Plasmid Sequence Analysis from Long Reads v2 (protocols.io.bqu3mwyn)

protocols.io ◽  
2020 ◽  
Author(s):  
David A
Keyword(s):  
Sequence Analysis ◽  
Plasmid Sequence ◽  
Long Reads

Plasmid Sequence Analysis from Long Reads v2

2021 ◽  
Author(s):  
David A Eccles
Keyword(s):  
Sequence Analysis ◽  
Plasmid Dna ◽  
De Novo ◽  
Consensus Sequence ◽  
Fasta File ◽  
High Quality ◽  
Plasmid Sequence ◽  
Long Reads ◽  
The Moment

This protocol demonstrates how to assemble reads from plasmid DNA, and generate a circularised and non-repetitive consensus sequence At the moment, this protocol uses Canu to de-novo assemble high-quality single-cut reads. Input(s): demultiplexed fastq files (see protocol Demultiplexing Nanopore reads with LAST). I've noticed that the default demultiplexing carried out by Guppy (at least up to v4.2.2, as used in the first version of this protocol) has issues with chimeric reads, which can affect assembly. Output(s): Consensus sequence per barcode as a fasta file

Plasmid Sequence Analysis from Long Reads v2

2021 ◽  
Author(s):  
David A Eccles
Keyword(s):  
Sequence Analysis ◽  
Plasmid Dna ◽  
De Novo ◽  
Consensus Sequence ◽  
Fasta File ◽  
High Quality ◽  
Plasmid Sequence ◽  
Long Reads ◽  
The Moment

This protocol demonstrates how to assemble reads from plasmid DNA, and generate a circularised and non-repetitive consensus sequence At the moment, this protocol uses Canu to de-novo assemble high-quality single-cut reads. Input(s): demultiplexed fastq files (see protocol Demultiplexing Nanopore reads with LAST). I've noticed that the default demultiplexing carried out by Guppy (at least up to v4.2.2, as used in the first version of this protocol) has issues with chimeric reads, which can affect assembly. Output(s): Consensus sequence per barcode as a fasta file

scholarly journals Kloning gen LEAFY kakao dari jaringan bantalan bunga aktif Cloning of cacao LEAFY gene from the active flower cushions

2016 ◽  
Vol 77 (2) ◽  
Author(s):  
Tetty CHAIDAMSARI ◽  
Rita HAYATI ◽  
Auzar SYARIEF ◽  
Aswaldi ANWAR ◽  
Djoko SANTOSO
Keyword(s):  
Sequence Analysis ◽  
Regulatory Gene ◽  
Rt Pcr ◽  
Plasmid Sequence ◽  
Perennial Crops ◽  
E Coli ◽  
Apical Meristems ◽  
Competent Cells ◽  
Leafy Gene ◽  
Alignment Analysis

SummaryAttempts to improve productivity of cacaoplantations lead us to study the molecularmechanism of flowering. In the model speciesArabidopsis thaliana as well as some otherspecies, LEAFY is a central regulatory gene forthe transition of shoot apical meristems toflowering meristems. Different from that ofArabidopsis, cacao inflorescence is acauliflorous type, by which flowers can developrepeatedly from the same flower cushion on thetrunk. In this research, a LEAFY homolog wasisolated from active flower cushion with RT-PCRusing a pair of DNA primer specifically designedto isolate its complete cds. Gel electrophoresisexamination indicated the presence of a 1.2 kbamplicon. Purified from the gel, this DNAfragment was cloned into competent cells ofE. coli XL1 Blue using pGEM-T Easy cloningvector at an orientation according to the T7promoter of the plasmid. Sequence analysis usingBLASTX, showed that the amplicon was LEAFY(LFY) homolog. Alignment analysis using ClustalW indicated that the cTcLFY highly homologousto those from other perennial crops such ascitrus, grape, apple and poplar. The highesthomology (conserved region) was found in the Cterminal of the encoded proteins.RingkasanUsaha untuk meningkatkan produktivitasperkebunan kakao telah mendorong penelitianmolekuler tentang mekanisme pembungaankakao. Pada tanaman model Arabidopsis thalianadan lainnya, LEAFY merupakan gen kunci dalamtransisi meristem tunas jadi meristem bunga.Berbeda dengan sistem pada Arabidopsis,pembungaan kakao termasuk tipe cauliflorous,bunga dapat muncul dari bantalan bunga yangsama sepanjang tahun. Dalam penelitian inihomolog LFY diisolasi dari bantalan bunga aktifmenggunakan RT-PCR dengan sepasang primerspesifik yang dirancang berdasarkan sekuenDNA di kedua ujung gen tersebut. Pemeriksaangel elektroforesis menunjukkan adanya amplikontunggal berukuran 1,2 kb. Setelah dimurnikandari gel, amplikon dapat diklon ke dalam selkompeten E. coli galur XL1 Blue menggunakanvektor pGEM-T Easy dengan orientasi yangsesuai dengan promoter T7 dari vektor. AnalisisBLASTX sekuen DNA membuktikan bahwaamplikon tersebut adalah homolog dari genLEAFY. Analisis penjajaran dengan mengguna-kan ClustalW menunjukkan bahwa gen cTcLFYtersebut memiliki homologi yang tinggi dengangen sejenis dari tanaman keras lainnya sepertitanaman jeruk, anggur, apel dan poplar.Homologi tertinggi (daerah terkonservasi)terdapat pada ujung (terminal) C dari proteinyang disandinya.

