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| Status |
Public on Jun 25, 2013 |
| Title |
C22 |
| Sample type |
SRA |
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| Source name |
Treponema spp.
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| Organism |
Bos taurus |
| Characteristics |
tissue: Digital dermatitis lesion
breed: holstein
sampling date: 2011-02-22
herd no.: 7
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Skin biopsies of 6 mm were taken from the centre of the lesion using a sterile biopsy punch. The biopsies were cut in small pieces, suspended in 0.5 ml of sterile PBS and frozen at -20 ºC. Bacterial DNA was extracted from tissue samples (approximately 2mm2) using the DNeasy Tissue Kit (Qiagen, Hilden, Germany). First, the biopsy samples were incubated for 1.5 h at 55°C in 180 µl lysis buffer with 20 µl proteinase K provided in the kit. All subsequent steps were performed according to the instructions provided by the manufacturer. For each sample, 2 µl of template DNA was applied for the construction of sequencing libraries.
DNA was amplified by PCR using the Treponema specific primers designed for this study. Each sample was amplified with a unique primer with an added hexamer barcode at the 5’ end of the forward and reverse primer. Amplification of the target region was carried out in a 50 µl reaction mix which contained 1 × AmpliTaq buffer (Applied Biosystems, Carlsbad, CA), 100 µM of each deoxynucleoside triphophate (Amersham Biosciences, Piscataway, NJ), 0.2 pmol of each primer, 2.5 U of AmpliTaq DNA polymerase (Applied biosystems), and 2 µl of template DNA. Thermal cycling using a T3 Thermocycler (Biometram Göttingen, Germany) was performed as follows: denaturation at 94°C for 3 min and 30 s, followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 59°C for 45 s, and extension at 72°C for 1 min, followed by a final elongation step of 5 min. Equal amounts of all 40 amplicons were pooled (final concentration of 5 µg) and purified with the Qiagen Mini Elute kit (Qiagen), according to the protocol provided by the manufacturer. Next generation sequencing was performed by LCG Genomics (Teddington, Middlesex, UK) using Roche / 454 Genome Sequencer FLX + Titanium according to their standard procedure (1/4 PLP).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
454 GS FLX Titanium |
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| Description |
16S rRNA
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| Data processing |
The 454 pyrosequencing reads were processed using the Ribosomal Database Project’s (RDP) Pyrosequencing Pipeline (Cole et al. 2005; Cole et al. 2009).
The raw sequences were parsed by barcodes using the Pipeline Initial Process.
The amplicons were screened for the presence of both forward and reverse primers, allowing 2 or fewer mismatches to the primer sequences, no ambiguous base calls and a 200-base minimum sequence length.
Supplementary_files_format_and_content: tab-delimited text files listing phylotype abundance for each sample
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| Submission date |
Nov 20, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Kirstine Klitgaard |
| E-mail(s) |
kksc@vet.dtu.dk
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| Phone |
0045886265
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| Organization name |
National Veterinary Laboratory
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| Street address |
Bulowsvej 27
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| City |
Frederiksberg |
| ZIP/Postal code |
1870 |
| Country |
Denmark |
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| Platform ID |
GPL16308 |
| Series (1) |
| GSE42426 |
Targeting the treponemal microbiome of digital dermatitis infections by deep coverage pyrosequencing |
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| Relations |
| SRA |
SRX205619 |
| BioSample |
SAMN01818004 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1039900_C22.txt.gz |
1.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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