All scripts in this section are found in Scripts/Bash/.
CZID.org (Kalantar et al 2020) has different "Host" genome options that the user selects to map their nucleotide sequences against, with the end goal of mining the unmapped reads for microbial identification. CZID has a "Bee" option on its webpage at the time of writing (February 2023) which aligns given reads against the Apis mellifera genome. For the many non-A.mellifera samples in this study there were a number of preprocessing steps that had to take place. The script Process_PreMapper_2.sh runs these processes which essentially consists of:
- Downloading the given SRA sample using
SRA Toolkit(Katz et al 2022) - Unpacking the sample
- Mapping it against the nearest host genome using
STAR(Dobin et al 2013) - Retaining unmapped reads
- Putting reads that mapped at < 50% to the side for further processing
This script requires a list of SRA sample accessions (one per line) and a directory of genomes as input, as well as the above packages installed, to work.
The genomes used in this analysis were:
- Andrena spp. : GCA_929108735.1
- Andrena camellia : GCA_910592295.1
- Andrena cineraria : GCA_944738655.1
- Andrena fulva : GCA_910592295.1
- Andrena haemorrhoa : GCA_910592295.1
- Andrena vaga : GCA_944738655.1
- Anthophora plumipes : GCA_001263275.1
- Apis cerana : GCA_001442555.1
- Bombus spp. : GCA_910591885.2
- Bombus breviceps : GCA_014825925.1
- Bombus confusus : GCA_014737475.1
- Bombus consobrinus : GCA_014737475.1
- Bombus difficillimus : GCA_014737525.1
- Bombus haemorrhoidalis : GCA_014825975.1
- Bombus ignitus : GCA_014825875.1
- Bombus lucorum : GCA_910591885.2
- Bombus opulentus : GCA_014737405.1
- Bombus pascuorum : GCA_905332965.1
- Bombus picipes : GCA_014737485.1
- Bombus pyrosoma : GCA_014825855.1
- Bombus rupestris : GCA_014737355.1
- Bombus sibiricus : GCA_014737505.1
- Bombus soroeensis : GCA_014737365.1
- Bombus superbus : GCA_014737385.1
- Bombus terrestris : GCA_910591885.2
- Bombus terricola : GCA_910591885.2
- Bombus turneri : GCA_014825825.1
- Bombus waltoni : GCA_014737395.1
- Ceratina australensis : GCA_004307685.1
- Dufourea novaeangliae : GCA_001272555.1
- Epeolus variegatus : GCA_907165295.1
- Eufriesea mexicana : GCA_001483705.1
- Euglossa dilemma : GCA_001483705.1
- Euglossa viridissima : GCA_001483705.1
- Exoneura spp. : GCA_019453415.1
- Habropoda laboriosa : GCA_001263275.1
- Halictus sexcinctus : GCA_916610255.1
- Lasioglossum spp. : GCA_916610255.1
- Megalopta genalis : GCA_011865705.1
- Nomada lathburiana : GCA_907165295.1
- Osmia bicornis : GCA_907164935.1
- Osmia cornuta : GCA_907164935.1
- Tetragonisca angustula : GCA_011634685.1
- Tetragonula carbonaria : GCA_011634685.1
Any samples that had less than 50% reads mapped were re-mapped using more relaxed parameters and ReProcess.sh. All unmapped reads were uploaded to CZID using CZidUpload.sh, which requires the installation of the CZID command-line interface (CLI).
As there is already an A. mellifera genome available on CZID.org, then these samples were downloaded from the SRA, unpacked and uploaded to CZID using the single script ProcessSRA_v3.sh. This requires similar input and the same dependencies as the above scripts.
The CZID pipeline runs automatically from the moment samples are uploaded (see the website for details / instructions). Once all are complete, taxon reports can be downloaded in bulk for the next stage of the anlaysis.
All of these scripts are ran in R and can be found in Scripts/R. Required R packages are indicated in the "Libraries" sections of the scripts at the outset.
Metadata is found in input/Metadata/, taxonomic classifications in input/Phylo_Misc/, CZID statistics in input/CZ_Overview/, CZID taxon reports in input/TaxonReports/ and counts are saved in input/Counts/ once produced.
All taxonomic classifications in this analysis were taken from a .dmp file from NCBI Taxonomy accessed 18th October 2022 (Schoch et al 2020). The taxonomy used throughout this analysis was made using Scripts/Bash/SplitTax.sh and MakeTaxa.R (instructions are within the annotations of the latter).
ReadnFilt_Dec22.R takes a folder of CZID taxon report files as input and produces count tables. This is tailored specifically for these data and requires the prefiltered sample metadata and a text file on the discrepancies between the taxon report taxa and the current taxonomy (according to NCBI, October 2022). As output it produces count tables with samples as columns and microbial hit as rows. This is done for 1) Prokaryotes 2) Eukaryotes and 3) Viruses.
Analysis_Prokaryote3.R uses vegan (Dixon et al 2003) packages to compute dissimilarity matrices, produce NMDS and use PERMANOVA (adonis2) to assess the impact of phylogeny, location and social lifestyle on prokaryote (bacterial) community composition. Analysis_Eukaryote3.R and Analysis_Viral3.R do the same for eukaryotic and viral counts, respectively.
To see if there are candidates for microbes associated with particular tribes, DescribeTribe.R identifies microbes found in over 50% of tribal members at an average relative abundance above 0.01%.
CZID CLI v4.1.2
CZID Pipeline v7.1
R v4.2.2
SRA Toolkit v3.0.0
vegan v2.6-4
Dixon, P., 2003. VEGAN, a package of R functions for community ecology. Journal of vegetation science, 14(6), pp.927-930.
Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M. and Gingeras, T.R., 2013. STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 29(1), pp.15-21.
Kalantar, K.L., Carvalho, T., de Bourcy, C.F., Dimitrov, B., Dingle, G., Egger, R., Han, J., Holmes, O.B., Juan, Y.F., King, R. and Kislyuk, A., 2020. IDseq—An open source cloud-based pipeline and analysis service for metagenomic pathogen detection and monitoring. Gigascience, 9(10), p.giaa111.
Katz, K., Shutov, O., Lapoint, R., Kimelman, M., Brister, J.R. and O’Sullivan, C., 2022. The sequence read archive: a decade more of explosive growth. Nucleic acids research, 50(D1), pp.D387-D390.
Schoch, C.L., Ciufo, S., Domrachev, M., Hotton, C.L., Kannan, S., Khovanskaya, R., Leipe, D., Mcveigh, R., O’Neill, K., Robbertse, B. and Sharma, S., 2020. NCBI Taxonomy: a comprehensive update on curation, resources and tools. Database, 2020.