GRAde: a long-read sequencing approach to efficiently identifying the CYP11B1/CYP11B2 chimeric form in patients with glucocorticoid-remediable aldosteronism

Abstract Background Glucocorticoid-remediable aldosteronism (GRA) is a form of heritable hypertension caused by a chimeric fusion resulting from unequal crossing over between 11β‐hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), which are two genes with similar sequences. Different crossover patterns of the CYP11B1 and CYP11B2 chimeric genes may be associated with a variety of clinical presentations. It is therefore necessary to develop an efficient approach for identifying the differences between the hybrid genes of a patient with GRA. Results We developed a long-read analysis pipeline named GRAde (GRA deciphering), which utilizes the nonidentical bases in the CYP11B1 and CYP11B2 genomic sequences to identify and visualize the chimeric form. We sequenced the polymerase chain reaction (PCR) products of the CYP11B1/CYP11B2 chimeric gene from 36 patients with GRA using the Nanopore MinION device and analyzed the sequences using GRAde. Crossover events were identified for 30 out of the 36 samples. The crossover sites appeared in the region exhibiting high sequence similarity between CYP11B1 and CYP11B2, and 53.3% of the cases were identified as having a gene conversion in intron 2. More importantly, there were six cases for whom the PCR products indicated a chimeric gene, but the GRAde results revealed no crossover pattern. The crossover regions were further verified by Sanger sequencing analysis. Conclusions PCR-based target enrichment followed by long-read sequencing is an efficient and precise approach to dissecting complex genomic regions, such as those involved in GRA mutations, which could be directly applied to clinical diagnosis. The scripts of GRAde are available at https://github.com/hsu-binfo/GRAde.

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Genome assembly and characterization of a complex zfBED-NLR gene-containing disease resistance locus in Carolina Gold Select rice with Nanopore sequencing

10.1101/675678 ◽

2019 ◽

Author(s):

Andrew C. Read ◽

Matthew J. Moscou ◽

Aleksey V. Zimin ◽

Geo Pertea ◽

Rachel S. Meyer ◽

...

Keyword(s):

Disease Resistance ◽

Genome Assembly ◽

Sequence Similarity ◽

Rice Variety ◽

Gene Families ◽

Resistance Locus ◽

High Sequence Similarity ◽

Long Read ◽

Genomic Regions

AbstractBackgroundLong-read sequencing facilitates assembly of complex genomic regions. In plants, loci containing nucleotide-binding, leucine-rich repeat (NLR) disease resistance genes are an important example of such regions. NLR genes make up one of the largest gene families in plants and are often clustered, evolving via duplication, contraction, and transposition. We recently mapped the Xo1 locus for resistance to bacterial blight and bacterial leaf streak, found in the American heirloom rice variety Carolina Gold Select, to a region that in the Nipponbare reference genome is rich in NLR genes.ResultsToward identification of the Xo1 gene, we combined Nanopore and Illumina reads to generate a high-quality genome assembly for Carolina Gold Select. We identified 529 full or partial NLR genes and discovered, relative to the reference, an expansion of NLR genes at the Xo1 locus. One NLR gene at Xo1 has high sequence similarity to the cloned, functionally similar Xa1 gene. Both harbor an integrated zfBED domain and near-identical, tandem, C-terminal repeats. Across diverse Oryzeae, we identified two sub-clades of such NLR genes, varying in the presence of the zfBED domain and the number of repeats.ConclusionsWhole genome sequencing combining Nanopore and Illumina reads effectively resolves NLR gene loci, providing context as well as content. Our identification of an Xo1 candidate is an important step toward mechanistic characterization, including the role(s) of the zfBED domain. Further, the Carolina Gold Select genome assembly will facilitate identification and exploitation of other useful traits in this historically important rice variety.

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Limited Genetic Diversity of blaCMY-2-Containing IncI1-pST12 Plasmids from Enterobacteriaceae of Human and Broiler Chicken Origin in The Netherlands

Microorganisms ◽

10.3390/microorganisms8111755 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1755

Author(s):

Evert Drijver ◽

Joep Stohr ◽

Jaco Verweij ◽

Carlo Verhulst ◽

Francisca Velkers ◽

...

