Pan-cancer mycobiome atlas

This is the GitHub repository for all TCGA, Hopkins, and UCSD analyses of the pan-cancer mycobiome atlas paper by Narunsky-Haziza, Sepich-Poore, Livyatan et al., 2022. Data and code provided herein cover the fungal and bacterial analysis of 17,401 patient tissue, blood, and plasma samples across 35 cancer types in four independent cohorts.

Please note that this repository is divided into multiple subdirectories. They are detailed below in the Organization of files section.

Related repositories

There are several other repositories related to this paper, including the following:

• The ITS2 pipeline for amplicon-based analyses: https://github.com/microbiofunc/ITS2-pipeline
• Fungal phylogenomics of reconstructed bins: https://github.com/stajichlab/Tumor_mycobiome_2022
• Aggregated and per-sample genome coverage calculations: https://github.com/ucsd-cmi/zebra_filter

Installation

On the top right of the main repository page, click the Code ▼ button followed by the Download ZIP button to download the entire repository.

Alternatively, or if using R, you can download the repository using:

library(devtools)
install_github("knightlab-analyses/mycobiome")

Download final TCGA mycobiome files

The final raw and batch-corrected TCGA files that were used for machine learning are located in Final_files subfolder. This includes fungal data for 14,495 samples. The files are the following:

• A taxonomy table for the rep200 database used throughout out paper (taxonomy_table_rep200.tsv).
• A metadata table for the 14,495 TCGA samples (metadata_fungi_14495samples.tsv). Note that the TCGA case IDs are under the tcga_case_id column.
• A raw count table for 224 decontaminated fungi (count_data_fungi_decontaminated_raw.tsv).
• A batch-corrected (using Voom-SNM) table for 224 decontaminated fungi (count_data_fungi_decontaminated_voom_snm_corrected.tsv).

Download TCGA fungi+bacterial data

Since the release of the paper, we have received requests to release the bacterial data alongside the fungi information. For now, we are conservatively posting abundances, in the Final_files subfolder, of Weizmann-overlapping bacteria (cf. Nejman et al. 2020. Science) and fungi (this paper, Narunsky-Haziza et al. 2022. Cell) at the genus and species levels, since these microbes have multiple layers of evidence in independent cohorts. These count files incorporate the updated host-depletion and read QC methods described in this paper. The following files contain Weizmann-overlapping bacteria and fungi in TCGA:

• TCGA raw genus-level abundances for WIS-overlapping bacteria and fungi (14,494 non-zero samples) (count_data_genus_raw_WIS_overlapping_fungi_bacteria_14494samples.tsv).
• Matching TCGA metadata for genus-level data (14,494 non-zero samples) (metadata_genus_WIS_overlapping_fungi_bacteria_14494samples.tsv). Note that the TCGA case IDs are under the tcga_case_id column.
• TCGA raw species-level abundances for WIS-overlapping bacteria and fungi (12,773 non-zero samples) (count_data_species_raw_WIS_overlapping_fungi_bacteria_12773samples.tsv).
• Matching TCGA metadata for species-level data (12,773 non-zero samples) (metadata_species_WIS_overlapping_fungi_bacteria_12773samples.tsv). Note that the TCGA case IDs are under the tcga_case_id column.
• Matching taxonomy table at species-level for WIS-overlapping bacteria and fungi (taxonomy_table_WIS_overlapping_fungi_bacteria.tsv)

Note #1: Subsetting TCGA samples to WIS-overlapping microbes leads to some sample dropout, which is why the sample counts in these files are slightly lower than the 14,495 number.

Note #2: The raw counts/abundances posted above do not correct for batch effects in TCGA. One should either mitigate the batch effects using batch correction (e.g., Voom-SNM, ComBat-Seq, etc.) or subset the samples to groups not affected by individual batches (i.e., a single sequencing center, experimental strategy [WGS or RNA-Seq], and sequencing platform).

Note #3: If using any of these files, please cite both Nejman et al. 2020. Science and Narunsky-Haziza et al. 2022. Cell (this paper).

Note #4: As noted in the manuscript's methods, completely raw TCGA data, including the host-depleted fastq files, can be accessed at the respective Qiita project study IDs: 13722 (TCGA WGS), 13767 (TCGA RNA-Seq).

Organization of files

R files, scripts, and subfolders:

The R files are enumerated and provided approximately in the same order as TCGA analyses detailed in the paper. The following subdirectories contain important information for running the R analyses:

• All of the input data used for the 17 main R scripts in the base directory are provided in the /Input_data subfolder.
• Supporting data files (e.g., taxonomy tables) are provided under the /Supporting_data subfolder.
• Data generated by earlier scripts that are used by later scripts are saved under /Interm_data.
• Scripts that were run on a Slurm compute cluster using processed versions of the data are located under /Supporting_scripts.
• Figures that were generated while processing the scripts were saved under /Figures (note the multiple subdirectories therein).
• In certain cases (e.g., ANCOM-BC differential abundance tables), data underlying certain Figures were saved under /Figures_data.

