dThis readme.txt file was generated on 2015/05/20 by Fernando Leite 

GENERAL INFORMATION

Title of Dataset: Changes in the Porcine Intestinal Microbiome in Response to Infection with Salmonella Enterica and Lawsonia Intracellularis

File Information:
   Filename: Salmonella.Lawsonia Fasta Files.zip
   Short description: ZIP directory containing 16S rRNA sequences from Porcine gut samples
	* Also contains Excel spreadsheet showing sample collection information

Principal Investigator Contact Information
   A. Name:Richard Isaacson
   B. Institution: University of Minnesota
   C. Address: 1971 Commonwealth Avenue, St. Paul, MN 55108
   D. Email:isaac015@umn.edu 
Associate or Co-investigator Contact Information
   A. Name: Klaudyna A. Borewicz
   B. Institution: Laboratory of Microbiology, Wageningen University, Wageningen, Netherlands
   C. Address: Dreijenplein 10 6703 HB Wageningen, The Netherlands
   D. Email: kaborewicz@gmail.com 
Associate or Co-investigator Contact Information
   A. Name: Hyeun B. Kim
   B. Institution: Department of Animal Resources Science, Dankook University, South Korea
   C. Address: Dandae-ro 119, Cheonan 330-714, South Korea
   D. Email: hbkim@dankook.ac.kr

Date of data collection (approximate date): 05/2011
Date files were created: 05/2011
Information about funding sources that supported the collection of the data:


This work was supported by a grant from the Emerging Infectious Diseases signature research program, College of Veterinary Medicine, 
University of Minnesota and by a grant from the USDA/AFRI #2007-35212-18046.

METHODOLOGICAL INFORMATION

Description of methods used for collection/generation of data: 

For experimental challenges a total of 28 4-week old crossbred, weaned pigs were obtained and randomly allocated into four pens (7 pigs per group). Empty pens were between each of the pens used for the pigs so that the pigs in a group did not have direct contact with pigs in another group. At five weeks of age, four pigs (1 per pen) were removed and necropsied. Ten-centimeter segments of jejunum, ileum (approximately 10 centimeters from the ileocecal junction), cecum and ascending colon were removed. The remaining pigs were treated as follows: pigs in pen numbers three and four were challenged orally with 1 x 1010 CFU L. intracellularis strain PHE/MN1-00. One week later (when the pigs were six weeks of age) pigs in pen numbers 2 and 4 were challenged orally with 2 x 108 CFU of nalidixic acid resistant S. Typhimurium strain 798. Pigs in pen number 1 remained unchallenged and served as controls. When the pigs were 7, 9, and 11 weeks of age two pigs from each pen were euthanatized with an overdose of sodium pentobarbital and necropsied. The 10 cm of cecum and colon samples were opened longitudinally and the mucosal layer scraped with a microscope slide to remove loosely associated contents and most of the mucosal tissue. The fibrous serosal tissue was discarded. The DNA extraction protocol employed two rounds of bead beating in the presence of NaCl and sodium dodecyl sulfate, followed by sequential ammonium acetate and isopropanol precipitations. The precipitated nucleic acids were then treated with RNase A and proteinase K, and the DNA purified using a QIAgen DNA Mini Stool Kit (Valencia, CA), according to manufacturer's recommendations. PCR primers that flanked the V3 hypervariable region of bacterial 16S rRNAs were designed based on the blueprint for barcodes. The oligonucleotide primers included Roche A or B sequencing adapters at the 5' ends and template specific sequences at the 3' end. Barcodes were located in between the sequencing adapter and the template specific sequences of the forward primer. The primer sequences were: 5' (sequence adapter) – 10-base barcode - CCTACGGGAGGCAGCAG 3' (forward) and 5' (sequence adapter) – ATTACCGCGGCTGCTGG 3' (reverse). The amplification mix contained 2.5 units of Taq polymerase, 0.2mM of dNTPs, 0.4µM of each fusion primer, and 50ng of DNA in a reaction volume of 50µl. Cycling conditions started with an initial denaturation at 95°C for 2 minutes followed by 20 cycles at 95oC for 30 seconds, 60oC for 30 seconds, and 72oC for 30 seconds. A final 7 minute extension was incubated at 72°C. For the jejunal and ileal samples, the number of PCR cycles was increased to 25 to compensate for lower bacterial numbers in these samples compared to the cecal and colonic samples. The PCR amplicon products were separated on a 1.5% agarose gels, extracted from the gels and then were cleaned using QIAgen DNA Mini Stool Kits (Valencia, CA). The quality of the product was assessed on a Bioanalyzer 2100 (Agilent, California, USA). Only PCR products without primer dimers and contaminant bands were used for sequencing. The PCR amplicons were sequenced using a Roche 454 GS-FLX sequencer (454 Life Sciences, Branford, CT).

Describe any quality-assurance procedures performed on the data:

To minimize effects of random sequencing errors, we eliminated (i) sequences that did not appropriately match the PCR primer and the barcode at the beginning of a read, (ii) sequence reads with <50 bases after the proximal PCR primer if they terminated before reaching the distal primer, and (iii) sequences that contained more than one undetermined nucleotide (N). Mothur implemented in Galaxy was used for quality assessments including sequence trimming and assignment of operational taxonomic units using a 97% similarity cutoff. Chimeras were removed using Chimera Slayer implemented in UCHIME. A phylogenetic assessment was conducted using RDP classifier with a bootstrap cutoff of 50%.

People involved with sample collection, processing, analysis and/or submission:

  Borewicz, Klaudyna; Kim, Hyeun; Singer, Randall; Gebhart, Connie; Sreevatsan, Srinand; Johnson, Timothy; Isaacson, Richard

DATA-SPECIFIC INFORMATION

Column headings for tabular data
   Full name: Sample Name
   Definition: Unique sample identifier
   Full name: Host
   Definition: Species name from which sample was collected
   Full name: Sample Size
   Definition: Number of replicates taken 
   Full name: Host Age
   Definition: Age of host at time of sample collection
   Full name: Treatment
   Definition: Experimental group (Either infected with Salmonella, Lawsonia, neither, or both)
   Full name: Host Tissue Sampled
   Definition: Tissue from which sample was collected


Additional related data collected that was not included in the current data package:



SHARING/ACCESS INFORMATION 

Licenses/restrictions placed on the data: CC0 1.0 Universal - Public Domain Dedication

Links to publications that cite or use the data:

Links to other publicly accessible locations of the data:

Links/relationships to ancillary data sets:

Was data derived from another source?
   * List source(s): no
Recommended citation for the data: Borewicz, Klaudyna; Kim, Hyeun; Singer, Randall; Gebhart, Connie; Sreevatsan, Srinand; Johnson, Timothy; Isaacson, Richard. (2015). Changes in the Porcine Intestinal Microbiome in Response to Infection with Salmonella Enterica and Lawsonia Intracellularis [dataset]. Retrieved from the Data Repository for the University of Minnesota, http://hdl.handle.net/11299/172268.



Credits: Template provided by the University of Minnesota Libraries, http://lib.umn.edu/datamanagement