scholarly journals A Statistical Method for Observing Personal Diploid Methylomes and Transcriptomes with Single-Molecule Real-Time Sequencing

Genes ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 460 ◽  
Author(s):  
Yuta Suzuki ◽  
Yunhao Wang ◽  
Kin Au ◽  
Shinichi Morishita
Keyword(s):  
Statistical Model ◽  
Real Time ◽  
Single Molecule ◽  
Error Rate ◽  
Methylation Pattern ◽  
Specific Expression ◽  
Long Reads ◽  
Allele Specific ◽  
Complex Locus ◽  
Allele Specific Methylation

We address the problem of observing personal diploid methylomes, CpG methylome pairs of homologous chromosomes that are distinguishable with respect to phased heterozygous variants (PHVs), which is challenging due to scarcity of PHVs in personal genomes. Single molecule real-time (SMRT) sequencing is promising as it outputs long reads with CpG methylation information, but a serious concern is whether reliable PHVs are available in erroneous SMRT reads with an error rate of ∼15%. To overcome the issue, we propose a statistical model that reduces the error rate of phasing CpG site to 1%, thereby calling CpG hypomethylation in each haplotype with >90% precision and sensitivity. Using our statistical model, we examined GNAS complex locus known for a combination of maternally, paternally, or biallelically expressed isoforms, and observed allele-specific methylation pattern almost perfectly reflecting their respective allele-specific expression status, demonstrating the merit of elucidating comprehensive personal diploid methylomes and transcriptomes.

scholarly journals Genome-wide detection of cytosine methylation by single molecule real-time sequencing

2021 ◽  
Vol 118 (5) ◽  
pp. e2019768118
Author(s):  
O. Y. Olivia Tse ◽  
Peiyong Jiang ◽  
Suk Hang Cheng ◽  
Wenlei Peng ◽  
Huimin Shang ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Cytosine Methylation ◽  
Epigenetic Modification ◽  
Methylation Status ◽  
Area Under The Curve ◽  
Buffy Coat ◽  
Genome Wide ◽  
Allele Specific ◽  
Allele Specific Methylation

5-Methylcytosine (5mC) is an important type of epigenetic modification. Bisulfite sequencing (BS-seq) has limitations, such as severe DNA degradation. Using single molecule real-time sequencing, we developed a methodology to directly examine 5mC. This approach holistically examined kinetic signals of a DNA polymerase (including interpulse duration and pulse width) and sequence context for every nucleotide within a measurement window, termed the holistic kinetic (HK) model. The measurement window of each analyzed double-stranded DNA molecule comprised 21 nucleotides with a cytosine in a CpG site in the center. We used amplified DNA (unmethylated) and M.SssI-treated DNA (methylated) (M.SssI being a CpG methyltransferase) to train a convolutional neural network. The area under the curve for differentiating methylation states using such samples was up to 0.97. The sensitivity and specificity for genome-wide 5mC detection at single-base resolution reached 90% and 94%, respectively. The HK model was then tested on human–mouse hybrid fragments in which each member of the hybrid had a different methylation status. The model was also tested on human genomic DNA molecules extracted from various biological samples, such as buffy coat, placental, and tumoral tissues. The overall methylation levels deduced by the HK model were well correlated with those by BS-seq (r = 0.99; P < 0.0001) and allowed the measurement of allele-specific methylation patterns in imprinted genes. Taken together, this methodology has provided a system for simultaneous genome-wide genetic and epigenetic analyses.

scholarly journals Single-copy detection of somatic variants from solid and liquid biopsy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana-Luisa Silva ◽  
Paulina Klaudyna Powalowska ◽  
Magdalena Stolarek ◽  
Eleanor Ruth Gray ◽  
Rebecca Natalie Palmer ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Clinical Decision Making ◽  
High Sensitivity ◽  
Clinical Decision ◽  
Single Copy ◽  
Turnaround Time ◽  
Copy Detection ◽  
Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

scholarly journals The complexity of alternative splicing and landscape of tissue-specific expression in lotus (Nelumbo nucifera) unveiled by Illumina- and single-molecule real-time-based RNA-sequencing

DNA Research ◽  
2019 ◽  
Vol 26 (4) ◽  
pp. 301-311 ◽  
Author(s):  
Yue Zhang ◽  
Tonny Maraga Nyong'A ◽  
Tao Shi ◽  
Pingfang Yang
Keyword(s):  
Alternative Splicing ◽  
Real Time ◽  
Single Molecule ◽  
Intron Retention ◽  
Critical Role ◽  
Full Length ◽  
Specific Expression ◽  
Splice Isoforms ◽  
Tissue Specific ◽  
Genomic Studies

