scholarly journals SMRT Genome Assembly Corrects Reference Errors, Resolving the Genetic Basis of Virulence in Mycobacterium tuberculosis

2016 ◽  
Author(s):  
Afif Elghraoui ◽  
Samuel J Modlin ◽  
Faramarz Valafar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Single Molecule ◽  
Genetic Basis ◽  
Reference Genome ◽  
Reference Sequence ◽  
Smrt Sequencing ◽  
Virulence Attenuation ◽  
Sequencing Platforms ◽  
Genome Comparisons ◽  
Reference Genomes

AbstractThe genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of its virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication. Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. We discuss how our results change the picture of virulence attenuation and the power of SMRT sequencing for producing high-quality reference genomes.

scholarly journals Improved maize reference genome with single molecule technologies

2016 ◽  
Author(s):  
Yinping Jiao ◽  
Paul Peluso ◽  
Jinghua Shi ◽  
Tiffany Liang ◽  
Michelle C. Stitzer ◽  
...  
Keyword(s):  
Single Molecule ◽  
Reference Genome ◽  
Optical Mapping ◽  
Fold Increase ◽  
Smrt Sequencing ◽  
Functional Variation ◽  
Density Region ◽  
Assembly Features ◽  
Reference Genomes

ABSTRACTComplete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate elucidation of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here, we report the assembly and annotation of maize, a genetic and agricultural model species, using Single Molecule Real-Time (SMRT) sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and significant improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed over 130,000 intact transposable elements (TEs), allowing us to identify TE lineage expansions unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by SMRT sequencing. In addition, comparative optical mapping of two other inbreds revealed a prevalence of deletions in the low gene density region and maize lineage-specific genes.

AStrap: identification of alternative splicing from transcript sequences without a reference genome

2018 ◽  
Vol 35 (15) ◽  
pp. 2654-2656 ◽  
Author(s):  
Guoli Ji ◽  
Wenbin Ye ◽  
Yaru Su ◽  
Moliang Chen ◽  
Guangzao Huang ◽  
...  
Keyword(s):  
Machine Learning ◽  
Alternative Splicing ◽  
Single Molecule ◽  
Reference Genome ◽  
De Novo ◽  
Supplementary Information ◽  
Model Organisms ◽  
Sequencing Data ◽  
Extensive Evaluation ◽  
Reference Genomes

Abstract Summary Alternative splicing (AS) is a well-established mechanism for increasing transcriptome and proteome diversity, however, detecting AS events and distinguishing among AS types in organisms without available reference genomes remains challenging. We developed a de novo approach called AStrap for AS analysis without using a reference genome. AStrap identifies AS events by extensive pair-wise alignments of transcript sequences and predicts AS types by a machine-learning model integrating more than 500 assembled features. We evaluated AStrap using collected AS events from reference genomes of rice and human as well as single-molecule real-time sequencing data from Amborella trichopoda. Results show that AStrap can identify much more AS events with comparable or higher accuracy than the competing method. AStrap also possesses a unique feature of predicting AS types, which achieves an overall accuracy of ∼0.87 for different species. Extensive evaluation of AStrap using different parameters, sample sizes and machine-learning models on different species also demonstrates the robustness and flexibility of AStrap. AStrap could be a valuable addition to the community for the study of AS in non-model organisms with limited genetic resources. Availability and implementation AStrap is available for download at https://github.com/BMILAB/AStrap. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Full-Length Transcriptome Sequences by A Combination of Sequencing Platforms Applied to Isoflavonoid and Triterpenoid Saponin Biosynthesis of Astragalus Mongholicus Bunge

2020 ◽  
Author(s):  
Minzhen Yin ◽  
Shanshan Chu ◽  
Tingyu Shan ◽  
Liangping Zha ◽  
Huasheng Peng
Keyword(s):  
Single Molecule ◽  
Molecular Genetic ◽  
Gene Families ◽  
Triterpenoid Saponin ◽  
Smrt Sequencing ◽  
Triterpenoid Saponins ◽  
Saponin Biosynthesis ◽  
Medicinal Value ◽  
Long Time ◽  
Sequencing Platforms