The 17 main R scripts in the base directory have the following brief descriptions:

• 00-Functions.R → This script contains all major functions used throughout the rest of the R scripts
• 01R-Merge-WGS-RNA-data-decontaminate-batch-correct.R → This script merged the TCGA WGS and RNA-Seq datasets (based on their Qiita outputs), followed by data decontamination and batch correction.
• 02R-Calculate-fungi-vs-bacteria-read-distributions.R → This script calculated the relative abundances of fungi vs. bacteria with and without genome size correction, and the fungal vs. bacterial read percentages.
• 03R-Intersect-TCGA-data-with-Weizmann-data.R → This script intersects the TCGA and WIS fungal and bacterial data at every taxa level.
• 04R-Prepare-TCGA-data-for-Qiime-and-plot-alpha-diversity.R → This script saves data for Qiime 2 alpha and beta diversity analyses of TCGA fungal data. Note: The data saved here were processed using /Qiime_data_and_scripts_resubmission_version/qiime2_mycobiome_tcga_analyses_resubmission_version.ipynb.
• 05R-Prepare-TCGA-data-for-machine-learning.R → This script does a lot of data formatting for most of the machine learning analyses presented in the paper. Typically, the formatted data are saved as .RData files, which are then called by Slurm-based scripts (stored under /Supporting_scripts), and the results of those Slurm-based runs are then plotted in this script.
• 06R-Perform-machine-learning-on-Weizmann-data.R → This script formats the WIS data for machine learning and does a preliminary version of it (although the main version of the machine learning is under /Supporting_scripts/S16R-ML-fungi-10k-rep1-weizmann.R.
• 07R-TCGA-compare-tumor-vs-normal-bray-curtis.R → This script calculates a Bray-Curtis-based PCoA for tumor vs. NAT samples using cancer types that overlapped with the WIS cohort. The resulting output comprises a 2D and 3D plot.
• 08R-TCGA-alpha-diversity-correlation-fungi-vs-bacteria.R → This script performs rarefaction on the entire dataset and calculates correlations among fungal and bacterial diversities across all cancer types.
• 09R-Prepare-TCGA-data-for-MMvec-and-ANCOM-BC.R → This script formats the data for running MMvec to identify fungal-bacterial-immune co-occurrences (see /MMvec-cooccurrence-analyses-resubmission for more) and runs ANCOM-BC differential abundance on fungi and bacteria in TCGA.
• 10R-Control-validation-analyses-TCGA.R → This script performs control analyses, including stratified splits in TCGA with cross-testing and permuted machine learning data analyses.
• 11R-Plasma-validation-cohort-UCSD.R → This script re-analyses plasma microbiome data generated by Poore and Kopylova et al. 2020 Nature for fungal analytes.
• 12R-Plasma-validation-cohort-Hopkins.R → This script re-analyses shallow WGS multi-cancer data generated by Cristiano et al. 2020 Nature for fungal and other microbial analytes.
• 13R-ML-fungi-vs-bacteria.R → This script examines the synergy of using fungal and bacterial biomarkers in combination for pan-cancer machine learning.
• 14R-ML-fungi-cancer-stage.R → This script performs machine learning to discriminate early vs. late stage cancers.
• 15R-ML-WIS-samples.R → This script performs the remaining machine learning analyses on WIS data that were not covered in 06R-Perform-machine-learning-on-Weizmann-data.R.
• 16R-Addressing-remaining-reviewer-comments.R → This script performs several analyses to respond to reviewer requests.

Qiime alpha and beta diversity analyses:

The Qiime-based analyses, including input data and script, are found under the /Qiime_data_and_scripts_resubmission_version subfolder. The CLI commands run are listed therein under qiime2_mycobiome_tcga_analyses_resubmission_version.ipynb.

MMvec fungal-bacterial-immune co-occurrence analyses:

The MMvec-based analyses, including input data and scripts, are found under the /MMvec-cooccurrence-analyses-resubmission subfolder. A separate README.md file is listed within that subdirectory explaining its contents.

Other directories:

• /EukDetect → This directory contains code and resultant tables for rerunning TCGA using EukDetect.
• /Coverage_analysis → This directory contains several scripts involved in calculating fungal genome coverages.

Docker host depletion pipeline (/Docker_host_depletion_pipeline)

For these analyses, we re-aligned all non-human reads against a uniform reference genome (GRCh38+PhiX). Due to the large amount of data being reprocessed, the host-depletion pipeline was optimized for speed. For others to use, we have packaged the host depletion steps into a Docker container, described below.