Keyword(s):

Escherichia Coli ◽

Sequence Data ◽

Sequence Similarity ◽

Sequencing Analysis ◽

Nucleotide Polymorphisms ◽

Pan Genome ◽

Next Generation Sequencing Analysis ◽

Long Read ◽

Short Read Sequence ◽

High Degree

Distinguishing epidemiologically related and unrelated plasmids is essential to confirm plasmid transmission. We compared IncI1–pST12 plasmids from both human and livestock origin and explored the degree of sequence similarity between plasmids from Enterobacteriaceae with different epidemiological links. Short-read sequence data of Enterobacteriaceae cultured from humans and broilers were screened for the presence of both a blaCMY-2 gene and an IncI1–pST12 replicon. Isolates were long-read sequenced on a MinION sequencer (OxfordNanopore Technologies). After plasmid reconstruction using hybrid assembly, pairwise single nucleotide polymorphisms (SNPs) were determined. The plasmids were annotated, and a pan-genome was constructed to compare genes variably present between the different plasmids. Nine Escherichia coli sequences of broiler origin, four Escherichia coli sequences, and one Salmonella enterica sequence of human origin were selected for the current analysis. A circular contig with the IncI1–pST12 replicon and blaCMY-2 gene was extracted from the assembly graph of all fourteen isolates. Analysis of the IncI1–pST12 plasmids revealed a low number of SNP differences (range of 0–9 SNPs). The range of SNP differences overlapped in isolates with different epidemiological links. One-hundred and twelve from a total of 113 genes of the pan-genome were present in all plasmid constructs. Next generation sequencing analysis of blaCMY-2-containing IncI1–pST12 plasmids isolated from Enterobacteriaceae with different epidemiological links show a high degree of sequence similarity in terms of SNP differences and the number of shared genes. Therefore, statements on the horizontal transfer of these plasmids based on genetic identity should be made with caution.

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In silico identification of off-target pesticidal dsRNA binding in honey bees (Apis mellifera)

PeerJ ◽

10.7717/peerj.4131 ◽

2017 ◽

Vol 5 ◽

pp. e4131 ◽

Cited By ~ 4

Author(s):

Christina L. Mogren ◽

Jonathan Gary Lundgren

Keyword(s):

Honey Bee ◽

Honey Bees ◽

In Silico ◽

Target Genes ◽

Sequence Similarity ◽

Phenotypic Effect ◽

High Sequence Similarity ◽

Dsrna Molecule ◽

Dsrna Binding ◽

Genomic Regions

Background Pesticidal RNAs that silence critical gene function have great potential in pest management, but the benefits of this technology must be weighed against non-target organism risks. Methods Published studies that developed pesticidal double stranded RNAs (dsRNAs) were collated into a database. The target gene sequences for these pesticidal RNAs were determined, and the degree of similarity with sequences in the honey bee genome were evaluated statistically. Results We identified 101 insecticidal RNAs sharing high sequence similarity with genomic regions in honey bees. The likelihood that off-target sequences were similar increased with the number of nucleotides in the dsRNA molecule. The similarities of non-target genes to the pesticidal RNA was unaffected by taxonomic relatedness of the target insect to honey bees, contrary to previous assertions. Gene groups active during honey bee development had disproportionately high sequence similarity with pesticidal RNAs relative to other areas of the genome. Discussion Although sequence similarity does not itself guarantee a significant phenotypic effect in honey bees by the primary dsRNA, in silico screening may help to identify appropriate experimental endpoints within a risk assessment framework for pesticidal RNAi.

