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11th Annual Sequencing, Finishing, and Analysis in the Future Meeting EXAMINATION OF WHOLE GENOME MULTI- LOCUS SEQUENCE TYPING (WGMLST) FOR CAMPYLOBACTER SURVEILLANCE Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.13) Lavin Joseph, Heather Carleton, Darlene Wagner, Michael Judd, Eija Trees, Vikrant Dutta, Janet Pruckler, Grant Williams, Kelley Hise, Collette Fitzgerald Centers for Disease Control and Prevention Background: Campylobacter species are a leading cause of bacterial foodborne illness in the United States. Pulsedfield gel electrophoresis (PFGE) is the current method used within PulseNet USA for surveillance and outbreak investigations. However, PulseNet USA is implementing whole genome sequencing (WGS) for surveillance of all the pathogens it tracks including Campylobacter. In this study, we examined the utility of whole genome multi‐locus sequence typing (wgMLST) to cluster or differentiate outbreak‐associated and sporadic Campylobacter isolates. Methods: A BioNumerics 7.5 wgMLST database was developed in collaboration with domestic and international partners for Campylobacter surveillance. Fourteen public health laboratories (PHL) performed real‐time WGS on 262 Campylobacter isolates (219 C. jejuni, 35 C. coli, 6 C. lari, 1 C. upsaliensis, and 1 C. fetus) isolates. In addition, 14 C. jejuni isolates from two temporarily associated outbreaks linked to a family reunion (two isolates) and church fundraiser (12 isolates) originating in the same state were sequenced by the local PHL. Sequencing was performed on the Illumina MiSeq using Nextera XT DNA libraries and 2x150 bp or 2x250 bp sequencing chemistry. Sequences with >20X coverage and average quality scores for R1 and R2 >30 were assembled using SPAdes and analyzed using wgMLST (2531 loci). For comparison, the C. jejuni sequences associated with the two outbreaks were also characterized with high quality single nucleotide polymorphism (hqSNP) analysis using the LYVE‐SET pipeline (github.com/lskatz/lyve‐SET) as well as K‐mer analysis using the NCBI Pathogen Detection Pipeline (http://www.ncbi.nlm.nih.gov/pathogens/). PFGE patterns for all isolates were generated using the PulseNet Campylobacter protocol, analyzed in BioNumerics 6.6.10, and named in accordance with PulseNet naming guidelines. Results: On average, 1547 (915‐1752), 573 (250‐737), 344 (305‐473), 371, and 453 wgMLST loci were identified within C. jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus sequences, respectively. C. jejuni and C. coli isolates presumed to be epidemiologically unrelated (n=20) formed 12 clusters containing indistinguishable SmaI/KpnI patterns; nine of these clusters contained isolates that were also indistinguishable or highly similar by wgMLST (0‐ 5 allele differences). The isolates in the remaining clusters were further differentiated by wgMLST (21‐98 allele differences). C. jejuni isolates from the two outbreaks clustered together by wgMLST (0‐3 allele differences), hqSNP (0‐3 SNP differences), and K‐mer analysis even though these outbreaks clustered separately by PFGE. Conclusion: The majority (82%) of the sporadic Campylobacter isolates indistinguishable by PFGE were not separated by wgMLST. The isolates within each cluster were from the same state and may be from patients exposed to a common source, whereas isolates differentiated by wgMLST could be epidemiologically unrelated. Interestingly, C. jejuni isolates from the two Campylobacter outbreaks clustered together by wgMLST, hqSNP, and K‐mer analysis, indicating that they may have origi‐ nated from the same source that was not discovered during the epidemiological investigations. It is also possible the sequence type associated with these isolates is relatively common. Our study shows that wgMLST appears to be a promising and user‐friendly high resolution tool for Campylobacter surveillance that can be implemented in local PHLs. 79
11th Annual Sequencing, Finishing, and Analysis in the Future Meeting AN IMPROVED ASSEMBLY ALGORITHM FOR DE NOVO CIRCULAR GENOME RECONSTRUCTION Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.14) Christian Olsen, Helen Shearman, Richard Moir, Matt Kearse, Simon Buxton, Matthew Cheung, Jonas Kuhn, Steven Stones Havas, Chris Duran 1 Biomatters, Inc. Circular chromosomes or genomes, such as viruses, bacteria, mitochondria and plasmids, are a common occurrence in nature, but despite the wide array of algorithms available for de novo assembly, the circularity of these DNA molecules is largely overlooked. There are some limitations of this oversight. Current NGS assembly algorithms assume a linear molecule and will result in a linearly represented genome, with a breakpoint in an arbitrary position. This is becoming increasingly problematic with increasing NGS read lengths resulting in fewer contig assemblies with less ambiguity. Additionally, long read sequencing technologies promise to provide sequences that may greatly span breakpoints at the expense of coverage resulting in a relatively large quanta of information loss. Although there are currently methods for the re‐circularisation of contigs post‐assembly by identifying common trailing/leading sequence motifs, we present a more robust approach of circularising during the assembly process whilst still allowing the merging of similar and sub‐contigs throughout the overlap‐based approach. This method can also combat the issue of chimeric sequence due to contamination, a common problem when sequencing bacterial cultures, due to decreased likelihood of conserved contig ends due to timely circularisation. We present results from both 28.6 million read Pan troglodytes illumina data set and 267,491 read Panthera leo persica (Asiatic Lion) mitochondrial NGS library produced using an Ion Torrent sequencing machine. These results are discussed and compared to some of the more popular linear assembly algorithms in common usage today. Geneious R8 is the first bioinformatics software package to offer a circular de novo assembly method. The Geneious Circular de novo assembler is developed by Biomatters and may be found at http://www.geneious.com 80
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Reliable solutions for focused NGS
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