Abstract Background: Astragalus mongholicus Bunge is an important medicinal plant and has been used in traditional Chinese medicine for a long history, which is rich in isoflavonoids and triterpenoid saponins. Although these active constituents in A. mongholicus have been discovered for a long time, the molecular genetic basis of the isoflavonoid and triterpenoid saponin biosynthesis pathways is virtually unknown due to the lack of a reference genome. The combination of next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to analyze genes involved in the biosynthetic pathways of secondary metabolites in medicinal plants has been widely recognized.Results: In this study, NGS, SMRT sequencing, and targeted compounds were combined to investigate the association between isoflavonoids and triterpenoid saponins and gene expression in roots, stems and leaves of A. mongholicus. A total of four main isoflavonoids and four astragalosides (belong to triterpenoid saponins) were measured, and 44 differentially expressed genes (DEGs) of nine gene families, 44 DEGs of 16 gene families that encode for enzymes involved in isoflavonoid and triterpenoid saponin biosynthesis were identified, separately. Additionally, transcription factors (TFs) associated with isoflavonoid and triterpenoid saponin biosynthesis were analyzed, including 72 MYBs, 53 bHLHs, 64 AP2-EREBPs and 11 bZIPs. The above transcripts exhibit different expression trends in different organs.Conclusions: Our study provides important genetic information for the essential genes of isoflavonoid and triterpenoid saponin biosynthesis in A. mongholicus, and provides a basis for developing its medicinal value.

scholarly journals Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana

2020 ◽  
Author(s):  
shaoshan zhang ◽  
Qiong Liu ◽  
Chengcheng Lyu ◽  
Jinsong chen ◽  
Renfeng xiao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Phylogenetic Trees ◽  
Leaf Tissue ◽  
Stevia Rebaudiana ◽  
Full Length ◽  
Steviol Glycosides ◽  
Smrt Sequencing ◽  
Leaf Tissues ◽  
Sequencing Platforms ◽  
Insight Into

Abstract Background: Stevia rebaudiana (Bertoni) is considered one of the most valuable plants because of the steviol glycosides (SGs) that can be extracted from its leaves. Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. rebaudiana and play important roles in the synthesis of steviol glycosides. Results: Six stevia genotypes with significantly different concentrations of SGs were obtained by induction through various mutagenic methods, and the contents of seven glycosides (stevioboside, Reb B, ST, Reb A, Reb F, Reb D and Reb M) in their leaves were considerably different. Then, NGS and single-molecule real-time (SMRT) sequencing were combined to analyse leaf tissue from these six different genotypes to generate a more complete and correct full-length transcriptome of S. rebaudiana. Two phylogenetic trees of glycosyltransferases (SrUGTs) were constructed by the neighbour-joining method and successfully predicted the functions of SrUGTs involved in SG biosynthesis. With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCNA) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and one SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results.Conclusion: Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. Moreover, the complete and correct full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.

scholarly journals Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana

2020 ◽  
Author(s):  
shaoshan zhang ◽  
Qiong Liu ◽  
Chengcheng Lyu ◽  
Jinsong chen ◽  
Renfeng xiao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Phylogenetic Trees ◽  
Leaf Tissue ◽  
Stevia Rebaudiana ◽  
Full Length ◽  
Steviol Glycosides ◽  
Smrt Sequencing ◽  
Leaf Tissues ◽  
Sequencing Platforms ◽  
Insight Into

Abstract Background: Stevia rebaudiana (Bertoni) is considered one of the most valuable plants because of the steviol glycosides (SGs) that can be extracted from its leaves. Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. rebaudiana and play important roles in the synthesis of steviol glycosides. Results: Six stevia genotypes with significantly different concentrations of SGs were obtained by induction through various mutagenic methods, and the contents of seven glycosides (stevioboside, Reb B, ST, Reb A, Reb F, Reb D and Reb M) in their leaves were considerably different. Then, NGS and single-molecule real-time (SMRT) sequencing were combined to analyse leaf tissue from these six different genotypes to generate a full-length transcriptome of S. rebaudiana. Two phylogenetic trees of glycosyltransferases (SrUGTs) were constructed by the neighbour-joining method and successfully predicted the functions of SrUGTs involved in SG biosynthesis. With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCNA) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and one SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results.Conclusion: Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. Moreover, the full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.

scholarly journals Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Shaoshan Zhang ◽  
Qiong Liu ◽  
Chengcheng Lyu ◽  
Jinsong Chen ◽  
Renfeng Xiao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Phylogenetic Trees ◽  
Leaf Tissue ◽  
Stevia Rebaudiana ◽  
Full Length ◽  
Steviol Glycosides ◽  
Smrt Sequencing ◽  
Leaf Tissues ◽  
Sequencing Platforms ◽  
Insight Into

Abstract Background Stevia rebaudiana (Bertoni) is considered one of the most valuable plants because of the steviol glycosides (SGs) that can be extracted from its leaves. Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. rebaudiana and play important roles in the synthesis of steviol glycosides. Results Six stevia genotypes with significantly different concentrations of SGs were obtained by induction through various mutagenic methods, and the contents of seven glycosides (stevioboside, Reb B, ST, Reb A, Reb F, Reb D and Reb M) in their leaves were considerably different. Then, NGS and single-molecule real-time (SMRT) sequencing were combined to analyse leaf tissue from these six different genotypes to generate a full-length transcriptome of S. rebaudiana. Two phylogenetic trees of glycosyltransferases (SrUGTs) were constructed by the neighbour-joining method and successfully predicted the functions of SrUGTs involved in SG biosynthesis. With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCNA) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results. Conclusion Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. Moreover, the full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.

