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Microbial Genomics Journey Workshop 2023

Session 13: Graduation Project

Description

  • There will be 2 teams. Each team will work together on a project with certain comparative genomics questions to try to answer. We talked about too many tools and approaches, this project will be the most important part of the workshop as you will be able to apply what you have learnt so far and put it all together.
  • The last session will be on 12/15 and each team will present their findings. Next Friday you will get more details.
  • You can access the dataset from Dataset of 20 genomes.
  • There is a fastq folder with 20 genomes and 1 file showing case and control designations.
  • Each team will have to present their methods, main commands and major findings on 12/15/2023.
  • The teaching team will be happy to answer all your questions.

Thoughts and Tips

  • Go through the material and you will be able to process these isolates from start to finish. You may not use all of the tools.
  • The readme file on github with the syllabus has a nice summary for all steps and tools we discussed.
  • Team should divide work. For instance, each one assemble 5 genomes...etc, using the same command.

Questions to answer

These 20 genomes are from a single species. There are two group of isolates (case and control), 10 each. You are provided with fastq reads files.

  1. What are the differences between the genomes in the case group vs control group?
  2. Build a phylogenetic tree for these 50 genomes. Describe your findings on the tree!

Solution

QC
FASTQC

cat GCA_000775395.1_R1.fastq.gz GCA_000775395.1_R2.fastq.gz > GCA_000775395.1_combined.fastq.gz
conda activate fastqc2
fastqc GCA_000775395.1_combined.fastq.gz

Assembly

../snippy_convert_list_input_tab.py ../accs.txt ../spades_bash.sh
chmod +x spades_bash.sh
./spades_bash.sh
spades.py --pe1-1 GCA_905128515.1_R1.fastq.gz --pe1-2 GCA_905128515.1_R2.fastq.gz -o assem_GCA_905128515.1

MLST

mlst --csv *.fasta > ../all_mlst.csv

There were an issue with the assembled genomes from the reads I gave you. Two sequence types (ST11/control and ST258/case) would have been identified if the right assemblies were used.

QC
BUSCO

conda activate busco
snippy_convert_list_input_tab.py accs.txt busco_bash.sh
chmod +x busco_bash.sh
cd assemblies
./busco_bash.sh

mash
good example

mash screen -w -p 8 /Users/moustafaam/Downloads/refseq.genomes.k21s1000.msh assemblies/GCA_000717515.1.fasta > GCA_000717515.1_mash.txt
sort -gr GCA_000717515.1_mash.txt > GCA_000717515.1_mash_sorted.txt

contamination example

mash screen -w -p 8 /Users/moustafaam/Downloads/refseq.genomes.k21s1000.msh assemblies/GCA_001068065.1.fasta > GCA_001068065.1_mash.txt
mash screen -w -p 8 /Users/moustafaam/Downloads/refseq.genomes.k21s1000.msh assemblies/GCA_905128515.1.fasta > GCA_905128515.1_mash.txt
sort -gr GCA_001068065.1_mash.txt > GCA_001068065.1_mash_sorted.txt
sort -gr GCA_905128515.1_mash.txt > GCA_905128515.1_mash_sorted.txt

These two genomes were excluded from the next steps.

Annotation

chmod +x copy_rename_files_prokka.py
./copy_rename_files_prokka.py
../snippy_convert_list_input_tab.py ../accs.txt prokka_bash.sh
conda activate prokka
chmod +x prokka_bash.sh
./prokka_bash.sh
prokka --outdir annotations/annot_GCA_003203435.1 --kingdom Bacteria --locustag GCA_003203435.1 --prefix GCA_003203435.1 assemblies/GCA_003203435.1.fasta

pangenome

find ./ -name '*.gff' -exec cp -prv '{}' '/Users/moustafaam/MGJW/final_project/fastq/gff' ';'
conda activate panaroo_m2
cd gff
panaroo -i *.gff -a core --aligner mafft -t 8 -o panaroo_results_align/ --clean-mode strict

GWAS
Scoary

scoary -t isolates.csv -g gene_presence_absence_roary.csv

There are 23 genes that are only present in the 24 isolates in the case group (100% specificity and sensitivity).

ABRICATE

for f in assemblies/*.fasta; do abricate $f > $f.tab ; done
abricate --summary *.tab > abricate_summary.tab

phylogeny

iqtree -s core_gene_alignment_filtered.aln -T 64 -m HKY -B 1000

There were an issue with the assembled genomes from the reads I gave you. Even with that, the tree clearly shows the two clades.

Visualization
The iqtree program outputs a bunch of files, the core_gene_alignment_filtered.aln.contree can be opened in Itol or Figtree, and it includes the branch support values. I used two genes from the Abricate output and ST designation from the MLST step to show on the tree. ML Tree

HPC

Request Access to CHOP Respublica HPC

Further Readings

  • PATRICBacterial and Viral Bioinformatics Resource Center (BV-BRC). BV-BRC is a joint effort among the University of Chicago (UChicago), J Craig Venter Institute (JCVI), Fellowship for Interpretation of Genomes (FIG), and University of Virginia (UVA) that provides a web-based information system designed to support the biomedical research community’s work on bacterial and viral infectious diseases via integration of vital pathogen information with rich data and analysis tools. BV-BRC combines the data, technology, and extensive user communities from two long-running centers: PATRIC, the bacterial system , and IRD/ViPR, the viral systems.
  • KBase is the first large-scale data science platform that enables users to upload their own data, analyze it alongside collaborator and public data, build increasingly realistic models, and share and publish reproducible workflows and conclusions.
  • Galaxy is an open source, web-based platform for data intensive biomedical research.