Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (azadirachta indica)

Abstract Background: Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The production of azadirachtin A depends on extraction from neem tissues, which is not an eco-friendly and sustainable process. The low yield and discontinuous supply of azadirachtin A impedes further applications. The biosynthetic pathway of azadirachtin A is still unknown and is the focus of our study. Results: We attempted to explore azadirachtin A biosynthetic pathway and identified the key genes involved by analyzing transcriptome data from five neem tissues through the hybrid-sequencing (Illumina HiSeq and Pacific Biosciences Single Molecule Real-Time (SMRT)) approach. Candidates were first screened by comparing the expression levels between the five tissues. After phylogenetic analysis, domain prediction, and molecular docking studies, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase, cytochrome P450 (CYP450), acyltransferase, and esterase were proposed to be potential genes involved in azadirachtin A biosynthesis. Among them, two unigenes encoding homologs of MaOSC1 and MaCYP71CD2 were identified. A unigene encoding the complete homolog of MaCYP71BQ5 was reported. Accuracy of the assembly was verified by quantitative real-time PCR (qRT-PCR) and full-length PCR cloning. Conclusions: By integrating and analyzing transcriptome data from hybrid-seq technology, 22 differentially expressed genes (DEGs) were finally selected as candidates involved in azadirachtin A pathway. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on understanding the biosynthesis of other triterpenoids in neem trees and provides a reference for exploring other valuable natural product biosynthesis in plants.

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Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (Azadirachta indica)

10.21203/rs.2.23446/v3 ◽

2020 ◽

Author(s):

Huiyan Wang ◽

Ning Wang ◽

Yixin Huo

Keyword(s):

Real Time ◽

Single Molecule ◽

Biosynthetic Pathway ◽

Docking Studies ◽

Transcriptome Data ◽

Sequencing Data ◽

Pcr Cloning ◽

Illumina Hiseq ◽

Oxidosqualene Cyclase ◽

Azadirachtin A

Abstract Background: Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The production of azadirachtin A depends on extraction from neem tissues, which is not an eco-friendly and sustainable process. The low yield and discontinuous supply of azadirachtin A impedes further applications. The biosynthetic pathway of azadirachtin A is still unknown and is the focus of our study. Results: We attempted to explore azadirachtin A biosynthetic pathway and identified the key genes involved by analyzing transcriptome data from five neem tissues through the hybrid-sequencing (Illumina HiSeq and Pacific Biosciences Single Molecule Real-Time (SMRT)) approach. Candidates were first screened by comparing the expression levels between the five tissues. After phylogenetic analysis, domain prediction, and molecular docking studies, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase, cytochrome P450 (CYP450), acyltransferase, and esterase were proposed to be potential genes involved in azadirachtin A biosynthesis. Among them, two unigenes encoding homologs of MaOSC1 and MaCYP71CD2 were identified. A unigene encoding the complete homolog of MaCYP71BQ5 was reported. Accuracy of the assembly was verified by quantitative real-time PCR (qRT-PCR) and full-length PCR cloning. Conclusions: By integrating and analyzing transcriptome data from hybrid-seq technology, 22 differentially expressed genes (DEGs) were finally selected as candidates involved in azadirachtin A pathway. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on understanding the biosynthesis of other triterpenoids in neem trees and provides a reference for exploring other valuable natural product biosynthesis in plants.

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Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (Azadirachta indica)

10.21203/rs.2.23446/v4 ◽

2020 ◽

Author(s):

Huiyan Wang ◽

Ning Wang ◽

Yixin Huo

Keyword(s):

