Marker gene sequencing of environmental sample - SRA

SRX22899528: Marker gene sequencing of environmental sample
1 ILLUMINA (Illumina MiSeq) run: 167,721 spots, 84.2M bases, 38.8Mb downloads

Design: The v4-v5 hypervariable regions of the bacterial SSU rRNA gene were amplified using degenerate primers. The primers were 518F (CCAGCAGCYGCGGTAAN) and 926R (CCGTCAATTCNTTTRAGT CCGTCAATTTCTTTGAGT CCGTCTATTCCTTTGANT). Amplification was done with fusion primers containing the 16S-only sequences fused to Illumina adapters. The forward primers included a 5 nt multiplexing barcode and the reverse a 6 nt index. We generated PCR amplicons in triplicate 33 uL reaction volumes with an amplification cocktail containing 0.67 U SuperFi Taq Polymerase (Invitrogen, Carlsbad, CA), 1X enzyme buffer (includes MgCl2), 200 uM dNTP PurePeak DNA polymerase mix (ThermoFisher), and 0.3 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, 57C for 45s, and 72C for 60s; final 2 minute extension at 72C. The triplicate PCR reactions were pooled after amplification, visualized with the negative controls on a Caliper LabChipGX or Agilent TapeStation 4200, and purified using Ampure followed by PicoGreen quantitation and Ampure size selection.

Submitted by: Marine Biological Laboratory

Study: Soudan Iron Mine marker gene sequencing

PRJNA1046457 • SRP478232 • All experiments • All runs

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We used marker gene sequencing of microbial community DNA retrieved from a Banded Iron Formation at the Soudan Iron Mine, Minnesota USA. The mine offers unique access to subsurface microbial communities across a wide range of geological, geochemical and hydrological conditions, including access to a deep subsurface, anoxic, iron rich, CaCl2 brine located beneath the lowest level of the mine, 713.5 m or 2341 feet. The brine seeps into the bottom level of the mine from a number of boreholes drilled just prior to its closing in the early 1960s. Obtaining a thorough inventory of the microbial communities across a range of geochemical and geological conditions will help develop hypotheses regarding species distribution with depth and along geochemical gradients in a deep terrestrial ecosystem. PI, Jeffrey Gralnick, University of Minnesota. See also PRJNA340294 and PRJNA248749.

Sample:

SAMN05712323 • SRS1650502 • All experiments • All runs

Organism: mine drainage metagenome

Library:

Name: 932W3_Bv4v5

Instrument: Illumina MiSeq

Strategy: AMPLICON

Source: METAGENOMIC

Selection: PCR

Layout: PAIRED

Runs: 1 run, 167,721 spots, 84.2M bases, 38.8Mb

Run	# of Spots	# of Bases	Size	Published
SRR27220819	167,721	84.2M	38.8Mb	2023-12-15

ID:: 30960367

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