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

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.

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High-throughput functional screening reveals low frequency of antibiotic resistance genes in DNA recovered from the Upper Mississippi River

Journal of Water and Health ◽

10.2166/wh.2014.215 ◽

2014 ◽

Vol 13 (3) ◽

pp. 693-703 ◽

Cited By ~ 9

Author(s):

Christopher Staley ◽

Trevor J. Gould ◽

Ping Wang ◽

Jane Phillips ◽

James B. Cotner ◽

...

Keyword(s):

Antibiotic Resistance ◽

Land Cover ◽

High Throughput ◽

Resistance Genes ◽

Mississippi River ◽

Selection Process ◽

Antibiotic Resistance Genes ◽

Nutrient Concentrations ◽

Functional Screening ◽

Upper Mississippi River

In this study, we determined the frequency of antibiotic resistance genes (ARGs) in the Upper Mississippi River using a high-throughput, functional, metagenomic screening procedure. Fosmid libraries containing ∼10,000 clones were screened for resistance to ampicillin, cephalothin, kanamycin, and tetracycline. We hypothesized that nutrient concentrations, land cover type, and taxonomic community composition may select for ARGs. Resistance to ampicillin, cephalothin, and kanamycin was low (<1.00%), and no resistance to tetracycline was detected. Ammonium and total dissolved solids (TDS) concentrations were correlated with kanamycin and cephalothin resistances (r = 0.617 and −0.449, P = 0.002 and 0.036, respectively). Cephalothin resistance was also positively correlated with the percentage of forested land cover (r = 0.444, P = 0.039). Only the candidate division OD1, among 35 phyla identified, was correlated with ampicillin resistance (r = 0.456, P = 0.033), suggesting that minority members of the community may be responsible for dissemination of ARGs in this ecosystem. Results of this study suggest that ammonium and TDS may be involved in a complex selection process for ARGs. Furthermore, we suggest that minority species, potentially contributed in low numbers from sediment and biofilm reservoirs, may be the primary carriers of ARGs in this riverine system.

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Construction of a Full-Length cDNA Library from Castor Endosperm for High-Throughput Functional Screening

Methods in Molecular Biology - cDNA Libraries ◽

10.1007/978-1-61779-065-2_3 ◽

2011 ◽

pp. 37-52 ◽

Cited By ~ 1

Author(s):

Chaofu Lu ◽

James G. Wallis ◽

John Browse

Keyword(s):

Cdna Library ◽

High Throughput ◽

Full Length ◽

Functional Screening ◽

Full Length Cdna

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A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG

mAbs ◽

10.1080/19420862.2017.1337617 ◽

2017 ◽

Vol 9 (6) ◽

pp. 996-1006 ◽

Cited By ~ 14

Author(s):

Xiaodong Xiao ◽

Julie A. Douthwaite ◽

Yan Chen ◽

Ben Kemp ◽

Sara Kidd ◽

...

Keyword(s):

Phage Display ◽

High Throughput ◽

Full Length ◽

Functional Screening ◽

Mammalian Expression ◽

Phage Display Libraries

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

10.1101/2020.07.27.222349 ◽

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.

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

Marine Drugs ◽

10.3390/md19070392 ◽

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.

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CRISPR/Cas9 high-throughput screening in cancer research

E3S Web of Conferences ◽

10.1051/e3sconf/202018503032 ◽

2020 ◽

Vol 185 ◽

pp. 03032

Author(s):

Zhuoxin Liu

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Genomic Library ◽

Gene Activation ◽

Library Screening ◽

Basic Principles ◽

Tumor Research ◽

Technology Strategies ◽

Target Effect ◽

Main Strategy

In recent years, CRISPR/Cas9 technology has developed rapidly. With its accurate, fast, and simple editing functions that can achieve gene activation, interference, knockout, and knock-in, it has become a powerful genetic screening tool that is widely used in various models, including cell lines of mice and zebrafish. The use of CRISPR system to construct a genomic library for high-throughput screening is the main strategy for research of disease, especially tumor target gene research. This article reviews the basic principles and latest developments of CRISPR/Cas9 library screening technology strategies to improve its off-target effect, the basic workflow of library screening, and its application in tumor research.

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