MedBioinfo 2023 Applied Bioinformatics

repo for re-analysis

this is for the assignment 9

Data source

Data is from Daniel Castañeda-Mogollón et al. Dec 2021 https://www.sciencedirect.com/science/article/pii/S1386653221002924

Samples (either Nasopharyngeal or Throat swabs) are from 125 patients, either COVID+ or COVID- by RT-PCR, were subjected to Illumina sequencing (one RNA and one DNA sequencing run for each patient).

Aknowledgment: kudos to the authors for providing all the required data to reproduce the analysis, both as online supplementary material to the published paper, as raw sequence data submissions to open international archives, and as as prompt replies to email queries.

Aim of the re-analysis

Use k-mer based binning of the raw reads to identify potential human pathogens in the samples (including SARS-CoV-2).

To investigate, amongst others:

• correlation between COVID+/- PCR status and SARS sequence presence in metatranscriptomes
• correlation between SARS2 infection and presence of known viral/bacterial co-pathogens
• effectiveness of SARS2 detection with regards swab type (Nasopharyngeal or Throat)

Each course participant is in charge of a subset of the 250 total samples sequenced. Results will then be compiled to allow overall analysis of the full dataset.

Structure of the shared repository

• data contains the original data from Castañeda-Mogollón (read only, ie file contents not to be modified!)
• docs contains documentation (eg papers, manuals etc.)
• scripts will contain any scripts used in the analysis workflow (eg Jupyter notebooks, SLURM bash scripts)
• analyses will contain results of the analyses, such as transformed datasets, output files, and figures

Because of their large size, the original raw FASTQ sequence files will be excluded from git tracking via the .gitignore file. This is reproducible-friendly because these FASTQ raw files will be considered strictly read only, and therefore can be re-instanciated simply by executing the download commands that will be documented in the scripts & notebooks.

Initial data

• the NCBI raw sample metadata annotation file (downloaded from NCBI SRA) is in data/SraRunTable.csv
• the EBI ENA sample metadata annotation file (downloaded from EBI ENA) is in data/filereport_read_run_PRJEB47870.tsv
• RT-PCR Ct (cycle threshold) values (obtained by contacting the authors by email) are in data/ct_values_covid19_metagenomics.xlsx
• IDseq (see ref below) processing metadata for 5'/forward reads (obtained by contacting the authors by email) are in data/sample_overviews 5.csv
• IDseq (see ref below) processing metadata for 3'/reverse reads (obtained by contacting the authors by email) are in data/sample_overviews 3.csv
• a copy of the Castañeda-Mogollón et al. paper is in docs/Castaneda-Mogollon_et_al_J_Clinical_Vir_Dec_2021.pdf
• a copy of the supplementary figures are in Castaneda-Mogollon_et_al_J_Clinical_Vir_Dec_2021_supp_figs.pdf
• a copy of the supplementary material & methods are in Castaneda-Mogollon_et_al_J_Clinical_Vir_Dec_2021_supp_mat_and_meth.pdf

Organisation of the collaborative analysis

• the raw sequencing files archived at the NCBI SRA https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=PRJEB47870 will be downloaded in data/
• data needed to collaborate will be stored in the SQLite database in analyses/sample_collab.db
  • table SraRunTable is a direct DB import of data/SraRunTable.csv
  • table sample_overviews is a direct DB import of concatenated files data/sample_overviews 5.csv plus data/sample_overviews 3.csv
  • table ct_values is a direct DB import of data/ct_values_covid19_metagenomics.xlsx
  • table users will hold the list of course participants (field username is your IFB account login)
  • samples to users dispatch to be made in table sample2user

References

• publication in Journal of Clinical Virology, Volume 145, December 2021: https://www.sciencedirect.com/science/article/pii/S1386653221002924
• referenced papers on the "spiked primer enrichment" technique (nothing to do with the SC2 Spike protein...):
  • https://www.nature.com/articles/s41564-019-0637-9
  • preprint applied to SC2: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217139/
  • with a corresponding "open reproducible protocol": https://www.protocols.io/view/sars-cov-2-enrichment-sequencing-by-spiked-primer-261geowedl47/v4
• paper describing the "IDSeq" (now morphed into https://czid.org/) metagenomic analysis protocol (used in the 2021 J. of Clinical Virol): https://academic.oup.com/gigascience/article/9/10/giaa111/5918865?login=true