleotide polymorphisms also were used. Marker segregation in the population was used to generate a high-density, gene-based genetic map. A total of 2841 genes were mapped to pine’s 12 linkage groups with an average of one marker every 0.58 cM. Capture data were used to detect gene presence/absence variations and position 65 genes on the map. We compared the marker order of genes previously mapped in loblolly pine and found high agreement. We estimated that 4123 genes had enough sequencing depth for reliable detection of markers, suggesting a high marker conversation rate of 92% (3787/4123). This is possible because a significant portion of the gene is captured and sequenced, increasing the chances of identifying a polymorphic site for characterization and mapping. This sub-centiMorgan genetic map provides a valuable resource for gene positioning on chromosomes and guide for the assembly of a reference pine genome.

Diverticulosis and Inflammatory Bowel Disease in DiGeorge Syndrome: A Double Whammy

Purpose: Diverticulosis is uncommon under the age of 40. Prevalence rate is approximately 5-10% in people younger than 50 years old, and increases with age thereafter. We present a case of an adolescent with DiGeorge syndrome who has inflammatory bowel disease (IBD) with co-occurring colonic diverticula. DiGeorge syndrome has a wide clinical spectrum, and its gastrointestinal manifestations are numerous and varied. However, inflammatory bowel disease as an association is quite rare. Moreover, diverticulosis linked to DiGeorge syndrome is unheard of. We present an 18-year-old Caucasian male with a complex past medical history, most notably DiGeorge syndrome, who presented with a 3-week history of loose, watery diarrhea with hematochezia. Associated symptoms included crampy abdominal pain, anorexia, 29% weight loss, and profound prostration. He also complained of tongue sores, odynophagia, and arthralgia. Pertinent physical examination findings showed patient to be severely cachectic and dehydrated, with multiple oral and tongue ulcers, scaphoid and diffusely tender abdomen on palpation, and the rectal exam had a grossly positive hemoccult stool. Blood work upon admission revealed anemia, elevated inflammatory markers (ESR 77, CRP 97), and severe electrolyte abnormalities. Patient was managed with bowel rest, rehydration, and empiric antibiotics. Parental nutrition was initiated with close monitoring for re-feeding syndrome. IBD serology 7 came back with multiple nucleotide polymorphism mutations detected. Magnetic resonance enterography showed abnormal colon with thickening, fat stranding, and significant fibrosis from chronic inflammatory process. Following improvement and stabilization of his clinical status, EGD and colonoscopy with biopsy was performed, which showed widespread inflammation and ulceration, findings consistent with IBD, with a surprising finding of multiple diverticula in the transverse colon (Figure 1). The patient was started on corticosteroid therapy, and was subsequently discharged improved and stable, on a long corticosteroid taper regimen. It is the intent of this report to describe a possible association between DiGeorge syndrome and the co-occurrence of diverticula in such patients.Figure: Diverticula in transverse colon.

Tool for rapid annotation of microbial SNPs (TRAMS): a simple program for rapid annotation of genomic variation in prokaryotes

Next generation sequencing (NGS) has been widely used to study genomic variation in a variety of prokaryotes. Single nucleotide polymorphisms (SNPs) resulting from genomic comparisons need to be annotated for their functional impact on the coding sequences. We have developed a program, TRAMS, for functional annotation of genomic SNPs which is available to download as a single file executable for WINDOWS users with limited computational experience and as a Python script for Mac OS and Linux users. TRAMS needs a tab delimited text file containing SNP locations, reference nucleotide and SNPs in variant strains along with a reference genome sequence in GenBank or EMBL format. SNPs are annotated as synonymous, nonsynonymous or nonsense. Nonsynonymous SNPs in start and stop codons are separated as non-start and non-stop SNPs, respectively. SNPs in multiple overlapping features are annotated separately for each feature and multiple nucleotide polymorphisms within a codon are combined before annotation. We have also developed a workflow for Galaxy, a highly used tool for analysing NGS data, to map short reads to a reference genome and extract and annotate the SNPs. TRAMS is a simple program for rapid and accurate annotation of SNPs that will be very useful for microbiologists in analysing genomic diversity in microbial populations.

Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift.

This paper describes a new program SnpSift for filtering differential DNA sequence variants between two or more experimental genomes after genotoxic chemical exposure. Here, we illustrate how SnpSift can be used to identify candidate phenotype-relevant variants including single nucleotide polymorphisms, multiple nucleotide polymorphisms, insertions, and deletions (InDels) in mutant strains isolated from genome-wide chemical mutagenesis of Drosophila melanogaster. First, the genomes of two independently isolated mutant fly strains that are allelic for a novel recessive male-sterile locus generated by genotoxic chemical exposure were sequenced using the Illumina next-generation DNA sequencer to obtain 20- to 29-fold coverage of the euchromatic sequences. The sequencing reads were processed and variants were called using standard bioinformatic tools. Next, SnpEff was used to annotate all sequence variants and their potential mutational effects on associated genes. Then, SnpSift was used to filter and select differential variants that potentially disrupt a common gene in the two allelic mutant strains. The potential causative DNA lesions were partially validated by capillary sequencing of polymerase chain reaction-amplified DNA in the genetic interval as defined by meiotic mapping and deletions that remove defined regions of the chromosome. Of the five candidate genes located in the genetic interval, the Pka-like gene CG12069 was found to carry a separate pre-mature stop codon mutation in each of the two allelic mutants whereas the other four candidate genes within the interval have wild-type sequences. The Pka-like gene is therefore a strong candidate gene for the male-sterile locus. These results demonstrate that combining SnpEff and SnpSift can expedite the identification of candidate phenotype-causative mutations in chemically mutagenized Drosophila strains. This technique can also be used to characterize the variety of mutations generated by genotoxic chemicals.