Marker gene sequencing of environmental sample - SRA

SRX22902469: Marker gene sequencing of environmental sample
1 ILLUMINA (Illumina MiSeq) run: 215,876 spots, 108.4M bases, 66.1Mb downloads

Design: The v4-v5 hypervariable regions of the archaeal SSU rRNA gene were amplified using degenerate primers. The primers were 517F (GCCTAAAGCATCCGTAGC GCCTAAARCGTYCGTAGC GTCTAAAGGGTCYGTAGC GCTTAAAGNGTYCGTAGC GTCTAAARCGYYCGTAGC) and 958R (CCGGCGTTGANTCCAATT). 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: 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

SAMN03246972 • SRS775467 • All experiments • All runs

Organism: aquatic metagenome

Library:

Name: KC_1_12_10_Av4v5

Instrument: Illumina MiSeq

Strategy: AMPLICON

Source: METAGENOMIC

Selection: PCR

Layout: PAIRED

Runs: 1 run, 215,876 spots, 108.4M bases, 66.1Mb

Run	# of Spots	# of Bases	Size	Published
SRR27223825	215,876	108.4M	66.1Mb	2023-12-15

ID:: 30963308

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