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Title: Practical applications of next-generation sequencing for food-safety research

item Oakley, Brian
item Line, John
item Hiett, Kelli
item MORALES, CESAR - Former ARS Employee
item Berrang, Mark
item JOHNSON, JESSICA - Former ARS Employee
item Buhr, Richard - Jeff
item Cox Jr, Nelson
item Seal, Bruce

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 12/15/2012
Publication Date: 3/11/2012
Citation: Oakley, B., Line, J.E., Hiett, K.L., Morales, C.A., Berrang, M.E., Johnson, J.M., Buhr, R.J., Cox Jr, N.A., Seal, B.S. 2012. Practical applications of next-generation sequencing for food-safety research. 2012 U.S. Japan Cooperative Program in Natural Resources (UNJR), Food and Agriculture Panel. March 5-6, 2012. Seiryo, Kaikan.

Interpretive Summary:

Technical Abstract: Next-generation sequencing (NGS) is a transformative technology that is revolutionizing the biological sciences. However, many researchers remain uncertain as to the best ways to harness the power of NGS and apply it to their own research questions. Here we highlight three case studies of how NGS can be applied to food safety research and provide background on analytical methodologies that should be generically useful to others in the field. Materials and Methods Samples were collected and sequenced as part of three distinct studies as previously described. First, to determine the best microbiological sampling method for poultry processing facilities, we used NGS to compare carcass rinses from individual birds versus drip samples collected cumulatively underneath the processing line [1]. Second, to quantitatively assess the selectivity of commonly-used growth media for the foodborne pathogen Campylobacter, we used NGS to exhaustively sequence colonies formed on plates and compared these data to the bacterial community structure of the poultry fecal samples used for plating [2]. Third, to empirically test the specificity and sensitivity of a putative Campylobacter-specific PCR assay, we used NGS to characterize the population of amplicons produced by this assay applied to a complex sample [3]. For each of these studies, sequence data were analyzed with an analysis pipeline we have constructed using the programming languages perl and R. Results and Discussion Comparison of individual carcass rinses vs. cumulative drip sampling: The whole carcass rinse (WCR) procedure is routinely used as a sampling method for determining the presence and number of quality-indicator organisms or pathogens associated with broiler chicken carcasses in processing facilities. For most bacterial taxa, relative abundance in individual WCR was correlated with cumulative drip samples, but some taxa were under-counted or missed entirely by individual WCR (Figure 1). Consequently, individual carcass rinses may not be representative of the flock microbial community. The cumulative drip sampling technique saves labor and can provide a more representative summary of process control in poultry processing facilities. Figure 1. Relative proportions of sequence types from cumulative drips versus individual bird rinses. Each point in the graph represents a unique taxon at the species level as determined by classification of sequences with the Silva reference database. Panel A shows all taxa for the two drip samples combined versus all three carcass rinses combined. The two solid points are shown in detail in Panels B and C for percentages of sequences classified as Gallibacterium anatis (B) and Klebsiella pneumoniae (C) for each sample type. Figure adapted from [1]. Comparison of Campylobacter-selective media: In this study, we used NGS to ask: 1) How selective are commonly-used Campylobacter cultivation methods, and 2) How do the specificity and sensitivity of these methods compare to one another? To answer these questions, we used 16S rRNA tagged-pyrosequencing to sequence directly from a pooled fecal sample representing a ca. 16,000 bird poultry flock, and compared these data to exhaustive sequencing of colonies formed after plating. We compared five commonly used media, two incubation atmospheres, and two incubation temperatures. Campylobacter represented only a small proportion (<0.04%) of sequences present in the feces, but 88 - 97% of sequences from each media type. The most common non-Campylobacter sequence type was Proteus, which ranged from 0.04% of sequences (mCCDA) to 10.8% (Cape Town). Incubation atmosphere had little effect on recovery, but a significant difference in media specificity (more non-Campylobacter OTUs; p=0.028) was found at 42° versus 37° C. NGS provides a novel and powerful approach to measure the performance of s