QX_bioinfo_analysis.Rmd

---
title: "Transcriptome analysis of _Marteilia sydneyi_ and _Saccostrea glomerata_ during QX infection in Sydney Rock Oysters"
author: "Niki Nenadic and Ido Bar"
date: "`r format(Sys.Date(), '%d %B %Y')`"
always_allow_html: yes
output: 
    bookdown::html_document2:
      df_print: paged
      theme: united
      highlight: pygments
      css: "style/style.css"
      toc: true
      toc_float: true
      toc_depth: 3
  #    highlight: pygments
      number_sections: false
      code_folding: hide
#      keep_md: true
#bibliography: style/comparative_genomics.bib
bibliography: ["style/QX_references.bib","style/comparative_genomics.bib", 'style/oyster_genomes.bib', 'style/pseudo_counts.bib', 'style/stLFR_assembers.bib', 'style/transcriptome_assembly_evaluation.bib', 'style/Metagenomes_assembly_papers.bib', 'style/NGS_QC.bib', 'style/RNAseq_tools.bib'] 
csl: style/springer-basic-improved-author-date-with-italic-et-al-period.csl
---

```{r setup, include=FALSE}
devtools::source_gist("7f63547158ecdbacf31b54a58af0d1cc", filename = "util.R")
knitr::opts_chunk$set(list(echo = TRUE, eval=FALSE, message=FALSE))
fav_packages <- c('tidyverse', 'rentrez', 'taxonomizr')
pak::pak(fav_packages)

pacman::p_load(char = basename(fav_packages))
```

# Experimental Design

## Aims

-   Transcriptomic characterisation of _Marteilia sydneyi_ and _Saccostrea glomerata_ during QX infection in Sydney Rock Oysters
-   Identify genes that play a role in the defence response of Sydney Rock Oysters (_Saccostrea glomerata_) during infection of _Marteilia sydneyi_ (QX disease)  

### Objectives

1. Assemble the _Marteilia sydneyi_ and _Saccostrea glomerata_ transcriptomes
2. Identify differential gene expression in infected and non-infected Sydney Rock Oysters (_Saccostrea glomerata_)

# Transcriptome assembly of _M. sydneyi_ and _Saccostrea glomerata_

## Analysis Pipeline

### General overview:

1.  Data pre-processing:
    a.  Quality check
    b.  Adaptor trimming
    c.  Post-trim quality check
    d.  Error correction  
    e.  Combine reads from control SRO samples and QX-infected SRO samples 
2.  Mapping reads to host reference genome
3.  _De Novo_ transcriptome assembly of the reads (for control and infected samples separately) 
4.  Homology-based taxonomic classification of transcripts  
5.  Separation of host (_S. glomerata_), _M. sydneyi_ and off-target (various contaminations) transcripts
6.  Annotation of host and pathogen transcripts
7.  Host transcriptome differential expression
8.  Summary statistics and visualisation

### Methods
#### RNA extraction
RNA was extracted from the digestive gland of 3 healthy Sydney Rock Oysters (SRO, _Saccostrea glomerata_) and 3 SRO infected with QX disease (as confirmed by PCR assay on DNA extracted from each tissue) using the Trizol method. The RNA was sent for sequencing on an Illumina NovaSeq 6000, generating 150 bp paired-end reads (Macrogen, Korea). 

