Clinical and molecular epidemiology of carbapenemase-producing Enterobacterales in hospitalized patients

# Wiki

# Data location:

The data was transferred from Athena medmicro) by e.g.:

```
rsync -avvP -e "ssh -i /home/katie/.ssh/id_rsa" /mnt/athena/medmicro/Clinton/CRE\ Pfizer\ Feb\ 2022/CRE\ study_4_results_11112022 katiel@transfer.ilifu.ac.za:/scratch3/users/katiel/Clinton/CRE_study_August_2022/raw/
```

to Ilifu:

```
/scratch3/users/katiel/Clinton/CRE_study_August_2022/
```

# Reference data:

Klebsiella pneumoniae – strain HS11286 (GenBank accession no. CP003200.1) (n=18);
Serratia marcescens – strain KS10 (GenBank accession no. CP027798.1) (n=3);
Escherichia coli – strain ATCC 25922 (GenBank accession no. CP009072.1) (n=1); and
Enterobacter cloacae – strain ATCC 13047 (GenBank accession no. NC_014121.1) (n=1).

```
/scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_genomes
```

# Objectives workflow:
![workflow.png]()

# QC:
11 sample from Run 1 failed QC and had to be rerun. Note that they accidentally reran these 11 (study1A) twice – once on 28 Feb and once on 22 September. These runs were merged by combining samples e.g.:

```
cat KLEB-CRE-GSH-0016_S11_L001_R2_001.fastq.gz >> merged_reads/G-16_S11_L001_R2_001.fastq.gz
```

file location:
```
/scratch3/users/katiel/Clinton/CRE_study_August_2022/raw/11_double_rerun_merged/merged_reads
```

Next these 11 merged-run samples were joined in one folder via symlinks with run B (passed QC):

```
/scratch3/users/katiel/Clinton/CRE_study_August_2022/raw/study_1A_B_combined
```

Filtering and trimming were executed as follows:

```
nextflow run kviljoen/fastq_QC --reads '/scratch3/users/katiel/Clinton/CRE_study_August_2022/raw/study_1A_B_combined/*_R{1,2}_001.fastq.gz' -profile ilifu
```
QC reports can be found in the 'files' tab

Runs 2 and 3 were combined with symlinks under:

```
/scratch3/users/katiel/Clinton/CRE_study_August_2022/raw/study_2_3_combined
```

FastQC was done (all samples passed):

```
nextflow run kviljoen/fastq_QC --reads '/scratch3/users/katiel/Clinton/CRE_study_August_2022/raw/study_2_3_combined/*_R{1,2}_001.fastq.gz' -profile ilifu
```

and can be found here:

```
/scratch3/users/katiel/Clinton/CRE_study_August_2022/2022-11-10-fastq_QC
```

Run 4 was added next and QCed:

Note: to agree with srst2 file naming specifications I renamed the trimmed files from e.g. *_R1.fq to *_1.fq (remove R) using e.g.
```
for f in *.fq; do mv -v "$f" "${f/_R/_}";done
```

# AMR profiling
The preference from Clinton is to do AMR profiling with the ResFinder DB. I'm getting errors there that I think relate to the header formatting though so in the interim have run with the ARG_annot DB that we used for previous projects as:

## ARGannot
```
nextflow run kviljoen/uct-srst2 --reads '/scratch3/users/katiel/Clinton/CRE_study_August_2022/2022-09-19-fastq_QC/bbduk/*_{1,2}.fq' -profile ilifu --gene_db /cbio/users/katie/Nicol/Ps_aerug_srst2_MLST/ARGannot_r3.fasta --outdir /scratch3/users/katiel/Clinton/CRE_study_August_2022/srst2_ARGannot/coverage_80_run --min_gene_cov 80
```
Individual results files compiled as:

```
srst2 --prev_output *results.txt --output ARGannot_AMRs
```