Real Time ◽

Single Molecule ◽

Biosynthetic Pathway ◽

Docking Studies ◽

Transcriptome Data ◽

Sequencing Data ◽

Pcr Cloning ◽

Illumina Hiseq ◽

Oxidosqualene Cyclase ◽

Azadirachtin A

Abstract Background: Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The production of azadirachtin A depends on extraction from neem tissues, which is not an eco-friendly and sustainable process. The low yield and discontinuous supply of azadirachtin A impedes further applications. The biosynthetic pathway of azadirachtin A is still unknown and is the focus of our study. Results: We attempted to explore azadirachtin A biosynthetic pathway and identified the key genes involved by analyzing transcriptome data from five neem tissues through the hybrid-sequencing (Illumina HiSeq and Pacific Biosciences Single Molecule Real-Time (SMRT)) approach. Candidates were first screened by comparing the expression levels between the five tissues. After phylogenetic analysis, domain prediction, and molecular docking studies, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase, cytochrome P450 (CYP450), acyltransferase, and esterase were proposed to be potential genes involved in azadirachtin A biosynthesis. Among them, two unigenes encoding homologs of MaOSC1 and MaCYP71CD2 were identified. A unigene encoding the complete homolog of MaCYP71BQ5 was reported. Accuracy of the assembly was verified by quantitative real-time PCR (qRT-PCR) and full-length PCR cloning. Conclusions: By integrating and analyzing transcriptome data from hybrid-seq technology, 22 differentially expressed genes (DEGs) were finally selected as candidates involved in azadirachtin A pathway. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on understanding the biosynthesis of other triterpenoids in neem trees and provides a reference for exploring other valuable natural product biosynthesis in plants.

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Multi-tissue transcriptome analysis using hybrid-seq revealed potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (Azadirachtin indica)

10.21203/rs.2.23446/v2 ◽

2020 ◽

Author(s):

Huiyan Wang ◽

Ning Wang ◽

Yixin Huo

Keyword(s):

Real Time ◽

Single Molecule ◽

Biosynthetic Pathway ◽

Transcriptome Data ◽

Quantitative Real Time Pcr ◽

Sequencing Data ◽

Neem Tree ◽

Illumina Hiseq ◽

Oxidosqualene Cyclase ◽

Azadirachtin A

Abstract Background: Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The manufacture of azadirachtin A depends on extraction from neem tissues, which is not ecofriendly and sustainable. The low yield and discontinuous supply impeded the further application. The biosynthetic pathway of azadirachtin A is still unknown. Results: We attempted to explore azadirachtin A biosynthetic pathway and identified key involved genes by analyzing transcriptome data of five neem tissues through hybrid-seq (Illumina HiSeq and Pacific Biosciences Single Molecule Real Time (PacBio SMRT)) approach. Candidates were firstly screened by comparing expression level within five tissues. After phylogenetic analysis, domain prediction and molecular docking, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase (ADH), cytochrome P450 (CYP450), acyltransferase (ACT) and esterase (EST) were proposed to be potential genes involved in azadirachtin A biosynthesis. Among them, two unigenes encoding homolog of MaOCS1 and MaCYP71CD2 were identified. An unigene encoding complete homolog of MaCYP71BQ5 was first reported. Accuracy of the assemblies were verified by Quantitative Real-Time PCR (qRT-PCR) and full-length cloning PCR. Conclusions: By integrating and analysis transcriptome data from hybrid-seq technology, 22 DEGs were finally selected as candidates involved in azadirachtin A pathway. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on the understanding of other triterpenoids biosynthesis in neem trees and provide reference for exploring other valuable natural product biosynthesis in plants.

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Multi-tissue transcriptome analysis using hybrid-seq revealed potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (Azadirachtin indica)

10.21203/rs.2.23446/v1 ◽

2020 ◽

Author(s):

Huiyan Wang ◽

Ning Wang ◽

Yixin Huo

Keyword(s):