The reads were downloaded to the Griffith University High Performance Computing cluster 'Gowonda' and the University of Queensland High Performance Computing cluster 'Bunya' (access provided by The Queensland Cyber Infrastructure Foundation (QCIF)) for bioinformatics processing and analyses, following the guidelines by Harvard Informatics (see [link](https://github.com/harvardinformatics/TranscriptomeAssemblyTools)) . 
These included error-correction with Rcorrector v1.5.0 [@songRcorrectorEfficientAccurate2015a] of the reads from each set (healthy SRO and QX-infected SRO) and removal of "unfixable" reads.

```{bash prep-rnaseq-reads}
# setup environment
CONDA_NAME="genomics"
mamba install -n $CONDA_NAME rcorrector agat # can be in the base environment
cd $HOME/etc/tools/Trinity # ~/aDNA/tools 
# install additional utilities
git clone https://github.com/harvardinformatics/TranscriptomeAssemblyTools.git
WORKDIR=/scratch/aDNA/niki_qx
mkdir -p $WORKDIR
cd $WORKDIR
# Prepare a general array PBS script
echo '#!/bin/bash 
#PBS -V

cd $WORKDIR
>&2 echo Current working directory: $PWD
source ~/.bashrc
conda activate $CONDA_NAME 
set -Eeo pipefail'"
gawk -v ARRAY_IND=\$PBS_ARRAY_INDEX 'NR==ARRAY_IND' \$CMDS_FILE | bash" > ${WORKDIR}/array.pbspro
# download reads
rclone copy -P GU_owncloud:Shared/Oyster_QX_Disease/QX_RNA-Seq ./QX_RNA-Seq
cd QX_RNA-Seq/data && mkdir corrected_reads

# prepare commands to run Rcorrector for each sample in parallel
# ls -1 NN01-5*_1.fastq.gz | parallel -k --dry-run --rpl "{infile2} s:_1:_2:;" "run_rcorrector.pl -1 {} -2 {infile2} -verbose -t \$NCPUS -od corrected_reads"  > rcorrector.cmds
# submit the jobs
# qsub -J1-$(cat rcorrector.cmds | wc -l) -l select=1:ncpus=8:mem=16GB,walltime=10:00:00 -N rcorr_batch -v "CMDS_FILE=rcorrector.cmds","WORKDIR=$PWD","CONDA_NAME=$CONDA_NAME" ${WORKDIR}/array.pbspro
# restart failed jobs at stage 3
# ls -1 NN01-5*_1.fastq.gz | parallel -k --dry-run --rpl "{infile2} s:_1:_2:;" "run_rcorrector.pl -1 {} -2 {infile2} -t \$NCPUS -od corrected_reads -stage 3" > rcorrector_s3.cmds
# submit the jobs
# qsub -J1-$(cat rcorrector_s3.cmds | wc -l) -l select=1:ncpus=8:mem=24GB,walltime=10:00:00 -N rcorr_batch_s3 -v "CMDS_FILE=rcorrector_s3.cmds","WORKDIR=$PWD","CONDA_NAME=$CONDA_NAME" ${WORKDIR}/array.pbspro
# didn't work for NN-567 for some reason
# restart job for NN-567 from stage 1
# echo "source ~/.bashrc; conda activate $CONDA_NAME; cd $PWD; set -Eeo pipefail; run_rcorrector.pl -1 NN01-567_1.fastq.gz -2 NN01-567_2.fastq.gz -verbose -t \$NCPUS -od corrected_reads" | qsub -l select=1:ncpus=12:mem=48gb,walltime=20:00:00 -N rcorrect_567 -q bigmem

# Run Rcorrect on Control samples
mkdir corrected_reads
R1_FILES_CON=$(seq 1 3 | parallel -k echo "NN01-56{}_1.fastq.gz" | paste -sd,)
R2_FILES_CON=$(seq 1 3 | parallel -k echo "NN01-56{}_2.fastq.gz" | paste -sd,)
# submit job
echo "source ~/.bashrc; conda activate $CONDA_NAME; cd $PWD; set -Eeo pipefail; run_rcorrector.pl -1 $R1_FILES_CON -2 $R2_FILES_CON -t \$NCPUS -od corrected_reads" | qsub -l select=1:ncpus=12:mem=48gb,walltime=50:00:00 -N rcorr_sro_control -q bigmem  

# combine and correct QX-infected samples
R1_FILES_QX=$(printf "67\n79\n80\n" | parallel -k echo "NN01-5{}_1.fastq.gz" | paste -sd,)
R2_FILES_QX=$(printf "67\n79\n80\n" | parallel -k echo "NN01-5{}_2.fastq.gz" | paste -sd,)
# submit job
echo "source ~/.bashrc; conda activate $CONDA_NAME; cd $PWD; set -Eeo pipefail; run_rcorrector.pl -1 $R1_FILES_QX -2 $R2_FILES_QX -t \$NCPUS -od corrected_reads" | qsub -l select=1:ncpus=12:mem=48gb,walltime=50:00:00 -N rcorr_sro_qx -q bigmem 

# combine reads from individually-corrected samples
cd corrected_reads
echo "source ~/.bashrc; cd $PWD; set -Eeo pipefail; seq 1 3 | parallel -k -j4 cat NN01-56{}_1.cor.fq.gz > SRO_control_R1.cor.fastq.gz ; seq 1 3 | parallel -k -j4 cat NN01-56{}_2.cor.fq.gz > SRO_control_R2.cor.fastq.gz ; "'printf "67\n79\n80\n" | parallel -k -j4 cat NN01-5{}_1.cor.fq.gz > SRO_QX_R1.cor.fastq.gz ; printf "67\n79\n80\n" | parallel -k -j4 cat NN01-5{}_2.cor.fq.gz > SRO_QX_R2.cor.fastq.gz' | qsub -l select=1:ncpus=8:mem=24gb,walltime=3:00:00 -N combine_corr_reads 

# remove unfixable reads
# SRO control
echo "source ~/.bashrc; cd $PWD; set -Eeo pipefail; python  ~/etc/tools/Trinity/TranscriptomeAssemblyTools/utilities/FilterUncorrectabledPEfastq.py -1 SRO_control_R1.cor.fastq.gz -2 SRO_control_R2.cor.fastq.gz -s SRO_control " | qsub -l select=1:ncpus=8:mem=24gb,walltime=5:00:00 -N rm_unfixable_control
# SRO-QX
echo "source ~/.bashrc; cd $PWD; set -Eeo pipefail; python  ~/etc/tools/Trinity/TranscriptomeAssemblyTools/utilities/FilterUncorrectabledPEfastq.py -1 SRO_QX_R1.cor.fastq.gz -2 SRO_QX_R2.cor.fastq.gz -s SRO_QX" | qsub -l select=1:ncpus=8:mem=24gb,walltime=5:00:00 -N rm_unfixable_qx

# compress files with genozip so we could upload them
ls -1 *.fastq.gz | parallel --dry-run genozip -@ \$NCPUS -f {} > genozip_files.cmds
# submit the jobs
qsub -J1-$(cat genozip_files.cmds | wc -l) -l select=1:ncpus=8:mem=16GB,walltime=4:00:00 -N genozip_fastq -v "CMDS_FILE=genozip_files.cmds","WORKDIR=$PWD","CONDA_NAME=base" ${WORKDIR}/array.pbspro

# upload files to Research Space
rclone copy -P $WORKDIR/QX_RNA-Seq/data GU_owncloud:Shared/Oyster_QX_Disease/QX_RNA-Seq/data
```


```{bash rcorrect-bunya}
WORKDIR=/scratch/project/adna/
mkdir -p $WORKDIR/QX_RNA-Seq/data/combined_reads
cd $WORKDIR/QX_RNA-Seq/data
# prepare slurm script
echo '#!/bin/bash --login
#SBATCH --job-name=QX-combine-reads
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --output=%x.%j.log
#SBATCH --cpus-per-task=8
#SBATCH --mem=16G
#SBATCH --time=4:00:00
#SBATCH --account=a_agri_genomics
#SBATCH --partition=general

source ~/.bashrc
set -Eeo pipefail
cd $SLURM_SUBMIT_DIR
srun seq 1 3 | parallel -k cat NN01-56{}_1.fastq.gz > combined_reads/SRO_control_R1.fastq.gz; \
seq 1 3 | parallel -k cat NN01-56{}_2.fastq.gz > combined_reads/SRO_control_R2.fastq.gz; \
printf "67\n79\n80\n" | parallel -k cat NN01-5{}_1.fastq.gz > combined_reads/SRO_QX_R1.fastq.gz; \
printf "67\n79\n80\n" | parallel -k cat NN01-5{}_2.fastq.gz > combined_reads/SRO_QX_R2.fastq.gz' > combine_reads.slurm
# submit the job 
sbatch combine_reads.slurm

echo '#!/bin/bash --login
#SBATCH --job-name=QX-combine-rcorrect
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --output=%x.%j.log
#SBATCH --cpus-per-task=8
#SBATCH --mem=16G
#SBATCH --time=4:00:00
#SBATCH --account=a_agri_genomics
#SBATCH --partition=general

source ~/.bashrc
set -Eeo pipefail
cd $SLURM_SUBMIT_DIR
srun seq 1 3 | parallel -k cat NN01-56{}_1.cor.fq.gz > SRO_control_R1.cor.fastq.gz; \
seq 1 3 | parallel -k cat NN01-56{}_2.cor.fq.gz > SRO_control_R2.cor.fastq.gz; \
printf "67\n79\n80\n" | parallel -k cat NN01-5{}_1.cor.fq.gz > SRO_QX_R1.cor.fastq.gz; \
printf "67\n79\n80\n" | parallel -k cat NN01-5{}_2.cor.fq.gz > SRO_QX_R2.cor.fastq.gz' > combine_cor_reads.slurm
# submit the job 
sbatch combine_cor_reads.slurm

# remove unfixable reads
# SRO control
echo '#!/bin/bash --login
#SBATCH --job-name=rm_unfixable_control
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --output=%x.%j.log
#SBATCH --cpus-per-task=8
#SBATCH --mem=24G
#SBATCH --time=6:00:00
#SBATCH --account=a_agri_genomics
#SBATCH --partition=general

source ~/.bashrc
set -Eeo pipefail
cd $SLURM_SUBMIT_DIR
srun python  ~/etc/tools/TranscriptomeAssemblyTools/utilities/FilterUncorrectabledPEfastq.py -1 SRO_control_R1.cor.fastq.gz -2 SRO_control_R2.cor.fastq.gz -s SRO_control ' > rm_unfixable_control.slurm
# submit the job 
sbatch rm_unfixable_control.slurm

# SRO-QX
echo '#!/bin/bash --login
#SBATCH --job-name=rm_unfixable_qx
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --output=%x.%j.log
#SBATCH --cpus-per-task=8
#SBATCH --mem=24G
#SBATCH --time=6:00:00
#SBATCH --account=a_agri_genomics
#SBATCH --partition=general

source ~/.bashrc
set -Eeo pipefail
cd $SLURM_SUBMIT_DIR
srun python  ~/etc/tools/TranscriptomeAssemblyTools/utilities/FilterUncorrectabledPEfastq.py -1 SRO_QX_R1.cor.fastq.gz -2 SRO_QX_R2.cor.fastq.gz -s SRO_QX' > rm_unfixable_qx.slurm
# submit the job 
sbatch rm_unfixable_qx.slurm