Abstract Alternative splicing (AS) plays a critical role in regulating different physiological and developmental processes in eukaryotes, by dramatically increasing the diversity of the transcriptome and the proteome. However, the saturation and complexity of AS remain unclear in lotus due to its limitation of rare obtainment of full-length multiple-splice isoforms. In this study, we apply a hybrid assembly strategy by combining single-molecule real-time sequencing and Illumina RNA-seq to get a comprehensive insight into the lotus transcriptomic landscape. We identified 211,802 high-quality full-length non-chimeric reads, with 192,690 non-redundant isoforms, and updated the lotus reference gene model. Moreover, our analysis identified a total of 104,288 AS events from 16,543 genes, with alternative 3ʹ splice-site being the predominant model, following by intron retention. By exploring tissue datasets, 370 tissue-specific AS events were identified among 12 tissues. Both the tissue-specific genes and isoforms might play important roles in tissue or organ development, and are suitable for ‘ABCE’ model partly in floral tissues. A large number of AS events and isoform variants identified in our study enhance the understanding of transcriptional diversity in lotus, and provide valuable resource for further functional genomic studies.

scholarly journals Investigation of Elements Sufficient To Imprint the Mouse Air Promoter

2001 ◽  
Vol 21 (15) ◽  
pp. 5008-5017 ◽  
Author(s):  
Frank Sleutels ◽  
Denise P. Barlow
Keyword(s):  
Antisense Rna ◽  
Cpg Island ◽  
Maternal Allele ◽  
Adenine Phosphoribosyltransferase ◽  
Specific Expression ◽  
Allele Specific ◽  
Factor Type ◽  
Allele Specific Methylation ◽  
Single Promoter

ABSTRACT 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 Igf2rCpG 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 ofAir have been transferred onto the AprtCpG 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.

scholarly journals Allele specific expression and methylation in the bumblebee, Bombus terrestris

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3798 ◽  
Author(s):  
Zoë Lonsdale ◽  
Kate Lee ◽  
Maria Kiriakidu ◽  
Harindra Amarasinghe ◽  
Despina Nathanael ◽  
...  
Keyword(s):  
Bombus Terrestris ◽  
Social Hymenoptera ◽  
Specific Expression ◽  
Allele Specific Expression ◽  
Epigenetic Marker ◽  
The Social ◽  
Allele Specific ◽  
Contradictory Evidence ◽  
Allele Specific Methylation

The social hymenoptera are emerging as models for epigenetics. DNA methylation, the addition of a methyl group, is a common epigenetic marker. In mammals and flowering plants methylation affects allele specific expression. There is contradictory evidence for the role of methylation on allele specific expression in social insects. The aim of this paper is to investigate allele specific expression and monoallelic methylation in the bumblebee, Bombus terrestris. We found nineteen genes that were both monoallelically methylated and monoallelically expressed in a single bee. Fourteen of these genes express the hypermethylated allele, while the other five express the hypomethylated allele. We also searched for allele specific expression in twenty-nine published RNA-seq libraries. We found 555 loci with allele-specific expression. We discuss our results with reference to the functional role of methylation in gene expression in insects and in the as yet unquantified role of genetic cis effects in insect allele specific methylation and expression.

scholarly journals Lineage and Parent-of-Origin Effects in DNA Methylation of Honey Bees (Apis mellifera) Revealed by Reciprocal Crosses and Whole-Genome Bisulfite Sequencing

2020 ◽  
Vol 12 (8) ◽  
pp. 1482-1492
Author(s):  
Xin Wu ◽  
David A Galbraith ◽  
Paramita Chatterjee ◽  
Hyeonsoo Jeong ◽  
Christina M Grozinger ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Honey Bees ◽  
Specific Expression ◽  
Africanized Honey Bee ◽  
Allele Specific ◽  
Parent Of Origin ◽  
Origin Effects ◽  
Genetic Background Effects ◽  
Allele Specific Methylation ◽  
Methylation Patterns

Abstract Parent-of-origin methylation arises when the methylation patterns of a particular allele are dependent on the parent it was inherited from. Previous work in honey bees has shown evidence of parent-of-origin-specific expression, yet the mechanisms regulating such pattern remain unknown in honey bees. In mammals and plants, DNA methylation is known to regulate parent-of-origin effects such as genomic imprinting. Here, we utilize genotyping of reciprocal European and Africanized honey bee crosses to study genome-wide allele-specific methylation patterns in sterile and reproductive individuals. Our data confirm the presence of allele-specific methylation in honey bees in lineage-specific contexts but also importantly, though to a lesser degree, parent-of-origin contexts. We show that the majority of allele-specific methylation occurs due to lineage rather than parent-of-origin factors, regardless of the reproductive state. Interestingly, genes affected by allele-specific DNA methylation often exhibit both lineage and parent-of-origin effects, indicating that they are particularly labile in terms of DNA methylation patterns. Additionally, we re-analyzed our previous study on parent-of-origin-specific expression in honey bees and found little association with parent-of-origin-specific methylation. These results indicate strong genetic background effects on allelic DNA methylation and suggest that although parent-of-origin effects are manifested in both DNA methylation and gene expression, they are not directly associated with each other.

scholarly journals Genome Sequence of Pantoea ananatis SGAir0210, Isolated from Outdoor Air in Singapore

