---
title: "FISH546 Week 6: Alignment Assignment"
author: "Sam Cryan"
date: "`r format(Sys.time(), '%d %B, %Y')`" 
output: 
  html_document:
    theme: journal
    toc: true
    toc_float: true
    number_sections: true
    code_folding: show
---

```{r, engine='bash'}         
cd ../data
curl -O https://gannet.fish.washington.edu/seashell/bu-mox/scrubbed/120321-cvBS/19F_R1_val_1_bismark_bt2_pe.deduplicated.sorted.bam
curl -O https://gannet.fish.washington.edu/seashell/bu-mox/scrubbed/120321-cvBS/19F_R1_val_1_bismark_bt2_pe.deduplicated.sorted.bam.bai
```

```{r, engine='bash'}         
cd ../data
curl -O https://gannet.fish.washington.edu/seashell/bu-mox/data/Cvirg-genome/GCF_002022765.2_C_virginica-3.0_genomic.fa
curl -O https://gannet.fish.washington.edu/seashell/bu-mox/data/Cvirg-genome/GCF_002022765.2_C_virginica-3.0_genomic.fa.fai
```
The interactive section - run in terminal

/home/shared/samtools-1.12/samtools tview \
../data/19F_R1_val_1_bismark_bt2_pe.deduplicated.sorted.bam \
../data/GCF_002022765.2_C_virginica-3.0_genomic.fa

PASTE IMAGE HERE

PART 2:
```{r, engine='bash'}
cd ../data
curl -O https://owl.fish.washington.edu/nightingales/C_gigas/F143n08_R2_001.fastq.gz
curl -O https://owl.fish.washington.edu/nightingales/C_gigas/F143n08_R1_001.fastq.gz
```

```{r, engine='bash'}
cd ../data
curl -O https://gannet.fish.washington.edu/panopea/Cg-roslin/cgigas_uk_roslin_v1_genomic-mito.fa
curl -O https://gannet.fish.washington.edu/panopea/Cg-roslin/cgigas_uk_roslin_v1_genomic-mito.fa.fai
curl -O https://gannet.fish.washington.edu/panopea/Cg-roslin/GCF_902806645.1_cgigas_uk_roslin_v1_genomic-mito.gtf
```
```{r, engine='bash'}
/home/shared/hisat2-2.2.1/hisat2-build \
-f ../data/cgigas_uk_roslin_v1_genomic-mito.fa \
../output/cgigas_uk_roslin_v1_genomic-mito.index
```

Index the fastq file to make a sam
```{r, engine='bash'}
/home/shared/hisat2-2.2.1/hisat2 \
-x ../output/cgigas_uk_roslin_v1_genomic-mito.index \
-p 4 \
-1 ../data/F143n08_R1_001.fastq.gz \
-2 ../data/F143n08_R2_001.fastq.gz \
-S ../output/F143_cgigas.sam
```


Convert the SAM to a BAM
```{r, engine='bash'}
# Convert SAM to BAM, using 4 additional threads
/home/shared/samtools-1.12/samtools view -@ 4 -bS \
../output/F143_cgigas.sam > ../output/F143_cgigas.bam
```

```{r, engine='bash'}
# Sort the BAM file, using 4 additional threads
/home/shared/samtools-1.12/samtools sort -@ 4 \
../output/F143_cgigas.bam -o ../output/F143_cgigas_sorted.bam

# Index the sorted BAM file (multi-threading is not applicable to this operation)
/home/shared/samtools-1.12/samtools index \
../output/F143_cgigas_sorted.bam
```


```{r, engine='bash'}
/home/shared/bcftools-1.14/bcftools mpileup --threads 4 --no-BAQ \
--fasta-ref ../data/cgigas_uk_roslin_v1_genomic-mito.fa \
../output/F143_cgigas_sorted.bam > ../output/F143_mpileup_output.txt
```

```{r, engine='bash'}
tail ../output/F143_mpileup_output.txt
```

```{r, engine='bash'}
cat ../output/F143_mpileup_output.txt | /home/shared/bcftools-1.14/bcftools call -mv -Oz > ../output/F143_mpile.vcf.gz
```


```{r, engine='bash'}
zgrep "^##" -v ../output/F143_mpile.vcf.gz | \
awk 'BEGIN{OFS="\t"} {split($8, a, ";"); print $1,$2,$4,$5,$6,a[1],$9,$10}' | head

```


```{r, engine='bash'}
/home/shared/bcftools-1.14/bcftools call \
-v -c ../output/F143_mpile.vcf.gz \
> ../output/F143_mpile_calls.vcf
```