```

#### Transcriptome assembly and Annotation with TransPi
A _De-Novo_ transcriptome was assembled from the combined error-corrected reads using [TransPi](https://palmuc.github.io/TransPi) [@rivera-vicensTransPiComprehensiveTRanscriptome2022]. In brief, TransPi performs a complete transcriptome assembly, basic annotation and assessment pipeline using well-established methods:  

 * Trimming of low-quality bases and removal of sequencing adaptors with Fastp [@chenFastpUltrafastAllone2018]  
 * Ribosomal RNA (rRNA) removal matching against the [SILVA rRNA database](https://www.arb-silva.de/) [@quastSILVARibosomalRNA2012]  
 * Transcriptome assembly using a range of tools, including Trinity, SPAdes, Trans-ABySS and SOAPdenovo-Trans, with a range of k-mers (see a comprehensive comparison of these tools at @holzerNovoTranscriptomeAssembly2019 and of k-mer selection at @haznedarogluOptimizationNovoTranscriptome2012)   
 * Collapsing redundant transcripts with [EvidentialGene](http://arthropods.eugenes.org/EvidentialGene/)  
 * Annotation of the assemblies against UniProt databases  
 * Building a [Trinotate](https://github.com/Trinotate/Trinotate/wiki) database for each assembly and its annotation  
 
TransPi needs to be installed and configured (download annotation databases and set their paths in a `nextflow.config` file). Unfortunately, a lot of the configuration for TransPi to work on GU Gowonda needs to be done manually, including downloading the Singularity containers and setting up the resources required for each process - see the current configuration and the following reported issues:  

1. Missing BUSCO container in the configuration - [Issue #53](https://github.com/PalMuc/TransPi/issues/53#issuecomment-1268051333)  
2. The precheck script fails to download the UniProt database due to change in the API - see [Issue #52](https://github.com/PalMuc/TransPi/issues/52)  
3. Use the single TransPiContainer profile to fix issues with some of the tools (see the [documentation](https://palmuc.github.io/TransPi/#_containers))  
4. Trinity and Velvet-Oases jobs are failing though they seem to complete without a problem (no output files are copied across from the compute nodes). It can be one of these possibilities:  
    a. Maybe I simply ran out of space (in `/scratch/project/adna`), I've requested additional storage on the `adna` folder on Bunya (now I have 10TB there)  
    b. It may be related to the use of `$TMPDIR` on the compute nodes (change the flag `scracth=true` into `scratch='/scratch/project/adna/tmp'` in `~/.nextflow/bunya.config`)  
    c. `$SINGULARITY_TMPDIR` that is running out of space or not properly configured, see this [thread on GitHub](https://github.com/nextflow-io/nextflow/issues/2786#issuecomment-1173975771)  
    d. Another option is that Bunya is using Apptainer and not Singularity, so maybe I need to configure a profile to use Apptainer instead (v22.11.1).  
    e. Found the problem!!! It appears that the way TransPi is writing the version numbers throws an error (when `$v` is defined multiple times). I fixed it by using `printf` statement instead of the multiple `echo` ones. 
    f. Some processes (such as `summary_custom_uniprot`) fail due to `set -o pipefail` directive (complex pipelines, including `grep`, etc.). It can be fixed by adding `set +o pipefail` to the top of the script.  
5. `Rnammer` fails - it needs to be setup to use `hmmsearch2` (which can be downloaded from [this link](http://eddylab.org/software/hmmer/hmmer-2.3.2.tar.gz) and remove the `--cpu 1` flag from `core-rnammer`, see details [here](https://groups.google.com/g/trinityrnaseq-users/c/WZjkGSMUT3I))  
6. `rnaQUAST` fails -- it requires an additional tool (GeneMark S-T) to be installed separately and put in the container's `$PATH` (add `export PATH=\$PATH:\$HOME/etc/tools/Annotation/GeneMarkST/` at the top of the script of the rnaquast process in the main nextflow). See details [here](https://github.com/ablab/rnaquast/issues/5#issuecomment-823996456).  
7. `signalp` fails -- need to edit the executable to allow it to find where it is being run from and increase the sequence limit (see [here](https://www.seqanswers.com/forum/bioinformatics/bioinformatics-aa/29132-how-to-increase-sequence-limit-in-signalp#post236326)) with the following command:  
`sed -ri.bak 's|SIGNALP} = '\''.+|SIGNALP} = "\$FindBin::RealBin"|; s/BEGIN/use FindBin\;
\nBEGIN/; s/MAX_ALLOWED_ENTRIES=.+/MAX_ALLOWED_ENTRIES=2000000;/' signalp`
8. Process `summary_custom_uniprot` fails because of weird table merging and file redundancies done in bash. I edited `Tranbspi/bin/custom_uniprot_hits.R` to perform these tasks and export the `csv` file with the results.  

```{bash setup-transpi}
# install TransPi
cd /home/ibar/adna/tools #Bunya
git clone https://github.com/idobar/TransPi.git # my version
bash ./TransPi/precheck_TransPi.sh $PWD/TransPi
sed -i "s/'singularity' || workflow.containerEngine == 'docker'/'singularity' || workflow.containerEngine == 'docker' || workflow.containerEngine == 'apptainer'/g" $PWD/TransPi/TransPi.nf # add support for Apptainer (it will also require NXF_VER=22.11.1-edge)  
# download the pipeline containers
grep "singularity_pull_docker_container" $PWD/TransPi/TransPi.nf | cut -f 2 -d '"' | sort | uniq | tr -d \' | parallel --dry-run --rpl  "{outfile} s=https://==; s=[:/]+=-=g;" aria2c -c {} -o $NXF_SINGULARITY_CACHEDIR/{outfile}.img 

# Install GeneMark
mkdir -p ~/etc/tools/Annotation/GeneMarkST
cd  !$
aria2c -x5 http://topaz.gatech.edu/GeneMark/tmp/GMtool_ZozF5/gmst_linux_64.tar.gz
tar xzf gmst_linux_64.tar.gz
ln -s $PWD/gmst.pl ~/bin/ # add it to the PATH
# need to apply for a licence at http://topaz.gatech.edu/GeneMark/license_download.cgi
aria2c http://topaz.gatech.edu/GeneMark/tmp/GMtool_ZozF5/gm_key_64.gz

# fix Rnammer
sed -i.bak 's/--cpu 1 //g' /home/ibar/scratch/tools/rnammer-1.2/core-rnammer
# fix signalp
sed -ri.bak 's|SIGNALP} = '\''.+|SIGNALP} = "\$FindBin::RealBin"|; s/BEGIN/use FindBin\;
\nBEGIN/; s/MAX_ALLOWED_ENTRIES=.+/MAX_ALLOWED_ENTRIES=2000000;/' /home/ibar/scratch/tools/signalp-4.1/signalp

cd ~/bin
aria2c -x5 https://github.com/nextflow-io/nextflow/releases/download/v21.10.4/nextflow-21.10.4-all # this is the original version in TransPi installer - it does not support Apptainer!
chmod +x nextflow-21.10.4-all
aria2c -x5 https://github.com/nextflow-io/nextflow/releases/download/v22.11.1-edge/nextflow-22.11.1-edge-all
chmod +x nextflow-22.11.1-edge-all # this is the only version that supports DSL1, Apptainer and has an update Tower plugin!
```