## CARD DB:

This database is the recommended by srst2 and has been formatted by them already. The DB was downloaded with:

```
wget https://github.com/katholt/srst2/blob/master/data/CARD_v3.0.8_SRST2.fasta?raw=true -O CARD_v3.0.8_SRST2.fasta
```

Pipeline execution as:

```
nextflow run kviljoen/uct-srst2 --reads '/scratch3/users/katiel/Clinton/CRE_study_August_2022/2022-09-19-fastq_QC/bbduk/*_{1,2}.fq' -profile ilifu --gene_db /scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/CARD_v3.0.8_SRST2.fasta --outdir /scratch3/users/katiel/Clinton/CRE_study_August_2022/srst2_CARD/coverage_80_run --min_gene_cov 80
```

Individual results files compiled as:

```
srst2 --prev_output *results.txt --output CARD_AMRs
```

# Virulence factors

Building the relevant VFDB for Klebsiella requires a python script that needs the biopython module (use the /cbio/users/katie/singularity_containers/srst2_v2.simg singularity container for this)
NB: in order to use the correct python version (2.7.5) for srst2 I first had to comment out the lines at the end of my .bashrc file relating to conda initialize

Build genus-specific DB:
```
python /cbio/users/katie/Nicol/Ps_aerug_srst2_MLST/VFDB/srst2/database_clustering/VFDBgenus.py --infile /scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/VFDB_setB_nt.fas --genus Klebsiella
```
was used to create the VF DB Klebsiella.fsa

The same procedure (as last year ;) was executed for Escherichia, Serratia and Enterobacter

cd-hit (needed to build vfdb as outlined here https://github.com/katholt/srst2#using-the-vfdb-virulence-factor-database-with-srst2) docker images was pulled from here https://quay.io/repository/biocontainers/cd-hit?tab=tags and converted to singularity image on BST server:
```
singularity exec /cbio/users/katie/singularity_containers/cd-hit.simg /bin/bash
```

then run CD-HIT to cluster the sequences for this genus, at 90% nucleotide identity:

```
cd-hit -i Klebsiella.fsa -o Klebsiella_cdhit90 -c 0.9 > Klebsiella_cdhit90.stdout
```

Repeat for other .fsa DBs

NExt parse the cluster output and tabulate the results using the specific Virulence gene DB compatible script (use srst2_v2.simg again)

```
python /cbio/users/katie/Nicol/Ps_aerug_srst2_MLST/VFDB/srst2/database_clustering/VFDB_cdhit_to_csv_KLedit.py --cluster_file Klebsiella_cdhit90.clstr --infile Klebsiella.fsa --outfile Klebsiella_cdhit90.csv
```

Next convert the resulting csv table to a SRST2-compatible sequence database using:

```
python /cbio/users/katie/Nicol/Ps_aerug_srst2_MLST/VFDB/srst2/database_clustering/csv_to_gene_db.py -t Klebsiella_cdhit90.csv -o Klebsiella_VF_clustered.fasta -s 5

```

The actual VF typing can now be done using this clustered DB:

```
nextflow run kviljoen/uct-srst2 --reads '/scratch3/users/katiel/Clinton/CRE_study_August_2022/2022-09-19-fastq_QC/bbduk/*_{1,2}.fq' -profile ilifu --gene_db /scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/Klebsiella_VF_clustered.fasta --outdir /scratch3/users/katiel/Clinton/CRE_study_August_2022/srst2_VFs/coverage_80_run --min_gene_cov 80
```

Same for other genera using:
```
/scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/Escherichia_VF_clustered.fasta
/scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/Serratia_VF_clustered.fasta
/scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/Enterobacter_VF_clustered.fasta
```

Again combine individual sample results files with e.g.
```
srst2 --prev_output *genes* --output Klebsiella_VFs
```

# MLST
MLST profiles were downloaded for E. coli and K. pneumoniae as:

```
python /cbio/users/katie/Nicol/Ps_aerug_srst2_MLST/VFDB/srst2/scripts/getmlst.py --species 'Escherichia coli#1'
python /cbio/users/katie/Nicol/Ps_aerug_srst2_MLST/VFDB/srst2/scripts/getmlst.py --species 'Escherichia coli#2'
python /cbio/users/katie/Nicol/Ps_aerug_srst2_MLST/VFDB/srst2/scripts/getmlst.py --species 'Klebsiella pneumoniae'

