Metatranscriptomes of two biological soil crust types from the Mojave desert in response to wetting

Biological soil crusts comprise diverse microbial communities that carry out vital ecological functions in dryland ecosystems (1). Under dry conditions, biocrust microbes primarily persist in dormancy (2–4). When water becomes available, they quickly respond by exploiting moisture to repair cell damage and synthesize new biomass (5, 6). Nevertheless, the specific gene expression and metabolic processes underlying these responses remain poorly understood.

We sought to compare two kinds of biocrust commonly found in the Sheephole Valley Wilderness (Mojave Desert): light algal crust (LAC) and cyanolichen crust (CLC). In all, 10 biocrust samples, each measuring 5 cm², were collected at GPS location 34.1736 N, 115.3888 W. Each sample was placed in a 10 cm petri dish with 2 mL of sterile ultrapure water added on top, covered with a petri dish cover, and incubated at ambient laboratory conditions. After 0.5, 6, 18, 30, and 50 h time points, an entire biocrust sample was transferred and stored at −80°C for subsequent total RNA extraction using a NucleoBond RNA Soil Midi kit (740140.20, Macherey-Nagel, Nordrhein-Westfalen, Germany). We pursued rRNA depletion of 100 ng of total RNA using a QIAseq FastSelect 5S/16S/23S kit for bacteria and FastSelect rRNA yeast and plant depletion for eukaryotes (335921, 334219, and 334319, QIAGEN, Germantown, MD) following the manufacturer’s instructions. The resulting RNA was reverse transcribed to create first-strand cDNA using a TruSeq Stranded mRNA Library prep kit (20020594, Illumina Inc., San Diego, CA). To synthesize second-strand cDNA, deoxyuridine triphosphate was incorporated in place of deoxythymidine triphosphate to quench the second strand during amplification and achieve strand specificity. Double-stranded cDNA fragments were A-tailed and ligated to JGI dual-indexed Y-adapters, followed by 10 cycles of PCR. The prepared libraries were quantified using KAPA Biosystems’ next-generation sequencing library qPCR kit and run on a LightCycler 480 real-time PCR instrument (Roche Diagnostics Corporation, Indianapolis, IN). NovaSeq sequencing (Illumina Inc., San Diego, CA) was performed using NovaSeq XP V1 reagent kits and an S4 flowcell following a 2 × 151 bp indexed run recipe. BBDuk version 38.87 (https://jgi.doe.gov/data-and-tools/bbtools/) was used to remove contaminants, trim adapters from Illumina raw sequencing reads, remove any reads that contained “N” bases, and were shorter than 51 bp. Filtered reads were assembled with MEGAHIT version v1.2.9 (7) and mapped back to the final transcriptome assembly and coverage determined using BBMap version 38.86 (8).

Nearly 95% of reads aligned to ribosomal reference sequences in the SILVA database (9) using BBDuk (version 38.87, default settings), suggesting that experimental rRNA depletion was not effective. Nevertheless, these rRNA reads could be assembled and used to comprehensively survey the taxonomic diversity contained within these biocrusts (10). We obtained at least 25 million mRNA reads per sample, of which 80% could be assembled into contigs; this represents an average transcriptome coverage of ~69× and should be sufficient depth for functional analyses of wetting the reanimation process.

We thank the BLM Needles CA office for their assistance with permitting at the Sheephole Valley Wilderness. This work was performed and supported in part by the Facilities Integrating Collaborations for User Science (FICUS) program (proposal: https://doi.org/10.46936/fics.proj.2018.50356/60000035) and used resources at the DOE Joint Genome Institute (JGI) (https://ror.org/04xm1d337) and the National Energy Research Scientific Computing Center (NERSC) (https://ror.org/05v3mvq14), which are DOE Office of Science User Facilities operated under Contract No. DE-AC02-05CH11231; Bureau of Land Management Cooperative Agreement L15AC00153 (NPi) and permit number 6850-CAD0000.06 (NPi and JES); the U.S. Department of Agriculture, National Institute of Food and Agriculture Hatch project CA-R-PPA-211–5062-H to NPo and JES; a Royal Thai Government Scholarship to NPo; and NSF GoLife grant DEB-1541538 and CAREER grant DEB-1846376 to EFYH. JES is a CIFAR fellow in the Fungal Kingdom: Threats and Opportunities program. This is UM’s Center for Biodiversity and Conservation Research Publication No. 39.