Real Time ◽

Single Molecule ◽

Biosynthetic Pathway ◽

De Novo ◽

Analysis Data ◽

Sequencing Data ◽

Neem Tree ◽

Illumina Hiseq ◽

Oxidosqualene Cyclase ◽

Azadirachtin A

Abstract Background: Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The manufacture of azadirachtin A depends on extraction from neem tissues, which is not ecofriendly and sustainable. The low yield and discontinuous supply impeded the further application. The biosynthetic pathway of azadirachtin A is still well-known.Results: We attempted to explore azadirachtin A biosynthetic pathway and identified key involved genes by analyzing transcriptome data of five neem tissues through hybrid-seq (Illumina HiSeq and Pacific Biosciences Single Molecule Real Time (PacBio SMRT)) technology. A total 219 and 397 up-regulated differentially expressed genes (DEGs) in leaf and fruit tissues than other tissues (root, stem and flower) were isolated. After phylogenetic analysis and domain prediction, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase (ADH), cytochrome P450 (CYP450), acyltransferase (ACT) and esterase (EST) proposed to be involved in azadirachtin A biosynthesis were finally selected. De novo assembled sequences were verified by Quantitative Real-Time PCR (qRT-PCR) analysis.Conclusions: By integrating and analysis data from Illumina HiSeq and PacBio SMRT platform, 22 DEGs were finally selected as candidates involved in azadirachtin A biosynthesis. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on the understanding of other triterpenoids biosynthesis in neem trees and provide reference for exploring other valuable natural product biosynthesis in plants.

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Chloroplast Genome Sequence ofArabidopsis thalianaAccession Landsbergerecta, Assembled from Single-Molecule, Real-Time Sequencing Data

Genome Announcements ◽

10.1128/genomea.00975-16 ◽

2016 ◽

Vol 4 (5) ◽

Cited By ~ 2

Author(s):

Kai Bernd Stadermann ◽

Daniela Holtgräwe ◽

Bernd Weisshaar

Keyword(s):

Arabidopsis Thaliana ◽

High Resolution ◽

Chloroplast Genome ◽

Real Time ◽

Genome Sequence ◽

Single Molecule ◽

Sequencing Data ◽

Data Set ◽

Chloroplast Genomes ◽

Chloroplast Genome Sequence

A publicly available data set from Pacific Biosciences was used to create an assembly of the chloroplast genome sequence of theArabidopsis thalianagenotype Landsbergerecta. The assembly is solely based on single-molecule, real-time sequencing data and hence provides high resolution of the two inverted repeat regions typically contained in chloroplast genomes.

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Single-Molecule Real-Time Transcript Sequencing of Turnips Unveiling the Complexity of the Turnip Transcriptome

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401434 ◽

2020 ◽

Vol 10 (10) ◽

pp. 3505-3514

Author(s):

Hongmei Zhuang ◽

Qiang Wang ◽

Hongwei Han ◽

Huifang Liu ◽

Hao Wang

Keyword(s):

Real Time ◽

Brassica Rapa ◽

Single Molecule ◽

Developmental Stages ◽

Full Length ◽

Sequencing Data ◽

Smrt Sequencing ◽

High Quality ◽

Transcript Structure ◽

Novel Transcripts

To generate the full-length transcriptome of Xinjiang green and purple turnips, Brassica rapa var. Rapa, using single-molecule real-time (SMRT) sequencing. The samples of two varieties of Brassica rapa var. Rapa at five developmental stages were collected and combined to perform SMRT sequencing. Meanwhile, next generation sequencing was performed to correct SMRT sequencing data. A series of analyses were performed to investigate the transcript structure. Finally, the obtained transcripts were mapped to the genome of Brassica rapa ssp. pekinesis Chiifu to identify potential novel transcripts. For green turnip (F01), a total of 19.54 Gb clean data were obtained from 8 cells. The number of reads of insert (ROI) and full-length non-chimeric (FLNC) reads were 510,137 and 267,666. In addition, 82,640 consensus isoforms were obtained in the isoform sequences clustering, of which 69,480 were high-quality, and 13,160 low-quality sequences were corrected using Illumina RNA seq data. For purple turnip (F02), there were 20.41 Gb clean data, 552,829 ROIs, and 274,915 FLNC sequences. A total of 93,775 consensus isoforms were obtained, of which 78,798 were high-quality, and the 14,977 low-quality sequences were corrected. Following the removal of redundant sequences, there were 46,516 and 49,429 non-redundant transcripts for F01 and F02, respectively; 7,774 and 9,385 alternative splicing events were predicted for F01 and F02; 63,890 simple sequence repeats, 59,460 complete coding sequences, and 535 long-non coding RNAs were predicted. Moreover, 5,194 and 5,369 novel transcripts were identified by mapping to Brassica rapa ssp. pekinesis Chiifu. The obtained transcriptome data may improve turnip genome annotation and facilitate further study of the Brassica rapa var. Rapa genome and transcriptome.

