---
title: "13-Hisat"
author: "Steven Roberts"
date: "`r format(Sys.time(), '%d %B, %Y')`"  
analyses: 
  github_document:
    toc: true
    toc_depth: 3
    number_sections: true
    html_preview: true
  html_document:
    theme: readable
    highlight: zenburn
    toc: true
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editor_options: 
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---

```{r setup, include=FALSE}
library(knitr)
library(tidyverse)
library(kableExtra)
library(DT)
library(Biostrings)
library(tm)
library(pheatmap)
library(DESeq2)
knitr::opts_chunk$set(
  echo = TRUE,         # Display code chunks
  eval = FALSE,         # Evaluate code chunks
  warning = FALSE,     # Hide warnings
  message = FALSE,     # Hide messages
  fig.width = 6,       # Set plot width in inches
  fig.height = 4,      # Set plot height in inches
  fig.align = "center", # Align plots to the center
  comment = ""         # Prevents appending '##' to beginning of lines in code analyses
)
```

# Differentially Expressed Genes

# Reads

```{r, engine='bash', eval=TRUE}
ls /home/shared/8TB_HDD_02/graceac9/data/pycno2021/*
```

```{r, engine='bash'}
/home/shared/FastQC-0.12.1/fastqc \
/home/shared/8TB_HDD_02/graceac9/data/pycno2021/*fq.gz \
-t 36 \
-o ../analyses/13-hisat-deseq2/
```

```{r, engine='bash'}
eval "$(/opt/anaconda/anaconda3/bin/conda shell.bash hook)"
conda activate
which multiqc


multiqc ../analyses/13-hisat-deseq2/ \
-o ../analyses/13-hisat-deseq2/
```

# Genome

<https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_032158295.1/>

![](http://gannet.fish.washington.edu/seashell/snaps/Monosnap_Pycnopodia_helianthoides_genome_assembly_ASM3215829v1_-_NCBI_-_NLM_2024-05-25_15-32-13.png)

```{r, engine='bash'}
cd ../data

/home/shared/datasets download genome accession GCA_032158295.1 --include gff3,rna,cds,protein,genome,seq-report
```

```{r, engine='bash'}
cd ../data 
unzip ncbi_dataset.zip
```

```{r, engine='bash', eval=TRUE}
ls ../data/ncbi_dataset/data/GCA_032158295.1

```

## Annotation files

```{r, engine='bash', eval=TRUE}
head ../analyses/12-fix-gff/mod_augustus.gtf
head ../data/ncbi_dataset/data/GCA_032158295.1/GCA_032158295.1_ASM3215829v1_genomic.fna
```

# Hisat

```{r, engine='bash'}
/home/shared/hisat2-2.2.1/hisat2_extract_exons.py \
../analyses/12-fix-gff/mod_augustus.gtf \
> ../analyses/13-hisat-deseq2/m_exon.tab
```

```{r, engine='bash'}
/home/shared/hisat2-2.2.1/hisat2_extract_splice_sites.py \
../analyses/12-fix-gff/mod_augustus.gtf \
> ../analyses/13-hisat-deseq2/m_spice_sites.tab
```

```{r, engine='bash'}
echo "13-hisat-deseq2/GCF*" >> ../analyses/.gitignore
echo "13-hisat-deseq2/GCF**fastq" >> ../analyses/.gitignore
```

```{r, engine='bash'}
/home/shared/hisat2-2.2.1/hisat2-build \
../data/ncbi_dataset/data/GCA_032158295.1/GCA_032158295.1_ASM3215829v1_genomic.fna \
../analyses/13-hisat-deseq2/GCA_032158295.index \
--exon ../analyses/13-hisat-deseq2/m_exon.tab \
--ss ../analyses/13-hisat-deseq2/m_spice_sites.tab \
-p 20 \
../analyses/12-fix-gff/mod_augustus.gtf \
2> ../analyses/13-hisat-deseq2/hisat2-build_stats.txt
```

```{r, engine='bash'}
echo "13-hisat-deseq2/*sam" >> ../analyses/.gitignore
```

```{r, engine='bash', eval=TRUE}
find /home/shared/8TB_HDD_02/graceac9/data/pycno2021/*_R1_001.fastq.gz.fastp-trim.20220810.fq.gz | xargs basename -s _R1_001.fastq.gz.fastp-trim.20220810.fq.gz | xargs -I{} echo {}
```

