Alignment Data

SAMs and BAMs

Screen Recording

Assignment

Create and inspect and alignment files. Including visualizing and capturing “outside” graphics. Publish notebook in rpubs and provide link at top of code.

Task 1

Looking at Alignment Files

Download alignment data

Caution

Reminder - these are big files, be sure to ignore on commit.

{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

Visualize with tview

Important

Run the following in Terminal as is interactive

/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

Capture Image

Take a screen shot of the tview display and place in your notebook.

Task II

Aligning WGS data and visualizing in IGV

```{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
```

Alignment

{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

{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

Take a look

{r, engine='bash'} tail -1 ../output/F143_cgigas.sam

```{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
```

mpileup

Now bcftools is recommended for mpileup instead of samtools (which was described in textbook)

```{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

```


The code below might not work. That is fine. The VCF in the above chunk can be used for visualization in IGV.

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

Visualize

these data in IGV and get a few cool snapshots.

Minimally show bam file, and at least 2 genome feature files.

Bonus for annotating screenshots.

Useful link: https://robertslab.github.io/resources/Genomic-Resources/#crassostrea-gigas-cgigas_uk_roslin_v1