The contribution of this subcontract will broadly cover the sampling and analysis of the microbial communities from the stromatolites and adjacent waters in Obsidian Pool Prime at Yellowstone National Park. All samples will be taken in compliance with our permit from the National Park Service. Sampling will be coordinated with the PI and other Co-PIs. The Co-PI (Stevenson) will participate in the sampling and processing each time. As with the rest of the participants, the PI has extensive field and laboratory experience. The Stevenson lab has conducted the RNA extraction and processing of samples in coordination with the Spear lab (Co-PI). The Stevenson lab will use purified RNA for metatranscriptomic sequencing. These libraries will be sent to the NUSeq Core at Northwestern University’s Center for Genetic Medicine for sequencing as described below. The Stevenson lab will also be responsible for the management and curation of the data generated from these sequencing and subsequent bioinformatic analyses through their dedicated compute and storage nodes that are part of the high-performance computing resource maintained by the OU Supercomputing Center for Education and Research (Schooner; OSCER). Data will also be archived on the Sequence Read Archive (SRA) at the National Center for Biotechnology Information (NCBI). Metatrascriptomes of RNA from triplicate samples will be pooled and compared to each of 4 time points during the 4 sampling trips, totaling 16 metatranscriptomes. The pooled RNA samples will be ribodepeleted, removing the majority of the rRNA and then used to prepare the metatranscriptome libraries. Prior to metatranscriptome library preparation, first strand cDNA will be synthesized using the ProtoScript cDNA synthesis kit (New England Biolabs, Ipswitch, MA), followed by second strand synthesis using the NEBNext mRNA second strand synthesis module (New England Biolabs). A mixture of random hexamer and poly-A primers will be used during first strand synthesis. After conversion to cDNA, samples will be quantified and prepared for sequencing. All metagenomic and transcriptomic libraries will then be sequenced on the Illumina HiSeq 500 Instrument using PE150 chemistry (Illumina, Inc.). Metatranscriptome data will first be filtered for quality and adapters will be removed. After quality control, sequence files will be concatenated into a single set of paired-end reads in FASTQ format, assembled de novo. After assembly, the reads will be annotated, mapped against transcripts, and differential significance of gene expression will be assessed.
|Effective start/end date||9/1/22 → 8/31/23|
- University of Southern California (SCON-00004012//80NSSC19K0479)
- National Aeronautics and Space Administration (SCON-00004012//80NSSC19K0479)
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