454 amplicon sequencing of bacterial 16S from BLM_1 - SRA

SRX791551: 454 amplicon sequencing of bacterial 16S from BLM_1
1 LS454 (454 GS FLX Titanium) run: 24,912 spots, 13.9M bases, 27.8Mb downloads

Design: "Construction protocol: The v6-v4 hypervariable regions of the bacterial SSU rRNA gene were amplified using degenerate primers. The primers were 1064R (CGACRRCCATGCANCACCT) and 518F (CCAGCAGCYGCGGTAAN) fused to Roche GSFLX amplicon sequencing adapters and including 5 nt multiplexing barcodes. We generated PCR amplicons in triplicate 33 uL reaction volumes with an amplification cocktail containing 1.0 U Platinum Taq Hi-Fidelity Polymerase (Invitrogen, Carlsbad, CA), 1X Hi-Fidelity buffer, 200 uM dNTP PurePeak DNA polymerase mix (Pierce Nucleic Acid Technologies, Milwaukee, WI), 1.5 mM MgSO4 and 0.2 uM of each primer. We added approximately 10-25 ng template DNA to each PCR and ran a no-template control for each primer pair. Amplification conditions were: initial 94C, 3 minute denaturation step; 30 cycles of 94C for 30s, 60C for 60s, and 72C for 90s; final 10 minute extension at 72C. The triplicate PCR reactions were pooled after amplification and purified using Ampure beads. Purified DNA was eluted in 30 uL of Qiagen buffer EB. We assessed the quality, size and concentration of PCR products on a Perkin Elmer Caliper GX. Reads were demultiplexed and barcodes removed for submission."

Submitted by: Marine Biological Laboratory

Study: Deep Subsurface Targeted Locus (Loci)

PRJNA268940 • SRP050383 • All experiments • All runs

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This project targets deep microbial ecosystems of three significant, mostly unexplored (for microbiology) North American geological provinces: the Basin and Range, Black Hills, and Canadian Shield. In addition to their value for the exploration of novel deep habitats, these samples also allow us to address the following questions: (1) Is everything truly everywhere? (2) How does one recognize indigenous deep life? and (3) Is it possible via existing excavations to sample below the lower biosphere limit?

Sample: Novel Terrestrial Deep Subsurface Habitats of the North American Basin and Range, Black Hills, and Canadian Shield

SAMN03246970 • SRS775465 • All experiments • All runs

Organism: aquatic metagenome

Library:

Name: BLM_1_Bv6v4

Instrument: 454 GS FLX Titanium

Strategy: AMPLICON

Source: METAGENOMIC

Selection: PCR

Layout: SINGLE

Spot descriptor:

1 forward

Runs: 1 run, 24,912 spots, 13.9M bases, 27.8Mb

Run	# of Spots	# of Bases	Size	Published
SRR1686680	24,912	13.9M	27.8Mb	2014-12-02

ID:: 1133482

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