DOI: 10.5061/dryad.z08kprrp1
title: Data from: Shotgun microbiome analysis of two Schizaphis graminum biotypes with time with and without carried cereal yellow dwarf virus

description: Reads from a prior RNAseq study of gene expression in greenbug aphid
 (Schizaphis graminum (Rondani)) were used for shotgun metagenomic analysis
 in relation to two aphid biotypes, presence or absence of carried cereal
 yellow dwarf virus (CYDV), and five timepoints from zero to 20 days
 post-infestation. There were three biological replicates per condition.
 Reads were aligned with bwa mem to reference or representative genomes of
 47000 bacterial, 1218 archaeal, 14165 viral, 571 fungal, and 94 protozoan
 taxa, plus greenbug and wheat, and each read was credited to the
 highest-scoring taxon. Read counts by taxon were imported into QIIME2 for
 statistical analysis and display. There were 105635 to 5938545 microbial
 reads per sample. The reads matched 3348 genera. The ratio of total
 microbial counts to total greenbug counts peaked at day 5 and declined 50%
 by day 20. Barplotted relative frequencies indicated two major
 communities, one enriched in Shigella and Escherichia, the other depleted
 in Shigella and Escherichia but enriched in Acinetobacter and Gilbertella.
 With one exception, the depleted community was restricted to days 15 and
 20 and existed in both biotypes with and without carried CYDV. CYDV was
 not detected in any sample, but an aphid pathogen, Rhopalosiphum padi
 virus, was present in 20 samples and exceeded 4000 counts in four samples,
 of which two were enriched in Shigella and Escherichia. Two unrelated
 photosynthetic genera, Microcystis and Lamprocystis, were relatively
 abundant; the latter was positively correlated with Shigella. Oddly,
 Letharia (as an ascomycete) was the fifteenth most abundant hit overall,
 despite that Letharia itself is a lichen. Shigella-depleted communities in
 24 samples were significantly more alpha-diverse than communities in the
 remaining samples (Shannon entropy 5.06 in depleted versus 3.84 otherwise,
 t = -8.46, p = 1.8e-09). Principal-components projection of Bray-Curtis
 dissimilarity showed two distinct clusters whose membership conformed to
 the two groups distinguishable in the barplotted relative frequencies.
 Sample B02 lay between the clusters. The first three principal components
 accounted for 60.54% of the variation. Permanova of Bray-Curtis distances
 with Adonis confirmed that only time (p = 0.001) and the interaction of
 time with biotype (p = 0.035) significantly affected beta diversity. In
 conclusion, two distinct microbiome communities existed in the aphids,
 where the Shigella-depleted community accompanied yellowing and death of
 the wheat host as it succumbed to aphid feeding and yellow dwarf disease.
description: An RNAseq study was conducted using whole <em>Schizaphis
 graminum</em> aphids without surface sterilization. Reads were
 mapped to reference genomes of <em>Schizaphis graminum</em>,
 its host <em>Triticum aestivum</em> (wheat), and all available
 reference or representative genomes of bacterial, archaeal, viral, fungal,
 and protozoan taxa available in NCBI as of 31 October 2023. It was
 necessary to divide these genomes among 61 separate databases and then
 merge the results of bwa mem runs because of the slowness of bwa indexing.
 A perl script, grouptaxa1211.pl, was written to collate closest hits in
 the resulting sam files. Other scripts were written to aggregate counts by
 taxon at different levels from species to phylum using files from NCBI
 taxonomy. The raw counts were subjected to DESeq2 to identify
 significantly responding taxa, since the data format was identical to the
 sister RNAseq data analyzed for aphid gene expression. The counts by taxon
 were also imported into QIIME2 for graphical presentation and analysis of
 community diversity. Of important note, the library preparation apparently
 selected RNA with a polyA tail. CYDV lacks a polyA tail and was not
 detected. RhPV has a polyA tail and was detected.
description: # RNAseq read counts by microbial taxon from *Schizaphis graminum* with
 and without carried Cereal Yellow Dwarf Virus
 [https://doi.org/10.5061/dryad.z08kprrp1](https://doi.org/10.5061/dryad.z08kprrp1) ## Description of the data and file structure To see the contents of this tar.gz file, type 'gunzip gbmbiomeItodatadryad.tar.gz; tar -xvf gbmbiomeItodatadryad.tar' at the Unix command line. This tar file contains two kinds of data files: all\*counttable0502.txt and \*usedsamcountsbytaxon\*.txt. The former were aggregated from the latter. The former has the taxon name in the first column and counts for each combination of biotype, viral carrier status, and timepoint in subsequent columns. The header row gives the codes for these combinations: biotype (B or H), viral carrier status (nothing or RPV), timepoint (integers from 0 through 7), and replicate (1, 2, or 3). The latter kind has two parts: an initial list of GenBank accessions grouped by NCBI taxonomic identities (taxid), followed by counts by taxon upon grouping GenBank accessions. There is one file of the second kind for each combination of biotype, viral carrier status, timepoint, and replicate. ## Sharing/Access information Reference and representative genomes were downloaded from subdirectories archaea, bacteria, viral, protozoa, and fungi in [https://ftp.ncbi.nlm.nih.gov/genomes/refseq/](https://ftp.ncbi.nlm.nih.gov/genomes/refseq/). Taxonomic files nucl_gb.accession2taxid, nucl_wgs.accession2taxid, nucl_wgs.accession2taxid.EXTRA, and rankedlineage.dmp were downloaded from [https://ftp.ncbi.nlm.nih.gov/pub/taxonomy/accession2taxid/](https://ftp.ncbi.nlm.nih.gov/pub/taxonomy/accession2taxid/) and [https://ftp.ncbi.nlm.nih.gov/pub/taxonomy/new_taxdump/](https://ftp.ncbi.nlm.nih.gov/pub/taxonomy/new_taxdump/). ## Code/Software Perl and bash scripts were written to download and manipulate files. Two scraping scripts, drillingwget.pl and drillingwget2.pl, were written to download files from NCBI; the second script was needed for the somewhat different naming convention for viruses. Bash scripts ran bwa index and bwa mem. Perl script maxscore1015.pl collated alignments for the same read among databases and found the highest-scoring hit for each read. Script grouptaxa1211.pl grouped counts by taxa at taxonomic levels from genus to phylum. Script buildtoptable.pl isolated the top-ranked taxa by overall count for importation into QIIME2 for community analysis and graphical presentation.