##### Test TransPi on subset of reads from one sample

```{bash transpi-test}
# install bbtools
mamba install -n gwas bbtools
mkdir -p ~/adna/sandbox/TransPi_test
cd !$

echo '#!/bin/bash --login
#SBATCH --job-name=downsample_reads
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --output=%x.%j.log
#SBATCH --cpus-per-task=8
#SBATCH --mem=24G
#SBATCH --time=6:00:00
#SBATCH --account=a_agri_genomics
#SBATCH --partition=general

source ~/.bashrc
conda activate gwas
set -Eeo pipefail
cd $SLURM_SUBMIT_DIR
srun reformat.sh  in=~/adna/Oyster_QX_Disease/QX_RNA-Seq/data/NN01-561_#.fastq.gz out=NN01-561_#.downsampled.fastq.gz samplerate=0.2' > downsample_reads.slurm
# submit the job 
sbatch downsample_reads.slurm
# conda activate nf21 # nextflow version 21.10.6
nextflow-22.11.1-edge-all run /home/ibar/adna/tools/TransPi/TransPi.nf -with-tower --all -profile apptainer,TransPiContainer,bunya -c /home/ibar/.nextflow/bunya.config --k 25,35 --maxReadLen 150 --pipeInstall /scratch/project/adna/tools/TransPi --allBuscos --buscoDist --reads './NN01-561_[1,2].downsampled.fastq.gz'

```
 
##### Run TransPi on the combined and corrected reads from each experimental group 

```{bash transpi-assemble}
# WORKDIR=/scratch/s2978925/aDNA/niki_qx #Gowonda
WORKDIR=/home/ibar/adna/Oyster_QX_Disease # Bunya
# mamba create -n nf-dsl1 nextflow=22.10.6 # need an older version (<22) to avoid Apptainer warnings
# export NXF_DEFAULT_DSL=1 # only needed if using nextflow version>22.03.0-edge and <22.12.0-edge
# NXF_VER=22.11.1-edge

# export TOWER_ACCESS_TOKEN=eyJ0aWQiOiA0NDYxfS5hNzkzY2M3NTI1YjZkZGFlMjVlZjJiZmIxNTU1MDAyMGE4YjNjZWQ0 # only needed if it is not already set in ~/.bashrc
# export NXF_VER=22.04.5
# prepare the folder and input files
mkdir -p $WORKDIR/SRO_TransPi_assembly/input_files && cd $WORKDIR/SRO_TransPi_assembly
ln -s $WORKDIR//QX_RNA-Seq/data/corrected_reads/unfixrm_SRO_*.gz input_files/
ln -s $WORKDIR//QX_RNA-Seq/data/combined_reads/*.fastq.gz input_files/
# start TransPi (Bunya) [optional tools and databases configured in nextflow.config]
nextflow-22.11.1-edge-all run /home/ibar/adna/tools/TransPi/TransPi.nf -with-tower --all -profile apptainer,TransPiContainer,bunya -c /home/ibar/.nextflow/bunya.config --k 25,35,41,53,65,71,83 --maxReadLen 150 --pipeInstall /scratch/project/adna/tools/TransPi --allBuscos --buscoDist --reads './input_files/*_R[1,2]*.fastq.gz' -resume

# start the run (Gowonda)
# nextflow run $WORKDIR/TransPi/TransPi.nf -with-tower --all --reads './input_files/*_R[1,2]*.fastq.gz'  -profile singularity,TransPiContainer,gowonda -c $HOME/.nextflow/GU.config --rRNAfilter --rRNAdb $WORKDIR/TransPi/DBs/rrna_db/SILVA_138.1_Small_and_Large_SURef_NR99_tax_silva_trunc.fasta --withSignalP --signalp $HOME/etc/tools/Annotation/signalp-4.1/signalp --withTMHMM --tmhmm $HOME/etc/tools/Annotation/tmhmm-2.0c/bin/tmhmm --withRnammer --rnam $HOME/etc/tools/Annotation/rnammer-1.2/rnammer --k 25,35,41,53,65,71,83 --maxReadLen 150 --pipeInstall $WORKDIR/TransPi --allBuscos --buscoDist -resume

```

##### Run EvidentialGene step of TransPi to collapse redundant transcripts

```{bash transpi-evigene}
JOBNAME="onlyEvigene"
NCORES=4
MEM=24
WALLTIME=20:00:00
mkdir -p $WORKDIR/SRO_TransPi_assembly/Evigene_analysis/
cd !$
ls -1 $WORKDIR/SRO_TransPi_assembly/results/assemblies/*SOAP.fa | parallel --dry-run --rpl "{ass} s:.+/(.+).SOAP.fa:\1:" --rpl "{ass_dir} s:.+/(.+).SOAP.fa:$WORKDIR/SRO_TransPi_assembly/Evigene_analysis/\1:" "source ~/.bash_profile; mkdir -p {ass_dir}/onlyEvi && cd {ass_dir}; ln -s $WORKDIR/SRO_TransPi_assembly/results/assemblies/{ass}*[!k0-9].fa {ass_dir}/onlyEvi/; nextflow-22.11.1-edge-all run /home/ibar/adna/tools/TransPi/TransPi.nf -name {ass}_evigene -with-tower --onlyEvi --outdir {ass}_evigene_results -profile apptainer,TransPiContainer,bunya -c /home/ibar/.nextflow/bunya.config" > $JOBNAME.cmds

# submit the job array to the cluster
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,PATH=$PATH,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=base ${WORKDIR}/array.slurm

# resume (if failed)
ls -1 $WORKDIR/SRO_TransPi_assembly/results/assemblies/*SOAP.fa | parallel --dry-run --rpl "{ass} s:.+/(.+).SOAP.fa:\1:" "source ~/.bash_profile; cd $WORKDIR/SRO_TransPi_assembly/Evigene_analysis/{ass};  nextflow-22.11.1-edge-all run /home/ibar/adna/tools/TransPi/TransPi.nf -name {ass}_evigene -with-tower --onlyEvi --outdir {ass}_evigene_results -profile apptainer,TransPiContainer,bunya -c /home/ibar/.nextflow/bunya.config -resume" > $JOBNAME.resume.cmds
# submit the job array to the cluster
sbatch -a 1-$(cat $JOBNAME.resume.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.resume.cmds,CONDA_NAME=base ${WORKDIR}/array.slurm

# Create 

```

##### Annotate chosen assemblies

The assemblies were annotated with TransPi, using homology searches (NCBI BLAST, @buchfinkFastSensitiveProtein2015a and DIAMOND) against the Uniprot_Sprot protein database, as well as using HMM search against the Pfam database of protein families. In addition, signal peptides, ribosomal genes and transmembrane topology were predicted with SignalP v4.1 [@petersenSignalPDiscriminatingSignal2011], Rnammer and TMHMM [@kroghPredictingTransmembraneProtein2001], respectively [@emanuelssonLocatingProteinsCell2007].

```{bash transpi-annot}
WORKDIR=/home/ibar/adna/Oyster_QX_Disease # Bunya
mkdir -p $WORKDIR/SRO_TransPi_assembly/Annotation/onlyAnn
ln -s $WORKDIR/SRO_TransPi_assembly/results/assemblies/*Trinity.fa $WORKDIR/SRO_TransPi_assembly/Annotation/onlyAnn/
cd $WORKDIR/SRO_TransPi_assembly/Annotation

nextflow-22.11.1-edge-all run /scratch/project/adna/tools/TransPi/TransPi.nf -with-tower --onlyAnn --outdir Annotation_results -profile apptainer,TransPiContainer,bunya -c /home/ibar/.nextflow/bunya.config --withSignalP --withTMHMM --withRnammer

# create gff and gtf files from selected assemblies
JOBNAME="gtf2gff"
NCORES=4
MEM=24
WALLTIME=20:00:00
cd $WORKDIR/SRO_TransPi_assembly/reslts/assemblies

find $WORKDIR/SRO_TransPi_assembly/results/assemblies -name "*Trinity.fa" | parallel --dry-run "perl ~/etc/tools/trinityrnaseq-v2.11.0/util/misc/cdna_fasta_file_to_transcript_gtf.pl {} > {.}.gtf; agat_convert_sp_gxf2gxf.pl -g {.}.gtf -o {.}.gff" > $JOBNAME.cmds

# submit the job array to the cluster
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,PATH=$PATH,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=genomics ${WORKDIR}/array.slurm 

rclone copy -P --dry-run --include "*Trinity*" $WORKDIR/SRO_TransPi_assembly/results/assemblies GURS_shared:Oyster_QX_Disease/QX_RNA-Seq/SRO_TransPi_assembly/results/assemblies
```

