---
title: "kallisto_pgenerosa_pipeline"
author: "Olivia"
date: "2/1/2022"
output: html_document
---
#This is for juv p.generosa from Trueseq-stranded-mRNA-libraries-GeoRNA7-G1-NR006_S4_L001_R1_001.fastq.gz
library(credentials)
set_github_pat()

In this little workflow, we will be using a relatively new technology, pseudoalignment and quantification to deal with RNA-seq data from two samples. The technical steps are:

1. Use the SRA SDK to download FASTQ files for each sample
2. Build a transcriptome index for Kallisto
3. Pseudoalignment and quantification with Kallisto
4. Read Kallisto output into a SummarizedExperiment object

##Robets lab: Take our RNA-seq data, map onto genome, and describe the gene repertoire expressed at different stages. Do everthing on Raven. 

Step 1: ID all relevant datasets

Step 2: QC data

Step 3: Map to genome to get expression / count data.

Step 4: Functional annotation of genes

```{bash}
#download geoduck transcriptome from genomic databank, do this only once
curl --insecure \
-O https://owl.fish.washington.edu/halfshell/genomic-databank/Pgenerosa_transcriptome_v5.fasta
```

```{bash}
pwd #find where you are in your directory, move data to better location
ls
#mv ../Pgenerosa_transcriptome_v5.fasta ../data/Pgenerosa_transcriptome_v5.fasta
mv Pgenerosa_transcriptome_v5.fasta 
```

```{bash}
#download Read 1 and 2 from nightingales spreadsheet 
cd ~/gitrepos/kallisto/data
curl --insecure \
-O http://owl.fish.washington.edu/nightingales/P_generosa/Trueseq-stranded-mRNA-libraries-GeoRNA1-A1-NR006_S1_L001_R1_001.fastq.gz
curl --insecure \
-O http://owl.fish.washington.edu/nightingales/P_generosa/Trueseq-stranded-mRNA-libraries-GeoRNA1-A1-NR006_S1_L001_R2_001.fastq.gz
```


```{bash}
#load kallisto
/home/shared/kallisto/kallisto \
index -i data/transcriptome_v5.idx data/Pgenerosa_transcriptome_v5.fasta
#press open when MacOs security message pops up
```

```{bash}
/home/shared/kallisto/kallisto quant \
-i /home/olivia/gitrepos/kallisto/data/transcriptome_v5.idx \
-o /home/olivia/gitrepos/kallisto/analyses/gonad-Trueseq-stranded-mRNA-libraries-NR006_1 \
-b 100 \
/home/olivia/gitrepos/kallisto/data/Trueseq-stranded-mRNA-libraries-GeoRNA1-A1-NR006_S1_L001_R1_001.fastq.gz \
/home/olivia/gitrepos/kallisto/data/Trueseq-stranded-mRNA-libraries-GeoRNA1-A1-NR006_S1_L001_R2_001.fastq.gz
```

```{bash}
rsync -avP ~/gitrepos/kallisto/analyses/gonad-Trueseq-stranded-mRNA-libraries-NR006_1  ocattau@gannet.fish.washington.edu:/volume2/web/gigas/analyses/
#push whole folder to gannet
```