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SRX22719004: Marker gene sequencing of environmental sample
1 ILLUMINA (Illumina MiSeq) run: 37,193 spots, 18.6M bases, 11.2Mb 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: Marum Crater Targeted loci environmental
show Abstracthide Abstract
Microbial life permeates our planet. From the atmosphere to the deep subsurface, a stunning diversity of organisms, employing a wide range of metabolisms, is able to persist, multiply, and evolve. But when does substrate colonization begin, and which organisms initiate the process? How does the community shift as ecological succession progresses in the deep subsurface? Due to the pervasiveness of microbial life on and within nearly any substrate, including the subsurface, acquiring sterile, time zero samples is a limiting factor in conducting subsurface colonization studies. Through the analysis of a unique sample set of basaltic substrate acquired from an active lava lake on Ambrym Island, Vanuatu, we will use subsurface analog material as a representative of colonization processes occurring across the bulk of the Earths habitable volume. With samples ranging in age from 30 seconds to 46 years, we are poised to address a series of fundamental questions about how life gains a foothold within our planet. PI, Jeffrey Marlow Harvard University.
Sample:
SAMN38574145 • SRS19709822 • All experiments • All runs
Library:
Name: 8B_Bv4v5
Instrument: Illumina MiSeq
Strategy: AMPLICON
Source: METAGENOMIC
Selection: PCR
Layout: PAIRED
Runs: 1 run, 37,193 spots, 18.6M bases, 11.2Mb
Run# of Spots# of BasesSizePublished
SRR2702716537,19318.6M11.2Mb2023-12-02

ID:
30752334

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