2018 ◽  
Vol 6 (27) ◽  
Author(s):  
Irvan Luhung ◽  
Ana Carolina M. Junqueira ◽  
Akira Uchida ◽  
Rikky W. Purbojati ◽  
James N. I. Houghton ◽  
...  
Keyword(s):  
Real Time ◽  
Genome Sequence ◽  
Single Molecule ◽  
Outdoor Air ◽  
Pantoea Ananatis ◽  
Content Type ◽  
Short Reads ◽  
Long Reads

Pantoea ananatis SGAir0210 was isolated from outdoor air collected in Singapore. The genome was assembled from long reads generated by single-molecule real-time sequencing complemented with short reads.

scholarly journals Allele-specific RNA imaging shows that allelic imbalances can arise in tissues through transcriptional bursting

2018 ◽  
Author(s):  
Orsolya Symmons ◽  
Marcello Chang ◽  
Ian A. Mellis ◽  
Jennifer M. Kalish ◽  
Marisa S. Bartolomei ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Cells ◽  
Gene Copy ◽  
Specific Expression ◽  
Rna Molecules ◽  
Gene Copies ◽  
Extensive Cell ◽  
Allele Specific ◽  
Transcriptional Bursting ◽  
Diploid Cells

AbstractExtensive cell-to-cell variation exists even among putatively identical cells, and there is great interest in understanding how the properties of transcription relate to this heterogeneity. Differential expression from the two gene copies in diploid cells could potentially contribute, yet our ability to measure from which gene copy individual RNAs originated remains limited, particularly in the context of tissues. Here, we demonstrate quantitative, single molecule allele-specific RNA FISH adapted for use on tissue sections, allowing us to determine the chromosome of origin of individual RNA molecules in formaldehyde-fixed tissues. We used this method to visualize the allele-specific expression of Xist and multiple autosomal genes in mouse kidney. By combining these data with mathematical modeling, we evaluated models for allele-specific heterogeneity, in particular demonstrating that apparent expression from only one of the alleles in single cells can arise as a consequence of low-level mRNA abundance and transcriptional bursting.

scholarly journals Complete Genome Sequence of Brachybacterium sp. Strain SGAir0954, Isolated from Singapore Air

2019 ◽  
Vol 8 (32) ◽  
Author(s):  
Phu Pwint Thin Hlaing ◽  
Ana Carolina M. Junqueira ◽  
Akira Uchida ◽  
Rikky W. Purbojati ◽  
James N. I. Houghton ◽  
...  
Keyword(s):  
Real Time ◽  
Genome Sequence ◽  
Single Molecule ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Smrt Sequencing ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Long Reads

Brachybacterium sp. strain SGAir0954 was isolated from tropical air collected in Singapore, and its genome was sequenced and assembled using long reads generated by single-molecule real-time (SMRT) sequencing. The complete genome has a size of 3.41 Mb and consists of 2,955 protein coding genes, 50 tRNAs, and 9 rRNAs.

scholarly journals Single molecule sequencing of nucleic acids in real time (SMRT) to characterize transcriptomes and mRNA isoforms

2020 ◽  
Vol 27 (1) ◽  
pp. 091-094
Author(s):  
F. Abel Ponce de León ◽  
Yue Guo ◽  
Brian Crooker
Keyword(s):  
Real Time ◽  
Y Chromosome ◽  
Single Molecule ◽  
Expression Profiles ◽  
Sex Differentiation ◽  
Expression Patterns ◽  
Single Copy ◽  
Mrna Isoforms ◽  
Specific Expression ◽  
Male Sex

Our efforts are oriented to assess bovine Y-chromosome gene expression patterns. One set of genes that are of interest are the so-called X-degenerate Y-chromosome genes that are located in the male-specific region of the Y-chromosome (MSY). This region contains 95% of the DNA of the Y chromosome. These genes are single copy and have an X-chromosome homolog. Both, the Y-encoded and X-encoded homologs have ubiquitous expression profiles. However, some genes, like SRY that regulates male sex determination, have functions that are more specific. Identifying DNA sequence differences between these homologs will allow evaluation of their spatial and temporal expression patterns. Identification of the Y-encoded mRNAs and their isoforms will allow our understanding of tissue specific expression of isoforms in male tissues. The latter will facilitate our evaluation of gene function in male sex differentiation and fertility. Hence, we hypothesized that each of these X-degenerate gene homologs generate isoforms and that differential expression patterns exist between sexes and across tissues. To investigate the latter we used a new generation sequencing (NGS) technology that generates long sequencing reads with a range between 1000 to 10,000 base pairs in length. Single molecule real time (SMRT) isoform sequencing (IsoSeq) of several tissues (liver, lung, adipose, muscle, hypothalamus and testis) was carried out. Transcript sequences were used for bioinformatics analysis and isoform characterization. Given the focus of this manuscript the SMRT technology we are only presenting results obtained with the analysis of the bUTY and bUTX genes.

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