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Transformation of Amorphadiene Synthase and Antisilencing P19 Genes into Artemisia annua L. and its Effect on Antimalarial Artemisinin Production

Advanced Pharmaceutical Bulletin ◽

10.34172/apb.2020.057 ◽

2020 ◽

Vol 10 (3) ◽

pp. 464-471

Author(s):

Elfahmi Elfahmi ◽

Fany Mutia Cahyani ◽

Tati Kristianti ◽

Sony Suhandono

Keyword(s):

Hairy Root ◽

Hairy Roots ◽

Artemisia Annua ◽

Pcr Analysis ◽

Sequencing Analysis ◽

Pcr Products ◽

Transformed Hairy Root ◽

Polymerase Chain ◽

Infiltration Method

Purpose : The low content of artemisinin related to the biosynthetic pathway is influenced by the role of certain enzymes in the formation of artemisinin. The regulation of genes involved in artemisinin biosynthesis through genetic engineering is a choice to enhance the content. This research aims to transform ads and p19 gene as an antisilencing into Artemisia annua and to see their effects on artemisinin production. Methods: The presence of p19 and ads genes was confirmed through polymerase chain reaction (PCR) products and sequencing analysis. The plasmids, which contain ads and/or p19 genes, were transformed into Agrobacterium tumefaciens, and then inserted into leaves and hairy roots of A. annua by vacuum and syringe infiltration methods. The successful transformation was checked through the GUS histochemical test and the PCR analysis. Artemisinin levels were measured using HPLC. Results: The percentages of the blue area on leaves by using vacuum and syringe infiltration method and on hairy roots were up to 98, 92.55%, and 99.00% respectively. The ads-p19 sample contained a higher level of artemisinin (0.18%) compared to other samples. Transformed hairy root with co-transformation of ads-p19 contained 0.095% artemisinin, where no artemisinin was found in the control hairy root. The transformation of ads and p19 genes into A. annua plant has been successfully done and could enhance the artemisinin content on the transformed leaves with ads-p19 up to 2.57 folds compared to the untransformed leaves, while for p19, cotransformed and ads were up to 2.25, 1.29, and 1.14 folds respectively. Conclusion: Antisilencing p19 gene could enhance the transformation efficiency of ads and artemisinin level in A. annua.

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Rapid characterization of complex genomic regions using Cas9 enrichment and Nanopore sequencing

10.1101/2021.03.11.434935 ◽

2021 ◽

Author(s):

Jesse Bruijnesteijn ◽

Marit van der Wiel ◽

Natasja G. de Groot ◽

Ronald E. Bontrop

Keyword(s):

Sequence Similarity ◽

Gene Clusters ◽

Oxford Nanopore ◽

Long Read ◽

Number Variation ◽

Rapid Characterization ◽

Multiple Species ◽

Genomic Regions ◽

Genome Assemblies

AbstractLong-read sequencing approaches have considerably improved the quality and contiguity of genome assemblies. Such platforms bear the potential to resolve even extremely complex regions, such as multigenic families and repetitive stretches of DNA. Deep sequencing coverage, however, is required to overcome low nucleotide accuracy, especially in regions with high homopolymer density, copy number variation, and sequence similarity, such as the MHC and KIR gene clusters of the immune system. Therefore, we have adapted a targeted enrichment protocol in combination with long-read sequencing to efficiently annotate complex genomic regions. Using Cas9 endonuclease activity, segments of the complex KIR gene cluster were enriched and sequenced on an Oxford Nanopore Technologies platform. This provided sufficient coverage to accurately resolve and phase highly complex KIR haplotypes. Our strategy facilitates rapid characterization of large and complex multigenic regions, including its epigenetic footprint, in multiple species, even in the absence of a reference genome.

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Exploring the diversity of marine-derived fungal polyketide synthases

Canadian Journal of Microbiology ◽

10.1139/w06-131 ◽

2007 ◽

Vol 53 (2) ◽

pp. 291-302 ◽

Cited By ~ 9

Author(s):

Kimberly M. Mayer ◽

Jermaine Ford ◽

Gordon R. Macpherson ◽

David Padgett ◽

Brigitte Volkmann-Kohlmeyer ◽

...