scholarly journals Kloning gen LEAFY kakao dari jaringan bantalan bunga aktif Cloning of cacao LEAFY gene from the active flower cushions

2016 ◽  
Vol 77 (2) ◽  
Author(s):  
Tetty CHAIDAMSARI ◽  
Rita HAYATI ◽  
Auzar SYARIEF ◽  
Aswaldi ANWAR ◽  
Djoko SANTOSO
Keyword(s):  
Sequence Analysis ◽  
Regulatory Gene ◽  
Rt Pcr ◽  
Plasmid Sequence ◽  
Perennial Crops ◽  
E Coli ◽  
Apical Meristems ◽  
Competent Cells ◽  
Leafy Gene ◽  
Alignment Analysis

SummaryAttempts to improve productivity of cacaoplantations lead us to study the molecularmechanism of flowering. In the model speciesArabidopsis thaliana as well as some otherspecies, LEAFY is a central regulatory gene forthe transition of shoot apical meristems toflowering meristems. Different from that ofArabidopsis, cacao inflorescence is acauliflorous type, by which flowers can developrepeatedly from the same flower cushion on thetrunk. In this research, a LEAFY homolog wasisolated from active flower cushion with RT-PCRusing a pair of DNA primer specifically designedto isolate its complete cds. Gel electrophoresisexamination indicated the presence of a 1.2 kbamplicon. Purified from the gel, this DNAfragment was cloned into competent cells ofE. coli XL1 Blue using pGEM-T Easy cloningvector at an orientation according to the T7promoter of the plasmid. Sequence analysis usingBLASTX, showed that the amplicon was LEAFY(LFY) homolog. Alignment analysis using ClustalW indicated that the cTcLFY highly homologousto those from other perennial crops such ascitrus, grape, apple and poplar. The highesthomology (conserved region) was found in the Cterminal of the encoded proteins.RingkasanUsaha untuk meningkatkan produktivitasperkebunan kakao telah mendorong penelitianmolekuler tentang mekanisme pembungaankakao. Pada tanaman model Arabidopsis thalianadan lainnya, LEAFY merupakan gen kunci dalamtransisi meristem tunas jadi meristem bunga.Berbeda dengan sistem pada Arabidopsis,pembungaan kakao termasuk tipe cauliflorous,bunga dapat muncul dari bantalan bunga yangsama sepanjang tahun. Dalam penelitian inihomolog LFY diisolasi dari bantalan bunga aktifmenggunakan RT-PCR dengan sepasang primerspesifik yang dirancang berdasarkan sekuenDNA di kedua ujung gen tersebut. Pemeriksaangel elektroforesis menunjukkan adanya amplikontunggal berukuran 1,2 kb. Setelah dimurnikandari gel, amplikon dapat diklon ke dalam selkompeten E. coli galur XL1 Blue menggunakanvektor pGEM-T Easy dengan orientasi yangsesuai dengan promoter T7 dari vektor. AnalisisBLASTX sekuen DNA membuktikan bahwaamplikon tersebut adalah homolog dari genLEAFY. Analisis penjajaran dengan mengguna-kan ClustalW menunjukkan bahwa gen cTcLFYtersebut memiliki homologi yang tinggi dengangen sejenis dari tanaman keras lainnya sepertitanaman jeruk, anggur, apel dan poplar.Homologi tertinggi (daerah terkonservasi)terdapat pada ujung (terminal) C dari proteinyang disandinya.