keeping unmapped reads

```{r, engine='bash'}
find /home/shared/8TB_HDD_02/graceac9/data/pycno2021/*_R1_001.fastq.gz.fastp-trim.20220810.fq.gz \
| xargs -I{} basename -s _R1_001.fastq.gz.fastp-trim.20220810.fq.gz {} \
| xargs -I{} sh -c '/home/shared/hisat2-2.2.1/hisat2 \
-x ../analyses/13-hisat-deseq2/GCA_032158295.index \
--dta \
-p 32 \
-1 /home/shared/8TB_HDD_02/graceac9/data/pycno2021/{}_R1_001.fastq.gz.fastp-trim.20220810.fq.gz \
-2 /home/shared/8TB_HDD_02/graceac9/data/pycno2021/{}_R2_001.fastq.gz.fastp-trim.20220810.fq.gz \
-S ../analyses/13-hisat-deseq2/{}_03.sam \
--un-conc ../analyses/13-hisat-deseq2/{}_unmapped_reads.fastq \
> ../analyses/13-hisat-deseq2/{}_hisat03.stdout 2> ../analyses/13-hisat-deseq2/{}_hisat03.stderr'
```

Explanation xargs -I{}: This option allows you to replace {} in the command with the output from the previous command (i.e., basename).
It's used twice: first, to strip the suffix from the filenames, and second, to construct and execute the hisat2 command.

sh -c: This is used to execute a complex command within xargs.
It's necessary because the output redirection (\>, 2\>) is shell functionality, and without sh -c, xargs wouldn't handle it correctly.

```{r, engine='bash'}
echo "13-hisat-deseq2/*bam" >> ../analyses/.gitignore
echo "13-hisat-deseq2/*bam*" >> ../analyses/.gitignore
```

```{r, engine='bash'}
for samfile in ../analyses/13-hisat-deseq2/*.sam; do
  bamfile="${samfile%.sam}.bam"
  sorted_bamfile="${samfile%.sam}.sorted.bam"
  /home/shared/samtools-1.12/samtools view -bS -@ 20 "$samfile" > "$bamfile"
  /home/shared/samtools-1.12/samtools sort -@ 20 "$bamfile" -o "$sorted_bamfile"
  /home/shared/samtools-1.12/samtools index -@ 20 "$sorted_bamfile"
done
```

,

```{r, engine='bash'}
rm ../analyses/13-hisat-deseq2/*sam
```

```{r, engine='bash'}
ls ../analyses/13-hisat-deseq2/*sorted.bam | wc -l
```

# Stringtie

```{r, engine='bash'}
echo "13-hisat-deseq2/*gtf" >> ../analyses/.gitignore
```

```{bash}
/home/shared/gffread-0.12.7.Linux_x86_64/gffread \
../analyses/12-fix-gff/mod_augustus.gtf \
-T \
-o ../analyses/13-hisat-deseq2/mod_augustus.gff

```

```{r, engine='bash'}
find ../analyses/13-hisat-deseq2/*sorted.bam \
| xargs basename -s .sorted.bam | xargs -I{} \
sh -c '/home/shared/stringtie-2.2.1.Linux_x86_64/stringtie \
-p 36 \
-eB \
-G ../analyses/13-hisat-deseq2/mod_augustus.gff \
-o ../analyses/13-hisat-deseq2/{}.gtf \
../analyses/13-hisat-deseq2/{}.sorted.bam'
```

```{r, engine='bash'}
eval "$(/opt/anaconda/anaconda3/bin/conda shell.bash hook)"
conda activate
which multiqc


multiqc ../analyses/13-hisat-deseq2/ \
-o ../analyses/13-hisat-deseq2/
```