Keyword(s):

Polyketide Synthase ◽

Sequence Similarity ◽

Three Dimensional ◽

Bioinformatic Analysis ◽

Sequence Information ◽

Fungal Isolates ◽

Specificity Determining Positions ◽

Pcr Products ◽

Polymerase Chain ◽

Similarity Searches

Using an approach based on polymerase chain reaction (PCR), we examined the diversity of polyketide synthase (PKS) genes present in 160 marine fungal isolates, representing 142 species. We obtained ketosynthase (KS) domain PCR products from 99 fungal isolates, representing Dothideomycetes, Sordariomycetes, Eurotiomycetes, and incertae sedis. Sequence similarity searches and phylogenetic analysis of 29 marine partial-KS-encoding sequences revealed domains predicted to encode reducing, nonreducing, and 6-methylsalicylic acid PKSs. Bioinformatic analysis of an alignment of the KS sequences from marine-derived fungi revealed no unique motifs in this region. However, several specificity-determining positions were apparent between fungal 6-methylsalicylic acid PKSs as compared with either reducing or nonreducing PKSs. Evaluation of these positions in the context of a modelled three-dimensional protein structure highlighted their potential use as PKS classification markers. Evaluating primer-binding sites was necessary to obtain KS domain fragments from putative PKSs while maintaining a level of sequence information adequate to properly classify and characterize them.

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Complete and haplotype-specific sequence assembly of segmental duplication-mediated genome rearrangements using CRISPR-targeted ultra-long read sequencing (CTLR-Seq)

10.1101/2020.10.23.349621 ◽

2020 ◽

Author(s):

Bo Zhou ◽

GiWon Shin ◽

Stephanie U. Greer ◽

Lisanne Vervoort ◽

Yiling Huang ◽

...

Keyword(s):

Segmental Duplication ◽

Base Pair ◽

De Novo ◽

Genome Rearrangements ◽

Pulse Field ◽

Sequencing Analysis ◽

Single Base ◽

Long Read ◽

Single Base Pair ◽

Genomic Regions

ABSTRACTWe have developed a generally applicable method based on CRISPR/Cas9-targeted ultra-long read sequencing (CTLR-Seq) to completely and haplotype-specifically resolve, at base-pair resolution, large, complex, and highly repetitive genomic regions that had been previously impenetrable to next-generation sequencing analysis such as large segmental duplication (SegDup) regions and their associated genome rearrangements that stretch hundreds of kilobases. Our method combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to haplotype-specifically isolate intact large (200-550 kb) target regions that encompass previously unresolvable genomic sequences. These target fragments are then sequenced (amplification-free) to produce ultra-long reads at up to 40x on-target coverage using Oxford nanopore technology, allowing for the complete assembly of the complex genomic regions of interest at single base-pair resolution. We applied CTLR-Seq to resolve the exact sequence of SegDup rearrangements that constitute the boundary regions of the 22q11.2 deletion CNV and of the 16p11.2 deletion and duplication CNVs. These CNVs are among the strongest known risk factors for schizophrenia and autism. We then perform de novo assembly to resolve, for the first time, at single base-pair resolution, the sequence rearrangements of the 22q11.2 and 16p11.2 CNVs, mapping out exactly the genes and non-coding regions that are affected by the CNV for different carriers.

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GB Virus C/Hepatitis G Virus Isolates in Japanese Haemophiliacs and their Origins

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615181 ◽

1998 ◽

Vol 80 (08) ◽

pp. 242-245 ◽

Cited By ~ 12

Author(s):

Yoshihide Fukuda ◽

Tetsuo Hayakawa ◽

Junki Takamatsu ◽

Hidehiko Saito ◽

Hiroaki Okamoto ◽

...