###### Annotate chosen assemblies with BLAST

In addition to the annotation performed with TransPi, the transcriptomes were annotated against the non-redundant nucleotide and protein databases of the NCBI (`nt` and `nr`, respectively) to achieve more accurate species-specific annotations.

```{bash trans-blast}
CONDA_NAME=genomics
mamba install -n $CONDA_NAME gsutil awscli blast
# download NCBI databases
mkdir -p ~/adna/tools/ncbi_db
cd ~/adna/tools/ncbi_db
WORKDIR=/scratch/project/adna/Oyster_QX_Disease
JOBNAME="update_blast_db"
NCORES=8
MEM=16
WALLTIME=20:00:00
parallel --dry-run "update_blastdb.pl --source ncbi --decompress --force --blastdb_version 5 --num_threads 0 {}" ::: nt nr taxdb > $JOBNAME.cmds
# or do it manually 
# curl -s -l ftp://ftp.ncbi.nlm.nih.gov/blast/db/v5/ | grep -E "^nr." | parallel --dry-run aria2c -x5 -c --auto-file-renaming=false https://ftp.ncbi.nlm.nih.gov/blast/db/v5/{} > download_blast_nr.cmds


# Prepare a general array Slurm script
echo '#!/bin/bash --login
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --output=%x.%A.%a.log'"
#SBATCH --account=a_agri_genomics
#SBATCH --partition=general

set -Eeo pipefail
source ~/.bashrc
conda activate \$CONDA_NAME
cd \$SLURM_SUBMIT_DIR
srun gawk -v ARRAY_IND=\$SLURM_ARRAY_TASK_ID 'NR==ARRAY_IND' \$CMDS_FILE | bash" > ${WORKDIR}/array.slurm


# send to the cluster
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=$CONDA_NAME   ${WORKDIR}/array.slurm 

# Blastn - this takes too long, see using blast-nf below
WORKDIR=/home/ibar/adna/Oyster_QX_Disease
cd $WORKDIR/SRO_TransPi_assembly/Annotation
mkdir -p $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/blastn_tax
JOBNAME="SRO_trans_blastn"
NCORES=24
MEM=96
WALLTIME=150:00:00
ls -1 $WORKDIR/SRO_TransPi_assembly/results/assemblies/*Trinity.fa | parallel --dry-run "blastn -task blastn -db /scratch/project/adna/tools/ncbi_db/nt -query {} -outfmt '6 std stitle staxids ssciname scomname' -evalue 1e-10 -max_target_seqs 20 -num_threads \$SLURM_CPUS_PER_TASK -out Annotation_results/blastn_tax/{/.}.nt_tax.outfmt6" > $JOBNAME.cmds

# submit the commands in the login node
bash $JOBNAME.cmds
# or send to the cluster as a job array
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=$CONDA_NAME --dependency=afterok:8070286  ${WORKDIR}/array.slurm 

# annotate proteins - this takes too long, see using blast-nf below
JOBNAME="SRO_trans_blastp"
ls -1 $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/transdecoder/*.transdecoder.pep | parallel --dry-run "blastp -task blastp -db /scratch/project/adna/tools/ncbi_db/nr -query {} -outfmt '6 std stitle staxids ssciname scomname' -evalue 1e-10 -max_target_seqs 20 -num_threads \$SLURM_CPUS_PER_TASK -out Annotation_results/blastp_tax/{/.}.nr_tax.outfmt6" > $JOBNAME.cmds

# submit the job array to the cluster
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=$CONDA_NAME --dependency=afterok:8070286  ${WORKDIR}/array.slurm

```

Regular BLAST is just too slow (mainly on the protein level), so we used a Nextflow implementation of BLAST that breaks down the input transcriptome into "chunks" and processes them in parallel on the HPC cluster.

```{bash blast-nf}
WORKDIR=/home/ibar/adna/Oyster_QX_Disease
cd $WORKDIR/SRO_TransPi_assembly/Annotation
JOBNAME=blastn_tax_nf
NCORES=4
MEM=24
WALLTIME=150:00:00
mkdir -p $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/$JOBNAME
ls -1 $WORKDIR/SRO_TransPi_assembly/results/assemblies/*Trinity.fa | parallel --dry-run --rpl "{sample} s:.+/(.+)\.Trinity.+:\1:" "~/bin/nextflow-22.11.1-edge-all run ~/etc/tools/blast-nf/main.nf --app blastn --dbDir /scratch/project/adna/tools/ncbi_db --dbName nt --query {} --outfmt \"'6 std stitle staxids ssciname scomname'\" --options '-evalue 1e-10 -max_target_seqs 20' --chunkSize 2500 --outdir  $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/$JOBNAME/{sample} --outfileName {/.}.nt_tax.outfmt6  -c ~/.nextflow/bunya.config -profile bunya,apptainer -with-tower" > $JOBNAME.cmds

# submit the job array to the cluster
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=base ${WORKDIR}/array.slurm

JOBNAME=blastp_tax_nf
mkdir -p $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/$JOBNAME
ls -1 $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/transdecoder/*.transdecoder.pep | parallel --dry-run --rpl "{sample} s:.+/(.+)\.Trinity.+:\1:" "~/bin/nextflow-22.11.1-edge-all run ~/etc/tools/blast-nf/main.nf --app blastp --dbDir /scratch/project/adna/tools/ncbi_db --dbName nr --query {} --outfmt \"'6 std stitle staxids ssciname scomname'\" --options '-evalue 1e-10 -max_target_seqs 20' --chunkSize 500 --outdir  $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/$JOBNAME/{sample} --outfileName {/}.nr_tax.outfmt6  -c ~/.nextflow/bunya.config -profile bunya,apptainer -with-tower" > $JOBNAME.cmds

# submit the job array to the cluster
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=base ${WORKDIR}/array.slurm

