scholarly journals Faculty Opinions recommendation of Flexible and Scalable Full-Length CYP2D6 Long Amplicon PacBio Sequencing.

2021 ◽  
Author(s):  
Michele Ramsay ◽  
David Twesigomwe
Keyword(s):  
Full Length ◽  
Pacbio Sequencing

scholarly journals Full-Length Transcriptome of Thalassiosira weissflogii as a Reference Resource and Mining of Chitin-Related Genes

Marine Drugs ◽  
2021 ◽  
Vol 19 (7) ◽  
pp. 392
Author(s):  
Haomiao Cheng ◽  
Chris Bowler ◽  
Xiaohui Xing ◽  
Vincent Bulone ◽  
Zhanru Shao ◽  
...  
Keyword(s):  
Cell Wall ◽  
Full Length ◽  
Biosynthetic Pathways ◽  
Glycosidic Linkage ◽  
Thalassiosira Weissflogii ◽  
Ecological Value ◽  
Pacbio Sequencing ◽  
Non Coding Rnas ◽  
Chitin And Chitosan ◽  
Simple Sequence

β-Chitin produced by diatoms is expected to have significant economic and ecological value due to its structure, which consists of parallel chains of chitin, its properties and the high abundance of diatoms. Nevertheless, few studies have functionally characterised chitin-related genes in diatoms owing to the lack of omics-based information. In this study, we first compared the chitin content of three representative Thalassiosira species. Cell wall glycosidic linkage analysis and chitin/chitosan staining assays showed that Thalassiosira weissflogii was an appropriate candidate chitin producer. A full-length (FL) transcriptome of T. weissflogii was obtained via PacBio sequencing. In total, the FL transcriptome comprised 23,362 annotated unigenes, 710 long non-coding RNAs (lncRNAs), 363 transcription factors (TFs), 3113 alternative splicing (AS) events and 3295 simple sequence repeats (SSRs). More specifically, 234 genes related to chitin metabolism were identified and the complete biosynthetic pathways of chitin and chitosan were explored. The information presented here will facilitate T. weissflogii molecular research and the exploitation of β-chitin-derived high-value enzymes and products.

scholarly journals Functional Genomic Identification of Cadmium Resistance Genes from a High GC Clone Library by Coupling the Sanger and PacBio Sequencing Strategies

Genes ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 7
Author(s):  
Jinghao Chen ◽  
Chao Xing ◽  
Xin Zheng ◽  
Xiaofang Li
Keyword(s):  
High Throughput ◽  
Resistance Genes ◽  
Sanger Sequencing ◽  
Genomic Library ◽  
Full Length ◽  
Host Cells ◽  
Full Genome Sequence ◽  
Functional Screening ◽  
Functional Genomic ◽  
Pacbio Sequencing

Functional (meta) genomics allows the high-throughput identification of functional genes in a premise-free way. However, it is still difficult to perform Sanger sequencing for high GC DNA templates, which hinders the functional genomic exploration of a high GC genomic library. Here, we developed a procedure to resolve this problem by coupling the Sanger and PacBio sequencing strategies. Identification of cadmium (Cd) resistance genes from a small-insert high GC genomic library was performed to test the procedure. The library was generated from a high GC (75.35%) bacterial genome. Nineteen clones that conferred Cd resistance to Escherichia coli subject to Sanger sequencing directly. The positive clones were in parallel subject to in vivo amplification in host cells, from which recombinant plasmids were extracted and linearized by selected restriction endonucleases. PacBio sequencing was performed to obtain the full-length sequences. As the identities, partial sequences from Sanger sequencing were aligned to the full-length sequences from PacBio sequencing, which led to the identification of seven unique full-length sequences. The unique sequences were further aligned to the full genome sequence of the source strain. Functional screening showed that the identified positive clones were all able to improve Cd resistance of the host cells. The functional genomic procedure developed here couples the Sanger and PacBio sequencing methods and overcomes the difficulties in PCR approaches for high GC DNA. The procedure can be a promising option for the high-throughput sequencing of functional genomic libraries, and realize a cost-effective and time-efficient identification of the positive clones, particularly for high GC genetic materials.

scholarly journals Flexible and Scalable Full-Length CYP2D6 Long Amplicon PacBio Sequencing

2017 ◽  
Vol 38 (3) ◽  
pp. 310-316 ◽  
Author(s):  
Henk P.J. Buermans ◽  
Rolf H.A.M. Vossen ◽  
Seyed Yahya Anvar ◽  
William G. Allard ◽  
Henk-Jan Guchelaar ◽  
...  
Keyword(s):  
Full Length ◽  
Pacbio Sequencing

scholarly journals Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification

2016 ◽  
Vol 16 (1) ◽  
Author(s):  
Josef Wagner ◽  
Paul Coupland ◽  
Hilary P. Browne ◽  
Trevor D. Lawley ◽  
Suzanna C. Francis ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Full Length ◽  
Rrna Gene ◽  
Pacbio Sequencing ◽  
Bacterial 16S Rrna Gene

scholarly journals HIT-scISOseq: High-throughput and High-accuracy Single-cell Full-length Isoform Sequencing for Corneal Epithelium

2020 ◽  
Author(s):  
Ying-Feng Zheng ◽  
Zhi-Chao Chen ◽  
Zhuo-Xing Shi ◽  
Kun-Hua Hu ◽  
Jia-Yong Zhong ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Cost Effective ◽  
Pcr Primers ◽  
Cell Types ◽  
High Accuracy ◽  
Full Length ◽  
Pacbio Sequencing ◽  
Simple Method ◽  
Long Read

AbstractSingle-cell isoform sequencing can reveal transcriptomic dynamics in individual cells invisible to bulk- and single-cell RNA analysis based on short-read sequencing. However, current long-read single-cell sequencing technologies have been limited by low throughput and high error rate. Here we introduce HIT-scISOseq for high-throughput single-cell isoform sequencing. This method was made possible by full-length cDNA capture using biotinylated PCR primers, and by our novel library preparation procedure that combines head-to-tail concatemeric full-length cDNAs into a long SMRTbell insert for high-accuracy PacBio sequencing. HIT-scISOseq yields > 10 million high-accuracy full-length isoforms in a single PacBio Sequel II 8M SMRT Cell, providing > 8 times more data output than the standard single-cell isoform PacBio sequencing protocol. We exemplified HIT-scISOseq by first studying transcriptome profiles of 4,000 normal and 8,000 injured corneal epitheliums from cynomolgus monkeys. We constructed dynamic transcriptome landscapes of known and rare cell types, revealed novel isoforms, and identified injury-related splicing and switching events that are previously not accessible with low throughput isoform sequencing. HIT-scISOseq represents a high-throughput, cost-effective, and technically simple method to accelerate the burgeoning field of long-read single-cell transcriptomics.

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

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