scholarly journals Understanding the diversity of DNA methylation in Mycobacterium tuberculosis

2020 ◽  
Author(s):  
Victor Ndhlovu ◽  
Anmol Kiran ◽  
Derek Sloan ◽  
Wilson Mandala ◽  
Marriot Nliwasa ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Single Molecule ◽  
Complete Loss ◽  
Phenotypic Heterogeneity ◽  
Epigenetic Variation ◽  
Smrt Sequencing ◽  
Underlying Mechanisms ◽  
First Time

AbstractAlthough Mycobacterium tuberculosis (Mtb) strains exhibit genomic homology of >99%, there is considerable variation in the phenotype. The underlying mechanisms of phenotypic heterogeneity in Mtb are not well understood but epigenetic variation is thought to contribute. At present the methylome of Mtb has not been completely characterized. We completed methylomes of 18 Mycobacterium tuberculosis (Mtb) clinical isolates from Malawi representing the largest number of Mtb genomes to be completed in a single study using Single Molecule Real Time (SMRT) sequencing to date. We replicate and confirm four methylation disrupting mutations in lineages of Mtb. For the first time we report complete loss of methylation courtesy of C758T (S253L) mutation in the MamB gene of Indo-oceanic lineage of Mtb. We also conducted a genomic and methylome comparison of the Malawian samples against a global sample. We confirm that methylation in Mtb is lineage specific although some unresolved issues still remain.

scholarly journals Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana

2020 ◽  
Author(s):  
shaoshan zhang ◽  
Qiong Liu ◽  
Chengcheng Lyu ◽  
Jinsong chen ◽  
Renfeng xiao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Phylogenetic Trees ◽  
Leaf Tissue ◽  
Stevia Rebaudiana ◽  
Full Length ◽  
Steviol Glycosides ◽  
Smrt Sequencing ◽  
Leaf Tissues ◽  
Sequencing Platforms ◽  
Insight Into

Abstract Background: Stevia rebaudiana (Bertoni) is considered one of the most valuable plants because of the steviol glycosides (SGs) that can be extracted from its leaves. Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. rebaudiana and play important roles in the synthesis of steviol glycosides. Results: Six stevia genotypes with significantly different concentrations of SGs were obtained by induction through various mutagenic methods, and the contents of seven glycosides (stevioboside, Reb B, ST, Reb A, Reb F, Reb D and Reb M) in their leaves were considerably different. Then, NGS and single-molecule real-time (SMRT) sequencing were combined to analyse leaf tissue from these six different genotypes to generate a full-length transcriptome of S. rebaudiana. Two phylogenetic trees of glycosyltransferases (SrUGTs) were constructed by the neighbour-joining method and successfully predicted the functions of SrUGTs involved in SG biosynthesis. With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCNA) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results.Conclusion: Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. Moreover, the full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.

scholarly journals SMRT Sequencing of the Full-Length Transcriptome of the Coelomactra antiquata

2021 ◽  
Vol 12 ◽  
Author(s):  
Aiping Deng ◽  
Jinpeng Li ◽  
Zebin Yao ◽  
Gyamfua Afriyie ◽  
Ziyang Chen ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Single Molecule ◽  
Reference Genome ◽  
Average Length ◽  
Full Length ◽  
Smrt Sequencing ◽  
Sequencing Technology ◽  
Medicinal Value ◽  
Biology Research

Coelomactra antiquata is an important aquatic economic shellfish with high medicinal value. However, because C. antiquata has no reference genome, a lot of molecular biology research cannot be carried out, so the analysis of its transcripts is an important step to study the regulatory genes of various substances in C. antiquata. In the present study, we conducted the first full-length transcriptome analysis of C. antiquata by using PacBio single-molecule real-time (SMRT) sequencing technology. The results identified a total of 39,209 unigenes with an average length of 2,732 bp, 23,338 CDSs, 251 AS events, 9,881 lncRNAs, 20,106 SSRs, and 2,316 TFs. Subsequently, 59.22% (23,220) of the unigenes were successfully annotated, of which 23,164, 18,711, 15,840, 13,534, and 13,474 unigenes could be annotated using NR, Swiss-prot, KOG, GO, and KEGG databases, respectively. This study lays the foundation for the follow-up research of molecular biology and provides a reference for studying the more medicinal value of C. antiquata.

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