|
| Status |
Public on Jul 16, 2012 |
| Title |
Environmental DNA from contaminated aquifer Feb. 06 6.9m rep.c + 20% qPCR quantified DNA from A. fisheri (rep. b) |
| Sample type |
SRA |
| |
|
| Source name |
Aquifer Sediment
|
| Organisms |
Aliivibrio fischeri; environmental samples |
| Characteristics |
sampling time: 23 February 2006
depth: 6.9 m below ground
replicate: 20%_b
sample type: Environmental sample + pure culture (A. fisheri) DNA
rna subtype: small RNA
file accn: HB93FIC08
barcode: ATCAGACACG
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Nucleic acids extracted with a bead beating-phenol/chloroform protocol from sediment samples were amplified for amplicon pyrosequencing using the primers Ba27f (5’-aga gtt tga tcm tgg ctc ag-3’) and Ba519r (5’- tat tac cgc ggc kgc tg-3’), extended as amplicon fusion primers with respective primer A or B adapters, key sequence and multiplex identifiers (MID) as recommended by 454/Roche. One of the samples (replicate c) from the quantitative assessment (via spiking of 16S rRNA-quantified A.fisheri sequences not originally present in the sample) was run as 2 steps PCR. Amplicons were purified and pooled as specified by the manufacturer. Emulsion PCR (emPCR), purification of DNA-enriched beads and sequencing run were performed following protocols and using a 2nd generation pyrosequencer (454 GS FLX Titanium, Roche) as recommended by the developer. Quality filtering of the pyrosequencing reads was performed using the automatic amplicon pipeline of the GS Run Processor (Roche), with a slight modification concerning the valley filter (vfScanAllFlows false instead of TiOnly) to extract the sequences. Demultiplexed raw reads from 2 runs were furhter trimmed for quality and lenght (>250 bp).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
454 GS FLX |
| |
|
| Description |
small rRNA were amplified using specific PCR primers from total nucleic acids extracted from environmental samples amended with increasing concentration of external DNA reference (A. fisheri)
|
| Data processing |
Trimming was performed with the "Trim" function of Greengenes web site, with the parameters: good quality score 20, window size 40 bp, window threshold 90%. Shorter reads (<250 bp) were excluded.
Genome Build:
Af20%_b.fas.txt: not applicable
|
| |
|
| Submission date |
Feb 08, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Giovanni Pilloni |
| Organization name |
Helmholtz-Centre Munich
|
| Street address |
Ingolstädter Landstr. 1
|
| City |
Neuherberg |
| ZIP/Postal code |
85764 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15201 |
| Series (1) |
| GSE35631 |
Testing the limits of 454 pyrotag sequencing: reproducibility, quantitative assessment and comparison to T-RFLP fingerprinting of aquifer microbes |
|
| Relations |
| SRA |
SRX120154 |
| BioSample |
SAMN00783995 |
