TY - JOUR
T1 - A Cautionary Report for Pathogen Identification Using Shotgun Metagenomics; A Comparison to Aerobic Culture and Polymerase Chain Reaction for Salmonella enterica Identification
AU - Doster, Enrique
AU - Rovira, Pablo
AU - Noyes, Noelle R.
AU - Burgess, Brandy A.
AU - Yang, Xiang
AU - Weinroth, Margaret D.
AU - Linke, Lyndsey
AU - Magnuson, Roberta
AU - Boucher, Christina
AU - Belk, Keith E.
AU - Morley, Paul S.
N1 - Publisher Copyright:
© Copyright © 2019 Doster, Rovira, Noyes, Burgess, Yang, Weinroth, Linke, Magnuson, Boucher, Belk and Morley.
PY - 2019/11/1
Y1 - 2019/11/1
N2 - This study was conducted to compare aerobic culture, polymerase chain reaction (PCR), lateral flow immunoassay (LFI), and shotgun metagenomics for identification of Salmonella enterica in feces collected from feedlot cattle. Samples were analyzed in parallel using all four tests. Results from aerobic culture and PCR were 100% concordant and indicated low S. enterica prevalence (3/60 samples positive). Although low S. enterica prevalence restricted formal statistical comparisons, LFI and deep metagenomic sequencing results were discordant with these results. Specifically, metagenomic analysis using k-mer-based classification against the RefSeq database indicated that 11/60 of samples contained sequence reads that matched to the S. enterica genome and uniquely identified this species of bacteria within the sample. However, further examination revealed that plasmid sequences were often included with bacterial genomic sequence data submitted to NCBI, which can lead to incorrect taxonomic classification. To circumvent this classification problem, we separated all plasmid sequences included in bacterial RefSeq genomes and reassigned them to a unique taxon so that they would not be uniquely associated with specific bacterial species such as S. enterica. Using this revised database and taxonomic structure, we found that only 6/60 samples contained sequences specific for S. enterica, suggesting increased relative specificity. Reads identified as S. enterica in these six samples were further evaluated using BLAST and NCBI’s nr/nt database, which identified that only 2/60 samples contained reads exclusive to S. enterica chromosomal genomes. These two samples were culture- and PCR-negative, suggesting that even deep metagenomic sequencing suffers from lower sensitivity and specificity in comparison to more traditional pathogen detection methods. Additionally, no sample reads were taxonomically classified as S. enterica with two other metagenomic tools, Metagenomic Intra-species Diversity Analysis System (MIDAS) and Metagenomic Phylogenetic Analysis 2 (MetaPhlAn2). This study re-affirmed that the traditional techniques of aerobic culture and PCR provide similar results for S. enterica identification in cattle feces. On the other hand, metagenomic results are highly influenced by the classification method and reference database employed. These results highlight the nuances of computational detection of species-level sequences within short-read metagenomic sequence data, and emphasize the need for cautious interpretation of such results.
AB - This study was conducted to compare aerobic culture, polymerase chain reaction (PCR), lateral flow immunoassay (LFI), and shotgun metagenomics for identification of Salmonella enterica in feces collected from feedlot cattle. Samples were analyzed in parallel using all four tests. Results from aerobic culture and PCR were 100% concordant and indicated low S. enterica prevalence (3/60 samples positive). Although low S. enterica prevalence restricted formal statistical comparisons, LFI and deep metagenomic sequencing results were discordant with these results. Specifically, metagenomic analysis using k-mer-based classification against the RefSeq database indicated that 11/60 of samples contained sequence reads that matched to the S. enterica genome and uniquely identified this species of bacteria within the sample. However, further examination revealed that plasmid sequences were often included with bacterial genomic sequence data submitted to NCBI, which can lead to incorrect taxonomic classification. To circumvent this classification problem, we separated all plasmid sequences included in bacterial RefSeq genomes and reassigned them to a unique taxon so that they would not be uniquely associated with specific bacterial species such as S. enterica. Using this revised database and taxonomic structure, we found that only 6/60 samples contained sequences specific for S. enterica, suggesting increased relative specificity. Reads identified as S. enterica in these six samples were further evaluated using BLAST and NCBI’s nr/nt database, which identified that only 2/60 samples contained reads exclusive to S. enterica chromosomal genomes. These two samples were culture- and PCR-negative, suggesting that even deep metagenomic sequencing suffers from lower sensitivity and specificity in comparison to more traditional pathogen detection methods. Additionally, no sample reads were taxonomically classified as S. enterica with two other metagenomic tools, Metagenomic Intra-species Diversity Analysis System (MIDAS) and Metagenomic Phylogenetic Analysis 2 (MetaPhlAn2). This study re-affirmed that the traditional techniques of aerobic culture and PCR provide similar results for S. enterica identification in cattle feces. On the other hand, metagenomic results are highly influenced by the classification method and reference database employed. These results highlight the nuances of computational detection of species-level sequences within short-read metagenomic sequence data, and emphasize the need for cautious interpretation of such results.
KW - PCR
KW - Salmonella enterica
KW - culture
KW - pathogen identification
KW - shotgun metagenomics
UR - http://www.scopus.com/inward/record.url?scp=85075355391&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85075355391&partnerID=8YFLogxK
U2 - 10.3389/fmicb.2019.02499
DO - 10.3389/fmicb.2019.02499
M3 - Article
C2 - 31736924
AN - SCOPUS:85075355391
SN - 1664-302X
VL - 10
JO - Frontiers in Microbiology
JF - Frontiers in Microbiology
M1 - 2499
ER -