The following files are listed in this subdirectory:

1. Dockerfile —> This text file is used to build the Docker container on your computer (instructions are below)
2. ebi_sra_importer.yml —> This yml file contains the conda environment and its dependences that are run in the Docker container
3. host_deplete_script.sh —> This bash script is used to run the host depletion within the Docker container on any new file. It accepts 3 arguments (in this order): (a) Original bam file to host deplete, (b) Minimap2 human database in .mmi format, (c) Integer number of cpus you would like to use on your server or machine.

Note: The minimap2 database (.mmi file) is located on figshare here: https://doi.org/10.6084/m9.figshare.19719418.v1

Building the Docker container:

You can build the Docker container with the following command (please substitute the <…> text with your own text; note the . at the end of the command as well): docker build --no-cache -t <MY_CONTAINER_NAME> .

It will take some time (~20-30 minutes) to build the container and occupy several gigabytes of space. Once the container has been built, you can interactively run it with the following command: docker run -ti <MY_CONTAINER_NAME>

Once the container is running, you can use the “host_deplete_script.sh” script to run the host depletion on any bam file you would like. It will then save the host depleted file as two fastq files with the following suffixes: <BASE_NAME>.R1.trimmed.fastq.gz, <BASE_NAME>.R2.trimmed.fastq.gz

The script within the Docker container is running the following one-liner piped command:

samtools view -f 4 -O BAM $in_dir/$filename |
samtools bam2fq - |
fastp -l 45 --stdin -w $cpus --stdout --interleaved_in |
minimap2 -ax sr -t $cpus $db - |
samtools fastq -@ $cpus -f 12 -F 256 - -1 $out_dir/$base_name.R1.trimmed.fastq.gz -2 $out_dir/$base_name.R2.trimmed.fastq.gz

where $cpus and $db denote the number of compute cores and a precomputed Minimap2 reference database (as a .mmi file), respectively.

Note 1: The minimap2 database for this study included the GRCh38.p7 human genome and the Phi X 174 viral genome.

Note 2: The host depletion automatically removes any reads less than 45 bp for quality control. If you would like to change this, you can alter the -l 45 argument to fastp on line 9 of the host_deplete_script.sh.

Note 3: If you prefer to avoid using a Docker container, you can create a new conda environment using the ebi_sra_importer.yml file on any machine or server with Conda installed. You can then adapt the host_deplete_script.sh script as you like to run within the Conda environment on any bam file that you would like to host deplete.

After the host depletion is complete: You should have paired R1 and R2 fastq files for each host depleted sample, which you can then upload to Qiita to perform taxonomy calling using Woltka.

Citation

If you use data or software from this repository, please cite the following three papers:

• Narunsky-Haziza, Sepich-Poore, Livyatan et al. 2022. Cell

@article{narunsky2022pan,
  title={Pan-cancer analyses reveal cancer-type-specific fungal ecologies and bacteriome interactions},
  author={Narunsky-Haziza, Lian and Sepich-Poore, Gregory D and Livyatan, Ilana and Asraf, Omer and Martino, Cameron and Nejman, Deborah and Gavert, Nancy and Stajich, Jason E and Amit, Guy and Gonz{\'a}lez, Antonio and others},
  journal={Cell},
  volume={185},
  number={20},
  pages={3789--3806},
  year={2022},
  publisher={Elsevier}
}

• Poore, Kopylova et al. 2020. Nature:

@article{poore2020microbiome,
  title={Microbiome analyses of blood and tissues suggest cancer diagnostic approach},
  author={Poore, Gregory D and Kopylova, Evguenia and Zhu, Qiyun and Carpenter, Carolina and Fraraccio, Serena and Wandro, Stephen and Kosciolek, Tomasz and Janssen, Stefan and Metcalf, Jessica and Song, Se Jin and others},
  journal={Nature},
  volume={579},
  number={7800},
  pages={567--574},
  year={2020},
  publisher={Nature Publishing Group}
}

• Nejman et al. 2020. Science

@article{nejman2020human,
  title={The human tumor microbiome is composed of tumor type--specific intracellular bacteria},
  author={Nejman, Deborah and Livyatan, Ilana and Fuks, Garold and Gavert, Nancy and Zwang, Yaara and Geller, Leore T and Rotter-Maskowitz, Aviva and Weiser, Roi and Mallel, Giuseppe and Gigi, Elinor and others},
  journal={Science},
  volume={368},
  number={6494},
  pages={973--980},
  year={2020},
  publisher={American Association for the Advancement of Science}
}

License

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Shield:

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.