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Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1601782113 ◽

2016 ◽

Vol 113 (19) ◽

pp. 5233-5238 ◽

Cited By ~ 79

Author(s):

Carl W. Fuller ◽

Shiv Kumar ◽

Mintu Porel ◽

Minchen Chien ◽

Arek Bibillo ◽

...

Keyword(s):

Dna Sequencing ◽

Real Time ◽

Lipid Bilayers ◽

Dna Polymerase ◽

Single Molecule ◽

High Throughput Sequencing ◽

Electrical Current ◽

Sequencing Data ◽

Sequencing By Synthesis ◽

Dna Strand

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

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Real-Time, Direct Classification of Nanopore Signals with SquiggleNet

10.1101/2021.01.15.426907 ◽

2021 ◽

Author(s):

Yuwei Bao ◽

Jack Wadden ◽

John R. Erb-Downward ◽

Piyush Ranjan ◽

Robert P. Dickson ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Bacterial Species ◽

Sequencing Data ◽

Base Calling ◽

Oxford Nanopore ◽

Order Of Magnitude ◽

Unseen Species ◽

Deep Learning Model

AbstractSingle-molecule sequencers made by Oxford Nanopore provide results in real time as DNA passes through a nanopore and can eject a molecule after it has been partly sequenced. However, the computational challenge of deciding whether to keep or reject a molecule in real time has limited the application of this capability. We present SquiggleNet, the first deep learning model that can classify nanopore reads directly from their electrical signals. SquiggleNet operates faster than the DNA passes through the pore, allowing real-time classification and read ejection. When given the amount of sequencing data generated in one second, the classifier achieves significantly higher accuracy than base calling followed by sequence alignment. Our approach is also faster and requires an order of magnitude less memory than approaches based on alignment. SquiggleNet distinguished human from bacterial DNA with over 90% accuracy across test datasets from different flowcells and sample preparations, generalized to unseen species, and identified bacterial species in a human respiratory meta genome sample.

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Phased Haplotype Resolution of the SLC6A4 Promoter Using Long-Read Single Molecule Real-Time (SMRT) Sequencing

Genes ◽

10.3390/genes11111333 ◽

2020 ◽

Vol 11 (11) ◽

pp. 1333

Author(s):

Mariana R. Botton ◽

Yao Yang ◽

Erick R. Scott ◽

Robert J. Desnick ◽

Stuart A. Scott

Keyword(s):

Real Time ◽

Single Molecule ◽

Variable Number ◽

Sequencing Data ◽

Smrt Sequencing ◽

Short Read ◽

Short Read Sequencing ◽

Sequencing Method ◽

Long Read ◽

S Allele

The SLC6A4 gene has been implicated in psychiatric disorder susceptibility and antidepressant response variability. The SLC6A4 promoter is defined by a variable number of homologous 20–24 bp repeats (5-HTTLPR), and long (L) and short (S) alleles are associated with higher and lower expression, respectively. However, this insertion/deletion variant is most informative when considered as a haplotype with the rs25531 and rs25532 variants. Therefore, we developed a long-read single molecule real-time (SMRT) sequencing method to interrogate the SLC6A4 promoter region. A total of 120 samples were subjected to SLC6A4 long-read SMRT sequencing, primarily selected based on available short-read sequencing data. Short-read genome sequencing from the 1000 Genomes (1KG) Project (~5X) and the Genetic Testing Reference Material Coordination Program (~45X), as well as high-depth short-read capture-based sequencing (~330X), could not identify the 5-HTTLPR short (S) allele, nor could short-read sequencing phase any identified variants. In contrast, long-read SMRT sequencing unambiguously identified the 5-HTTLPR short (S) allele (frequency of 0.467) and phased SLC6A4 promoter haplotypes. Additionally, discordant rs25531 genotypes were reviewed and determined to be short-read errors. Taken together, long-read SMRT sequencing is an innovative and robust method for phased resolution of the SLC6A4 promoter, which could enable more accurate pharmacogenetic testing for both research and clinical applications.

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