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2015 ; 5
(ä): 11047
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Preferential Amplification of Pathogenic Sequences
#MMPMID26067233
Ge F
; Parker J
; Chul Choi S
; Layer M
; Ross K
; Jilly B
; Chen J
Sci Rep
2015[Jun]; 5
(ä): 11047
PMID26067233
show ga
The application of next generation sequencing (NGS) technology in the diagnosis
of human pathogens is hindered by the fact that pathogenic sequences, especially
viral, are often scarce in human clinical specimens. This known disproportion
leads to the requirement of subsequent deep sequencing and extensive
bioinformatics analysis. Here we report a method we called "Preferential
Amplification of Pathogenic Sequences (PATHseq)" that can be used to greatly
enrich pathogenic sequences. Using a computer program, we developed 8-, 9-, and
10-mer oligonucleotides called "non-human primers" that do not match the most
abundant human transcripts, but instead selectively match transcripts of human
pathogens. Instead of using random primers in the construction of cDNA libraries,
the PATHseq method recruits these short non-human primers, which in turn,
preferentially amplifies non-human, presumably pathogenic sequences. Using this
method, we were able to enrich pathogenic sequences up to 200-fold in the final
sequencing library. This method does not require prior knowledge of the pathogen
or assumption of the infection; therefore, it provides a fast and
sequence-independent approach for detection and identification of human viruses
and other pathogens. The PATHseq method, coupled with NGS technology, can be
broadly used in identification of known human pathogens and discovery of new
pathogens.