```

Note: MLST profiles not available for Serratia marecescens or Enterobacter cloacae

MLST profiling execution:

```
nextflow run kviljoen/uct-srst2 --reads '/scratch3/users/katiel/Clinton/CRE_study_August_2022/2022-09-19-fastq_QC/bbduk/*_{1,2}.fq' -profile ilifu --mlst_definitions /scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/Klebsiella_definitions --mlst_db /scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/Klebsiella_pneumoniae.fasta --mlst_delimiter _ --outdir /scratch3/users/katiel/Clinton/CRE_study_August_2022/srst2_MLSTs/Klebsiella_MLSTs
```

```
nextflow run kviljoen/uct-srst2 --reads '/scratch3/users/katiel/Clinton/CRE_study_August_2022/2022-09-19-fastq_QC/bbduk/*_{1,2}.fq' -profile ilifu --mlst_definitions /scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/E_coli_1_definitions --mlst_db /scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/Escherichia_coli#1.fasta --mlst_delimiter _ --outdir /scratch3/users/katiel/Clinton/CRE_study_August_2022/srst2_MLSTs/E_coli1_MLSTs
```

```
nextflow run kviljoen/uct-srst2 --reads '/scratch3/users/katiel/Clinton/CRE_study_August_2022/2022-09-19-fastq_QC/bbduk/*_{1,2}.fq' -profile ilifu --mlst_definitions /scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/E_coli_2_definitions --mlst_db /scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/Escherichia_coli#2.fasta --mlst_delimiter _ --outdir /scratch3/users/katiel/Clinton/CRE_study_August_2022/srst2_MLSTs/E_coli2_MLSTs
```

# Combining runs

For prelim analysis run1 was combined with the output from runs 2-4 with e.g. (from directory /scratch3/users/katiel/Clinton/CRE_study_August_2022/srst2_combined_output_run1-4/CARD)

```
ln -s ../../srst2_CARD_run2to4/coverage_80_run/srst2/*genes* ./
ln -s ../../srst2_CARD_v3/coverage_80_run/srst2/*genes* ./
```

# Tychus alignment module

Note the ilifu branch of the Tychus repo should be used. Because we don't have a main.nf file we can't use the standard `nextflow pull` syntax so did a git clone instead
git clone --branch ilifu https://github.com/kviljoen/Tychus/

A list of fasata files for reference genomes was created here
NB: Error when trying to use E_coli_ATCC_25922_CP009072_1.fasta as --genome (but not e.g. Serratia)

NB: error in makephylogenies process:

``` .command.sh: 7: [: missing ]
mv: cannot stat 'kSNP3_results/*.tre': No such file or directory
```
If you go into the working directory you'll find a NameErrors.txt file which states that some of the genome file names are illegal. The one that applies here is: A file name may contian only one dot ('.') character, that which separates the file ID from the extension.
EcoSME175.fasta is legal, EcoSME17.5.fasta is not

So all files were renamed from e.g. E_coli_ATCC_25922_CP009072.1.fasta to E_coli_ATCC_25922_CP009072_1.fasta

```
/scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/full_fasta_list
```

Alignment run example against Serratia:

```
nextflow alignment.nf --alignment_out_dir /scratch3/users/katiel/Clinton/CRE_study_August_2022/Tychus_alignment_Serratia --genome /scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/Serratia_marescens_KS10_CP027798_1.fasta --read_pairs '/scratch3/users/katiel/Clinton/CRE_study_August_2022/2022-10-10-fastq_QC/bbduk/*_{1,2}.fq' --user_genome_paths /scratch3/users/katiel/Clinton/CRE_study_August_2022/ref_files/full_fasta_list -profile alignment
```