Keyword(s):

Sequence Similarity ◽

West African ◽

Blood Products ◽

Hepatitis G Virus ◽

Gb Virus C ◽

Route Of Transmission ◽

Polymerase Chain ◽

Hepatitis G ◽

Virus C ◽

Virus Isolates

SummaryJapanese haemophiliacs have been at high risk for infection with parenterally-transmissible viruses through the use of blood products, especially imported ones. Recently, novel transfusion-transmissible virus, GB virus C (GBV-C)/hepatitis G virus (HGV) were isolated. We investigated the origin and route of transmission of GBV-C/HGV isolates in haemophiliacs in Japan. GBV-C/HGV RNA was measured by nested reverse transcription polymerase chain reaction in 91 Japanese haemophiliacs. Phylogenetic analysis and genotypic grouping of GBV-C/HGV isolates in Japanese haemophiliacs were performed based on sequences in the 5’ untranslated region, and the characteristics were compared with those of reported isolates. GBV-C/HGV infection was present in 19 of 91 haemophiliacs (20.9%). Sequence analysis showed that 15 of the 19 isolates (78.9%) showed sequence similarity to a group in which mainly West African isolates have been reported. The other 4 isolates (21.1%) showed sequence similarity to Asian isolates. None of the GBV-C/HGV isolates showed sequences similar to those generally found in isolates from USA and Europe. The majority of GBV-C/HGV isolates found in Japanese haemophiliacs who are considered to have been infected by imported blood products were similar to those detected in West Africa.

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Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton

Plant Methods ◽

10.1186/s13007-021-00712-x ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Mohamed Ramadan ◽

Muna Alariqi ◽

Yizan Ma ◽

Yanlong Li ◽

Zhenping Liu ◽

...

Keyword(s):

Genetic Transformation ◽

Sequence Similarity ◽

High Specificity ◽

Transformation Method ◽

High Sequence Similarity ◽

Single Experiment ◽

Next Generation Sequencing Technology ◽

Cotton Transformation ◽

Assembly Method ◽

Multiple Genes

Abstract Background Upland cotton (Gossypium hirsutum), harboring a complex allotetraploid genome, consists of A and D sub-genomes. Every gene has multiple copies with high sequence similarity that makes genetic, genomic and functional analyses extremely challenging. The recent accessibility of CRISPR/Cas9 tool provides the ability to modify targeted locus efficiently in various complicated plant genomes. However, current cotton transformation method targeting one gene requires a complicated, long and laborious regeneration process. Hence, optimizing strategy that targeting multiple genes is of great value in cotton functional genomics and genetic engineering. Results To target multiple genes in a single experiment, 112 plant development-related genes were knocked out via optimized CRISPR/Cas9 system. We optimized the key steps of pooled sgRNAs assembly method by which 116 sgRNAs pooled together into 4 groups (each group consisted of 29 sgRNAs). Each group of sgRNAs was compiled in one PCR reaction which subsequently went through one round of vector construction, transformation, sgRNAs identification and also one round of genetic transformation. Through the genetic transformation mediated Agrobacterium, we successfully generated more than 800 plants. For mutants identification, Next Generation Sequencing technology has been used and results showed that all generated plants were positive and all targeted genes were covered. Interestingly, among all the transgenic plants, 85% harbored a single sgRNA insertion, 9% two insertions, 3% three different sgRNAs insertions, 2.5% mutated sgRNAs. These plants with different targeted sgRNAs exhibited numerous combinations of phenotypes in plant flowering tissues. Conclusion All targeted genes were successfully edited with high specificity. Our pooled sgRNAs assembly offers a simple, fast and efficient method/strategy to target multiple genes in one time and surely accelerated the study of genes function in cotton.

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Evolution of Chi motifs in Proteobacteria

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkaa054 ◽

2021 ◽

Author(s):

Angélique Buton ◽

Louis-Marie Bobay

Keyword(s):

Sequence Similarity ◽

Bacterial Species ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Large Dataset ◽

High Sequence Similarity ◽

Strand Breaks ◽

E Coli ◽

Dna Motif ◽

And Staphylococcus Aureus

Abstract Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex allows bacteria to repair DNA double strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis and Staphylococcus aureus. In this study we detected putative Chi motifs in a large dataset of Proteobacteria and we identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.

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