Severity: Warning
Message: file_get_contents(https://...@pubfacts.com&api_key=b8daa3ad693db53b1410957c26c9a51b4908&a=1): Failed to open stream: HTTP request failed! HTTP/1.1 429 Too Many Requests
Filename: helpers/my_audit_helper.php
Line Number: 176
Backtrace:
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 176
Function: file_get_contents
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 250
Function: simplexml_load_file_from_url
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 3122
Function: getPubMedXML
File: /var/www/html/application/controllers/Detail.php
Line: 575
Function: pubMedSearch_Global
File: /var/www/html/application/controllers/Detail.php
Line: 489
Function: pubMedGetRelatedKeyword
File: /var/www/html/index.php
Line: 316
Function: require_once
Invasive fungal infections caused by Candida species are life threatening with high mortality, posing a severe public health threat. New technologies for rapid, genome-wide identification of virulence genes and therapeutic targets are urgently needed. Our recent engineering of a piggyBac (PB) transposon-mediated mutagenesis system in haploid Candida albicans provides a powerful discovery tool, which we anticipate should be adaptable to other haploid Candida species. In this protocol, we use haploid C. albicans as an example to present an improved version of the mutagenesis system and provide a detailed description of the protocol for constructing high-quality mutant libraries. We also describe a method for quantitative PB insertion site sequencing, PBISeq. The PBISeq library preparation procedure exploits tagmentation to quickly and efficiently construct sequencing libraries. Finally, we present a pipeline to analyze PB insertion sites in a de novo assembled genome of our engineered haploid C. albicans strain. The entire protocol takes ~7 d from transposition induction to having a final library ready for sequencing. This protocol is highly efficient and less labor intensive than alternative approaches and significantly accelerates genetic studies of Candida.
Download full-text PDF |
Source |
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http://dx.doi.org/10.1038/s41596-020-0351-3 | DOI Listing |
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