```

###### Annotate predicted proteins with DIAMOND

Even when using a split-combine strategy using `blast-nf`, BLASTp is just too slow (it takes around 50 hours to process a chunk of 500 proteins). Therefore we also used DIAMOND, which can be x1,000 quicker than BLAST [@buchfinkFastSensitiveProtein2015a].


```{bash prot-diamond}
WORKDIR=/home/ibar/adna/Oyster_QX_Disease
cd /scratch/project/adna/tools
JOBNAME=prep_diamond_db
NCORES=8
MEM=48
WALLTIME=10:00:00

DB_PATH="/scratch/project/adna/tools/ncbi_db"
DB="nr"
# prepare script
echo '#!/bin/bash --login
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --output=%x.%A.%a.log'"
#SBATCH --account=a_agri_genomics
#SBATCH --partition=general

set -Eeo pipefail
source ~/.bashrc
conda activate \$CONDA_NAME
cd \$SLURM_SUBMIT_DIR

blastdbcmd -entry all -db ${DB_PATH}/${DB} -out ${DB_PATH}/${DB}.fasta 
printf \"Finished extracting sequences from ${DB} database as '${DB}.fasta'\n\"
aria2c -x5 -c --auto-file-renaming=false -d ${DB_PATH}  https://ftp.ncbi.nlm.nih.gov/pub/taxonomy/accession2taxid/prot.accession2taxid.FULL.gz
aria2c -x5 -c --auto-file-renaming=false -d ${DB_PATH}  https://ftp.ncbi.nlm.nih.gov/pub/taxonomy/taxdmp.zip
cd ${DB_PATH} && unzip taxdmp.zip
gzip -v -t ${DB_PATH}/prot.accession2taxid.FULL.gz
printf \"\$(date) - Finished validating 'prot.accession2taxid.FULL.gz'.\n\"
diamond makedb -p \$SLURM_CPUS_PER_TASK --in ${DB_PATH}/${DB}.fasta -d ${DB_PATH}/${DB} --taxonmap ${DB_PATH}/prot.accession2taxid.FULL.gz --taxonnodes ${DB_PATH}/nodes.dmp --taxonnames ${DB_PATH}/names.dmp
printf \"\$(date) - Finished preparing Diamond database '${DB}.dmnd'. \n\"
rm ${DB_PATH}/${DB}.fasta
printf \"\$(date) - Removed '${DB}.fasta', script completed. \n\"" > $JOBNAME.slurm
# send the job to the cluster
sbatch --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CONDA_NAME=genomics $JOBNAME.slurm

WORKDIR=/home/ibar/adna/Oyster_QX_Disease
JOBNAME=prot_diamond_tax
cd $WORKDIR/SRO_TransPi_assembly/Annotation
mkdir -p $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/$JOBNAME
NCORES=12
MEM=96
WALLTIME=50:00:00
ls -1 $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/transdecoder/*.transdecoder.pep | parallel --dry-run --rpl "{sample} s:.+/(.+)\.Trinity.+:\1:" "apptainer run docker://buchfink/diamond:latest blastp -d ~/adna/tools/ncbi_db/nr.dmnd -q {} -p \$SLURM_CPUS_PER_TASK -f 6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore stitle staxids sscinames skingdoms sphylums --sensitive -k 20 -e 0.00001 > $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/$JOBNAME/{sample}.diamond_blastp.nr_tax.outfmt6" > $JOBNAME.cmds


# run within an interactive job
grep "unfixrm_SRO_control" $JOBNAME.cmds | bash
# submit the job array to the cluster
sbatch -a 1-2,4 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=base ${WORKDIR}/array.slurm
# each job took roughly 3:30 hours
```

Using the Nextflow-enabled version of DIAMOND (reducing the time to 1.5h)

```{bash prot-diamond-nf}
start_long_interactive_job # start interactive job on Bunya
WORKDIR=/home/ibar/adna/Oyster_QX_Disease
JOBNAME=nf_diamond_blastp_tax
mkdir -p $WORKDIR/SRO_TransPi_assembly/Annotation/$JOBNAME
cd $WORKDIR/SRO_TransPi_assembly/Annotation/$JOBNAME

# mkdir -p $WORKDIR/Annotation/Annotation_results/$JOBNAME
ls -1 $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/transdecoder/*.transdecoder.pep | parallel --dry-run "~/bin/nextflow-22.11.1-edge-all run ~/adna/tools/nf-blast/nf-blast.nf -profile bunya,apptainer,diamond_tax --query {} --app 'diamond blastp' --db ~/adna/tools/ncbi_db/nr --diamondOpts '--very-sensitive -e 1e-10 -k 20' --chunkSize 10000 --outDir  $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/$JOBNAME --out {/}.dmnd_blastp.nr_tax.outfmt6  -c ~/.nextflow/bunya.config -with-tower" > $JOBNAME.cmds

# run SRO_control interactively
grep "unfixrm_SRO_control" $JOBNAME.cmds | bash
# or submit all the commands as a job array to the cluster

NCORES=4
MEM=24
WALLTIME=250:00:00
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=base ${WORKDIR}/array.slurm

```

###### Functional annotation of predicted proteins with InterPro

The predicted proteins in the transcriptomes were further annotated using InterProScan v5.66-98.0 (or [FA-nf](https://github.com/guigolab/FA-nf)) to assign protein families, motifs and ontologies to assist with transcript-to-gene annotation.
Notice the issues mentioned above with SignalP and TMHMM and a new one for Phobius (see discussions and solutions on [GitHub](https://github.com/nextgenusfs/funannotate/issues/696#issuecomment-1057663190) and [BioStar](https://www.biostars.org/p/238642/))

```{bash prot-annotation}
cd ~/adna/tools
start_interactive_job # start an interactive job to download interproscan
IPSCAN_VERSION=5.66-98.0
IPRO_DATA=interproscan-data-${IPSCAN_VERSION}.tar.gz
# http://ftp.ebi.ac.uk/pub/software/unix/iprscan/5/5.66-98.0/alt/interproscan-data-5.66-98.0.tar.gz
DATA_URL="https://ftp.ebi.ac.uk/pub/software/unix/iprscan/5/${IPSCAN_VERSION}/alt/${IPRO_DATA}"
aria2c -x 6 -c $DATA_URL && \
 aria2c -c $DATA_URL.md5 && \
 md5sum -c ${IPRO_DATA}.md5 && pigz -dc $IPRO_DATA | tar xf -
# download the interproscan container
apptainer pull docker://index.docker.io/interpro/interproscan:${IPSCAN_VERSION} --dir $NXF_APPTAINER_CACHEDIR
# create a link in SINGULARITY_CACHEDIR
ln -s interproscan_${IPSCAN_VERSION}.sif $NXF_APPTAINER_CACHEDIR/

# download the FA-nf pipeline
git clone --recursive https://github.com/guigolab/FA-nf
# copy external software and containers from Research Space
rclone copy -P  GURS_shared:Oyster_QX_Disease/FA-nf/containers FA-nf/containers
cd FA-nf/containers/interproscan && tar xzf external_tools.tar.gz && cd -
# ls -1  FA-nf/containers/interproscan/external/tmhmm-2.0c/bin/* | parallel chmod +x {}
# wget https://github.com/chefnb/ParseTargs/raw/master/tmhmm-2.0c/bin/decodeanhmm -O FA-nf/containers/interproscan/external/phobius/decodeanhmm
# chmod +x FA-nf/containers/interproscan/external/signalp-4.1/signalp FA-nf/containers/interproscan/external/phobius/decodeanhmm  FA-nf/containers/interproscan/external/phobius/phobius.pl

# copy user-defined settings
cp -r FA-nf/containers/interproscan/.interproscan-5 ~/
# edit path to external programs and databases
# nano ~/.interproscan-5/interproscan.properties
( echo "data.directory=$PWD/interproscan-${IPSCAN_VERSION}/data"; sed -r  "s|=.+/FA-nf/containers/interproscan/external|=/home/ibar/scratch/tools|g" $PWD/FA-nf/containers/interproscan/.interproscan-5/interproscan.properties ) > ~/.interproscan-5/interproscan.properties
# sed -i  "s|/home/ibar/etc|$PWD|g" ~/.interproscan-5/interproscan.properties
# echo "data.directory=$PWD/interproscan-${IPSCAN_VERSION}/data" >> ~/.interproscan-5/interproscan.properties
# test that interproscan is working
start_interactive_job # make sure it is run on a compute node
apptainer exec FA-nf/containers/interproscan/interproscan_${IPSCAN_VERSION}.sif /opt/interproscan/interproscan.sh
# press the HMM databases 

apptainer exec -B $PWD/interproscan-${IPSCAN_VERSION}/data:/opt/interproscan/data  $PWD/FA-nf/containers/interproscan/interproscan_${IPSCAN_VERSION}.sif /bin/sh -c "cd /opt/interproscan/; python3 setup.py -f interproscan.properties"
# if the code above isn't working then do it interactively
apptainer shell -B $PWD/interproscan-${IPSCAN_VERSION}/data:/opt/interproscan/data  $PWD/FA-nf/containers/interproscan/interproscan_${IPSCAN_VERSION}.sif
cd /opt/interproscan/; python3 setup.py -f interproscan.properties


# annotate proteins 
WORKDIR=/home/ibar/adna/Oyster_QX_Disease
cd $WORKDIR/SRO_TransPi_assembly/Annotation
mkdir -p $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/ipscan
JOBNAME="SRO_prot_ipscan"
# prepare the commands (don't forget to remove the asterisk at the end of the proteins!)
ls -1 $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/transdecoder/*.transdecoder.pep | parallel --dry-run "sed 's/[*]//g' {} > \$TMPDIR/{/.}.tmp ; apptainer exec -B /home/ibar/scratch/tools -B $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/ipscan:/output -B \$TMPDIR:/temp $NXF_SINGULARITY_CACHEDIR/interproscan_${IPSCAN_VERSION}.sif /opt/interproscan/interproscan.sh -i /temp/{/.}.tmp -d /output -pa -dp -goterms -f TSV -T /temp -cpu \$SLURM_CPUS_PER_TASK ; gawk '\$4~/PANTHER/' $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/ipscan/{/.}.tsv > $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/ipscan/{/.}.panther.tsv" > $JOBNAME.cmds

# submit the job array to the cluster
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=$CONDA_NAME  ${WORKDIR}/array.slurm

cd $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/ipscan
# remove the tmp from the filenames
rename -v 's/.tmp//' *
# extract PANTHER results
# ls -1 *.tsv | parallel  "gawk '\$4~/PANTHER/' {} > {.}.panther.tsv"
# send results back to Research Space
rclone copy -P $WORKDIR/SRO_TransPi_assembly/Annotation/Annotation_results/ipscan GURS_shared:Oyster_QX_Disease/QX_RNA-Seq/SRO_TransPi_assembly/Annotation_results/ipscan