scholarly journals HaplotypeTools: a toolkit for accurately identifying recombination and recombinant genotypes

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Rhys A. Farrer
Keyword(s):  
Sequence Analysis ◽  
Batrachochytrium Dendrobatidis ◽  
Fungal Isolate ◽  
Computer Cluster ◽  
Phylogenetic Placement ◽  
Long Reads ◽  
Illumina Data ◽  
Mitotic Gene Conversion ◽  
Downstream Analysis ◽  
Genomic Regions

Abstract Background Identifying haplotypes is central to sequence analysis in diploid or polyploid genomes. Despite this, there remains a lack of research and tools designed for physical phasing and its downstream analysis. Results HaplotypeTools is a new toolset to phase variant sites using VCF and BAM files and to analyse phased VCFs. Phasing is achieved via the identification of reads overlapping ≥ 2 heterozygous positions and then extended by additional reads, a process that can be parallelized across a computer cluster. HaplotypeTools includes various utility scripts for downstream analysis including crossover detection and phylogenetic placement of haplotypes to other lineages or species. HaplotypeTools was assessed for accuracy against WhatsHap using simulated short and long reads, demonstrating higher accuracy, albeit with reduced haplotype length. HaplotypeTools was also tested on real Illumina data to determine the ancestry of hybrid fungal isolate Batrachochytrium dendrobatidis (Bd) SA-EC3, finding 80% of haplotypes across the genome phylogenetically cluster with parental lineages BdGPL (39%) and BdCAPE (41%), indicating those are the parental lineages. Finally, ~ 99% of phasing was conserved between overlapping phase groups between SA-EC3 and either parental lineage, indicating mitotic gene conversion/parasexuality as the mechanism of recombination for this hybrid isolate. HaplotypeTools is open source and freely available from https://github.com/rhysf/HaplotypeTools under the MIT License. Conclusions HaplotypeTools is a powerful resource for analyzing hybrid or recombinant diploid or polyploid genomes and identifying parental ancestry for sub-genomic regions.

scholarly journals The Genomic Epidemiology of Carbapenemase-Producing Enterobacterales (CPE) at a Hospital System in Toronto, Ontario, Canada, 2007-2018

2021 ◽  
Author(s):  
Alainna J. Jamal ◽  
Laura F. Mataseje ◽  
Victoria Williams ◽  
Jerome A. Leis ◽  
Nathalie Tijet ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Index Case ◽  
Epidemiological Data ◽  
Population Based ◽  
Hospital System ◽  
Related Case ◽  
Plasmid Sequence ◽  
Genomic Epidemiology ◽  
Cluster A ◽  
And Control

Background: At a hospital system (H1) in Ontario, Canada, we investigated whether whole-genome sequencing (WGS) altered initial epidemiological interpretation of carbapenemase-producing Enterobacterales (CPE) transmission. Methods: We included patients with CPE colonization/infection identified by population-based surveillance from October 2007 to August 2018 who received healthcare at H1 in the year before/after CPE detection. H1 reported epidemiological transmission clusters. We combined single nucleotide variant (SNV) analysis, plasmid characterization, and epidemiological data. Results: Eighty-five patients were included. H1 identified 7 epidemiological transmission clusters A-G involving 24/85 (28%) patients. SNV analysis confirmed transmission clusters C, D, and G, and identified two additional cases belonging to cluster A. One was a travel-related case that was the likely index case (0-6 SNVs from other isolates); this case stayed on the same unit as the initially presumed index case 4 months prior to detection of the initially presumed index case on another unit. The second additional case occupied a room previously occupied by 5 cluster A cases. Plasmid sequence analysis excluded a case from cluster A and identified clusters E and F as possibly two parts of a single cluster. SNV analysis also identified a case without direct epidemiologic links that was 18-21 SNVs away from cluster B, suggesting possible undetected inter-hospital transmission. Conclusions: SNV and plasmid sequence analysis identified cases belonging to transmission clusters that conventional epidemiology missed and excluded other cases. Implementation of routine WGS to complement epidemiological transmission investigations has the potential to improve prevention and control of CPE in hospitals.

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