Sequencing

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11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting ALLELE-MINING RNA-SEQ DATA OF COTTON (GOSSYPIUM HIRSUTUM L.) ROOTS Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.11) Daojun Yuan 1 , Alex Freeman 1 , Christopher Hanson 1 , Aaron Sharp 1 , Sara Greenfiled 1 , Lori Hinze 2 , Richard Percy 2 , Joshua A Udall 1 1 Brigham Young University, 2 USDA‐ARS South Plains Agricultural Research Center Cotton genomic resources and nucleotide polymorphism information is useful to understand germplasm diversity, crop domestication, and crop improvement. There are many publicly available resources of cotton; however most resources of gene expression are from fiber tissue and roots have been less studied. In this study, we report individual transcriptomes of 108 G. hirsutum accessions of root tissue, including 41 improved (domesticated) and 67 wild accessions from the USDA Germplasm collection. More than 3.0 billion clean reads were generated. Of the 37,505 genes in the diploid cotton G. raimondii reference genome, 61.4% were expressed in root tissue. Of these, 1,648 and 1,487 genes in improved and wild accessions respectively which were differentially expressed between the two subgenomes. The RNA‐seq data was mined for alleles that were polymorphic among the diverse germplasm. The AT‐genome had 234,266 SNPs in 25,993 genes, while the DT‐genome had 169,123 SNPs in 25,836 genes (5x coverage). After a stricter filtering (
11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting COMPARISON OF STANDARD CULTURE AND SINGLE-COLONY DNA EXTRACTION METHODS FOR WHOLE-GENOME SEQUENCING OF CAMPYLOBACTER JEJUNI Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.12) Kun Liu, Karen Jinneman US FDA Introduction Campylobacter jejuni is the most common food‐borne bacteria causing diarrhea in the US and is most often associated with poultry, raw milk, unpasteurized eggs, contaminated water or produce. The technical advancement and decrease in cost of whole‐genome sequencing (WGS) have made WGS more widely available for rapid and accurate identification of pathogens such as C. jejuni from contaminated food. Since bacterial culture and DNA extraction are critical to WGS, we evaluated the effects of two available methods on WGS data quality and analysis. Purpose To compare the quality of C. jejuni genomes prepared from two extraction methods. The subculture method, currently used in FDA, requires more biomass and involves an automated extraction. In contrast, a more rapid protocol needs only a single colony and mechanically releases DNA, which shows potential to greatly reduce culture time and reagent cost. Here, DNA was prepared from both methods, data quality was compared and method applications were evaluated based on WGS results. Methods C. jejuni B9 strain was isolated by Pacific Laboratory Northwest in 1983. DNA was extracted using QIAcube (Qiagen Inc., Valencia, CA) from subculture 1 or single‐colony 2. DNA concentrations were determined by Qubit HS kit (Thermo Fisher Scientific Corp., Waltham, MA). Libraries were prepared with Nextera XT Kit (Illumina Inc., San Diego, CA). WGS experiments were performed on MiSeq (Illumina Inc., San Diego CA). Genomes were assembled with CLC genomics workbench (Qiagen Inc.). WGS data were evaluated using SpeciesFinder for 16S typing 3 and KmerFinder for speciation 3, 4. Genome annotations were performed with Rapid Annotation using Subsystem Technology (RAST) 5‐7. Results The subculture method took 48 hours to grow C. jejuni and 2 hours on extraction, whereas the single‐colony method took less than 30 minutes to finish all steps. The former yielded higher DNA concentration than the latter (11.4 vs. 0.5 ng/ul), but both were sufficient for library preparation which only requires 0.2 ng/ul. Qualitatively, B9 was correctly identified to be C. jejuni based on WGS data from both extraction methods and highly homologous to reference strains NCTC‐11168 and RM‐1221. Quantitatively, de novo assembly from the subculture method yielded an N50 length of 188 kb from 63 contigs. In comparison, that from the single‐colony method yielded an N50 length of 72 kb from 244 contigs. Average coverage rates were both greater than 270x. RAST annotations showed similar results. Thedraft genome from subculture was predicted to contain 1751 coding sequences, 315 subsystems, and 43 RNAs, while that from single‐colony was predicted with 1753 coding sequences, 312 subsystems, and 43 RNAs. Conclusions We compared two DNA preparation methods on a C. jejuni field isolate and evaluated WGS data generated to assess applications for regulatory food safety science. For qualitative analysis following WGS, such as confirmation and speciation, the single‐colony method is as good as subculture with a great time‐saving advantage (49.5 hours shorter) and reduced cost ($4.60 less per sample). However, for deeper genomics analysis, DNA from subculture exhibits better quality based on N50 and contig counts. 78
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Reliable solutions for focused NGS
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Sequencing

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