```

These annotation tables were added to the Trinotate database of the assemblies.

##### Assess the quality of assemblies

The transcriptome assemblies were assessed and compared using [rnaQuast](https://github.com/ablab/rnaquast) v2.2.3 [@bushmanovaRnaQUASTQualityAssessment2016], [TransRate](http://hibberdlab.com/transrate) v1.0.3 [@smith-unnaTransRateReferencefreeQuality2016] and [DETONATE](https://deweylab.biostat.wisc.edu/detonate/) v1.11 [@liEvaluationNovoTranscriptome2014b], as suggested in the Trinity [documentation](https://github.com/trinityrnaseq/trinityrnaseq/wiki/Transcriptome-Assembly-Quality-Assessment) and by @beheraPlantTranscriptomeAssembly2021.

The transcripts were compared to the Pacific Oyster (*Crassostrea gigas*) proteins, which were obtained from NCBI (Accession [GCA_902806645.1](https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_902806645.1/)) [@penalozaChromosomelevelGenomeAssembly2021] 

```{bash eval-assembly}
CONDA_NAME=rnaseq
mamba create -n $CONDA_NAME transrate ncbi-datasets-cli detonate
WORKDIR='/home/ibar/adna/Oyster_QX_Disease'
conda activate $CONDA_NAME

mkdir -p $WORKDIR/references && cd $WORKDIR/references
datasets download genome accession GCF_902806645.1 --include gff3,rna,cds,protein,genome --filename GCF_902806645.1.zip
unzip GCF_902806645.1.zip
REF_PROT=$WORKDIR/references/ncbi_dataset/data/GCF_902806645.1/protein.faa
# $WORKDIR/SRO_TransPi_assembly/results/assemblies
# install the ORP version of transrate
cd ~/etc/tools 
wget https://github.com/macmanes-lab/Oyster_River_Protocol/raw/master/software/orp-transrate.tar.gz
tar xzf orp-transrate.tar.gz

# or install the ORP container
apptainer pull docker://macmaneslab/orp:2.3.3 --dir $NXF_APPTAINER_CACHEDIR

cd $WORKDIR/SRO_TransPi_assembly/

NCORES=24
MEM=96
WALLTIME=100:00:00

#mkdir -p $WORKDIR/SRO_TransPi_assembly/results/transrate
# cp /home/ibar/adna/Oyster_QX_Disease/SRO_TransPi_assembly/work/27/72b07a2131e97971f644da74fc9dc6/unfixrm_SRO_QX_no_rRNA.R1.fq /home/ibar/adna/Oyster_QX_Disease/SRO_TransPi_assembly/work/2b/f20013a2ae897e646817eac5d0997c/SRO_QX_no_rRNA.R1.fq /home/ibar/adna/Oyster_QX_Disease/SRO_TransPi_assembly/work/e5/25ba5d1510380c3631ee6c18bf2e89/SRO_control_no_rRNA.R1.fq /home/ibar/adna/Oyster_QX_Disease/SRO_TransPi_assembly/work/81/5bd2ffe863bf0ee37388694b4ad254/unfixrm_SRO_control_no_rRNA.R1.fq $WORKDIR/SRO_TransPi_assembly/input_files/

# use no_rRNA.R1.fq files as input 
JOBNAME="transrate_no_rRNA"
OUTDIR=$WORKDIR/SRO_TransPi_assembly/results/$JOBNAME # or OUTDIR=~/scratch/sandbox/SRO_TransPi_assembly/$JOBNAME
mkdir -p $OUTDIR
parallel --dry-run --rpl "{sample} s:.+/(.+)_no_rRNA.R1.fq:\1:" --rpl "{read2} s:.R1:.R2:" "mkdir -p $OUTDIR/{sample}; ASSEMBLIES=\$(ls -1 $WORKDIR/SRO_TransPi_assembly/results/assemblies/{sample}*[!k0-9].fa | gawk -v ORS=\",\" '1' | sed 's/,$//'); $HOME/etc/tools/orp-transrate/transrate --assembly \$ASSEMBLIES --left {} --right {read2} --threads \$SLURM_CPUS_PER_TASK --reference $REF_PROT --output $OUTDIR/{sample}" ::: $(ls -1 $WORKDIR/SRO_TransPi_assembly/input_files/*SRO*no_rRNA.R1.fq) > $JOBNAME.cmds
# --merge-assemblies {sample}.merged.fa 
# submit the job array to the cluster
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=$CONDA_NAME ${WORKDIR}/array.slurm

# use normalised (.norm.fq) files as input 
JOBNAME="transrate_norm"
OUTDIR=$WORKDIR/SRO_TransPi_assembly/results/$JOBNAME # or OUTDIR=$HOME/scratch/sandbox/SRO_TransPi_assembly/$JOBNAME
mkdir -p $OUTDIR
parallel --dry-run --rpl "{sample} s:.+/\w+-(.+).norm.fq:\1:" --rpl "{read2} s:left-:right-:" "mkdir -p $OUTDIR/{sample}; ASSEMBLIES=\$(ls -1 $WORKDIR/SRO_TransPi_assembly/results/assemblies/{sample}*[!k0-9].fa | gawk -v ORS=\",\" '1' | sed 's/,$//'); $HOME/etc/tools/orp-transrate/transrate --assembly \$ASSEMBLIES --left {} --right {read2} --threads \$SLURM_CPUS_PER_TASK --reference $REF_PROT --output $OUTDIR/{sample}" ::: $(find $WORKDIR/SRO_TransPi_assembly/work -type f -name left-*SRO*.norm.fq) > $JOBNAME.cmds
# --merge-assemblies {sample}.merged.fa 
# submit the job array to the cluster
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=$CONDA_NAME ${WORKDIR}/array.slurm


```

<!-- ### SRO transcriptome assembly with ORP -->
<!-- The SRO transcriptome of non-infected (control) tissues was also assembled with the Oyster River Protocol pipeline [@macmanesOysterRiverProtocol2018a]. -->

<!-- ```{bash orp-assembly} -->
<!-- # prepare slurm script -->
<!-- WORKDIR='/scratch/project/adna/Oyster_QX_Disease' -->
<!-- JOBNAME="SRO_orp_pipeline" -->
<!-- NCORES=24 -->
<!-- MEM=150 -->
<!-- WALLTIME=200:00:00 -->
<!-- mkdir -p $WORKDIR/$JOBNAME && cd $WORKDIR/$JOBNAME -->

<!-- echo '#!/bin/bash --login'" -->
<!-- #SBATCH --job-name=$JOBNAME -->
<!-- #SBATCH --nodes=1 -->
<!-- #SBATCH --ntasks=1 -->
<!-- #SBATCH --output=%x.%j.log -->
<!-- #SBATCH --cpus-per-task=$NCORES -->
<!-- #SBATCH --mem=${MEM}G -->
<!-- #SBATCH --time=$WALLTIME -->
<!-- #SBATCH --account=a_agri_genomics -->
<!-- #SBATCH --partition=general -->

<!-- source ~/.bashrc -->
<!-- set -Eeo pipefail -->
<!-- cd \$SLURM_SUBMIT_DIR -->

<!-- apptainer exec -B $WORKDIR:/scratch --no-mount /home $NXF_APPTAINER_CACHEDIR/docker.io-macmaneslab-orp-2.3.3.img bash -c \"source /home/orp/Oyster_River_Protocol/software/anaconda/install/bin/activate orp && unset -f which && cd /scratch/$JOBNAME && /home/orp/Oyster_River_Protocol/oyster.mk TPM_FILT=1 CPU=\$SLURM_CPUS_PER_TASK MEM=$MEM  READ1=/scratch/SRO_TransPi_assembly/input_files/unfixrm_SRO_control_R1.cor.fastq.gz READ2=/scratch/SRO_TransPi_assembly/input_files/unfixrm_SRO_control_R2.cor.fastq.gz RUNOUT=SRO_trans_orp\" " >  $JOBNAME.slurm -->

<!-- # submit the job -->
<!-- sbatch $JOBNAME.slurm -->


<!-- ``` -->


#### Gene Expression Analysis

The chosen most representative transcriptome assembly for SRO (Rcorrected SRO non-infected assembly) was used as reference to quantify gene/transcript abundance using [Salmon](https://salmon.readthedocs.io/en/latest/salmon.html) [@patroSalmonProvidesFast2017; @loveSwimmingDownstreamStatistical2018].

```{bash salmon-quant}
CONDA_NAME=genomics
mamba install -n $CONDA_NAME kallisto salmon 
WORKDIR=/home/ibar/adna/Oyster_QX_Disease
mkdir -p $WORKDIR/SRO_TransPi_assembly/Gene_expression
cd !$

JOBNAME="Salmon_index"
NCORES=12
MEM=64
WALLTIME=10:00:00
parallel --dry-run "salmon index -t {} -i {/.}.index" ::: $(ls -1 $WORKDIR/SRO_TransPi_assembly/results/assemblies/*Trinity.fa ) > $JOBNAME.cmds 

# submit the job array to the cluster
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=$CONDA_NAME ${WORKDIR}/array.slurm

DATE=`date +%d_%m_%Y`
JOBNAME="Salmon_quant_$DATE"
RUNDIR=$WORKDIR/SRO_TransPi_assembly/Gene_expression/$JOBNAME
mkdir -p $RUNDIR && cd $RUNDIR
parallel --dry-run --rpl "{sample} s:.+/(.+)_1.fastq.gz:\1:" --rpl "{read2} s:_1:_2:" "salmon quant -i $WORKDIR/SRO_TransPi_assembly/Gene_expression/unfixrm_SRO_control.Trinity.index -l A -1 {} -2 {read2} -o {sample}_salmon_eq --numBootstraps 500 --useVBOpt --seqBias --dumpEq -p \$SLURM_CPUS_PER_TASK --validateMappings" ::: $(ls -1 $WORKDIR/QX_RNA-Seq/data/NN01-5*_1.fastq.gz ) > $JOBNAME.cmds

# submit the job array to the cluster
sbatch -a 1-$(cat $JOBNAME.cmds | wc -l)%10 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=$CONDA_NAME ${WORKDIR}/array.slurm


```

Expression-based clustering of transcripts was performed with [RapClust](https://github.com/COMBINE-lab/RapClust) [@srivastavaAccurateFastLightweight2016a]. 

```{bash rapclust}
CONDA_NAME=genomics
conda activate $CONDA_NAME
mamba install mcl # need to install mcl
pip install git+https://github.com/IdoBar/RapClust@patch-1
# mamba install -n $CONDA_NAME kallisto salmon rapclust
WORKDIR=/home/ibar/adna/Oyster_QX_Disease
cd $WORKDIR/SRO_TransPi_assembly/Gene_expression/Salmon_quant_14_06_2024

# unzip the eq_classes files
find . -name eq_classes.txt.gz | parallel  "gzip -cd {} > {.}"

JOBNAME="SRO_rapclust"
NCORES=12
MEM=64
WALLTIME=10:00:00
# prepare config file for RapClust
find . -maxdepth 1 -type d -name  "NN01-*salmon_eq" | sort | gawk -F "/" 'BEGIN{printf "conditions:\n  - SRO_Control\n  - SRO_QX\nsamples:\n  SRO_Control:"}{printf "\n    - %s", $2}NR==3{printf "\n  SRO_QX:"}END{printf "\noutdir: SRO_rapclust\n"}' > $JOBNAME.yaml
# prepare SLURM script 
echo "RapClust --config $JOBNAME.yaml" > $JOBNAME.cmds
# submit the job array to the cluster
sbatch -a 1 --job-name=$JOBNAME --cpus-per-task=$NCORES --mem=${MEM}G --time=$WALLTIME --export=ALL,CMDS_FILE=$JOBNAME.cmds,CONDA_NAME=$CONDA_NAME ${WORKDIR}/array.slurm

```


## General information

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------------------------------------------------------------------------

## Bibliography