---
title: "Blast"
author: "Chris Mantegna"
date: "`r format(Sys.time(), '%d %B, %Y')`"
output: 
  html_document:
  theme: cerulean
  highlight: 
    toc: true
    toc_float: true
    number_sections: true
    code_folding: show
    code_download: true
    callout-appearance: default
---

# Blast Off! ![ba](https://github.com/course-fish546-2023/chris-coursework1/blob/main/photos/fleetweek.jpeg?raw=true){width="20%"}

The workflow to BLAST genetic sequences is similar regardless of type (i.e., genome, transcriptome, protein, gene, etc.). 'Blasting' a sequence is a fun way to compare your genetic data against the largest 'crowd sourced' database of reference genetic data available for free. Here we will walk through the process and a few things you can do to use with the data you find. 

```{bash}
cd /home/shared/8TB_HDD_02/cnmntgna/Applications/bioinfo/
curl -O https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/ncbi-blast-2.13.0+-x64-linux.tar.gz
tar -xf ncbi-blast-2.13.0+-x64-linux.tar.gz
```

```{bash}
ls /home/shared/8TB_HDD_02/cnmntgna/Applications/bioinfo/

# trying to remove macos copy, receive an error message
# made the removal happen over in the 'Terminal' access tab
# rm -r ncbi-blast-2.13.0+-x64-macosx.tar.gz
```

```{bash}
/home/shared/8TB_HDD_02/cnmntgna/Applications/bioinfo/ncbi-blast-2.13.0+/bin/blastx -h
```

```{bash}
cd ../data
curl -O https://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/uniprot_sprot.fasta.gz
mv uniprot_sprot.fasta.gz uniprot_sprot_r2023_01.fasta.gz
gunzip -k uniprot_sprot_r2023_01.fasta.gz
ls ../data
```

```{bash}
# add r engine/ false echo code for rendering in 1.1
/home/shared/8TB_HDD_02/cnmntgna/Applications/bioinfo/ncbi-blast-2.13.0+/bin/makeblastdb \
-in /home/shared/8TB_HDD_02/cnmntgna/data/uniprot_sprot_r2023_01.fasta \
-dbtype prot \
-out /home/shared/8TB_HDD_02/cnmntgna/output/blastdb/uniprot_sprot_r2023_01
```

```{bash}
curl https://eagle.fish.washington.edu/cnidarian/Ab_4denovo_CLC6_a.fa \
-k \
> /home/shared/8TB_HDD_02/cnmntgna/data/Ab_4denovo_CLC6_a.fa
```

```{bash}
head /home/shared/8TB_HDD_02/cnmntgna/data/Ab_4denovo_CLC6_a.fa
echo "How many sequences are there?"
grep -c ">" /home/shared/8TB_HDD_02/cnmntgna/data/Ab_4denovo_CLC6_a.fa
```

```{bash}
#fix my paths for reproducibility purposes, the full path is incorrect for that purpose
/home/shared/8TB_HDD_02/cnmntgna/Applications/bioinfo/ncbi-blast-2.13.0+/bin/blastx \
-query /home/shared/8TB_HDD_02/cnmntgna/data/Ab_4denovo_CLC6_a.fa \
-db /home/shared/8TB_HDD_02/cnmntgna/output/blastdb/uniprot_sprot_r2023_01 \
-out /home/shared/8TB_HDD_02/cnmntgna/output/Ab_4-uniprot_blastx.tab \
-evalue 1E-20 \
-num_threads 20 \
-max_target_seqs 1 \
-outfmt 6

#code throws the following warning: Warning: [blastx] Examining 5 or more matches is recommended
```

```{bash}
head -2 /home/shared/8TB_HDD_02/cnmntgna/output/Ab_4-uniprot_blastx.tab
wc -l /home/shared/8TB_HDD_02/cnmntgna/output/Ab_4-uniprot_blastx.tab
```

```{bash}
curl -O "Accept: text/plain; format=tsv" "https://rest.uniprot.org/uniprotkb/search?query=reviewed:true+AND+organism_id:9606"

```

---
title: "blast"
output: md_document
date: "2023-03-30"
---



# Download Software


https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/ncbi-blast-2.13.0+-x64-linux.tar.gz


```bash
cd /home/jovyan/applications
curl -O https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/ncbi-blast-2.13.0+-x64-linux.tar.gz
```

```bash
cd /home/jovyan/applications
tar -xf ncbi-blast-2.13.0+-x64-linux.tar.gz
```

```bash
~/applications/ncbi-blast-2.13.0+/bin/blastx -h
```

# Make a database

```bash
cd ../data
curl -O https://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/uniprot_sprot.fasta.gz
mv uniprot_sprot.fasta.gz uniprot_sprot_r2023_01.fasta.gz
gunzip -k uniprot_sprot_r2023_01.fasta.gz
ls ../data

```

```bash
~/applications/ncbi-blast-2.13.0+/bin/makeblastdb \
-in ../data/uniprot_sprot_r2023_01.fasta \
-dbtype prot \
-out ../blastdb/uniprot_sprot_r2023_01
```


# download query

```bash
curl https://eagle.fish.washington.edu/cnidarian/Ab_4denovo_CLC6_a.fa \
-k \
> ../data/Ab_4denovo_CLC6_a.fa
```

```{bash}
head ../data/Ab_4denovo_CLC6_a.fa
echo "How many sequences are there?"
grep -c ">" ../data/Ab_4denovo_CLC6_a.fa
```





# blast

```bash
~/applications/ncbi-blast-2.13.0+/bin/blastx \
-query ../data/Ab_4denovo_CLC6_a.fa \
-db ../blastdb/uniprot_sprot_r2023_01 \
-out ../output/Ab_4-uniprot_blastx.tab \
-evalue 1E-20 \
-num_threads 20 \
-max_target_seqs 1 \
-outfmt 6
```


# Joining with Annotation information


First need to change formate of blast output in bash...

```bash
tr '|' '\t' < ../output/Ab_4-uniprot_blastx.tab \
> ../output/Ab_4-uniprot_blastx_sep.tab
```



## Now into R


```{{r}}
library(tidyverse)
```


read in data

```{{r}}
bltabl <- read.csv("../output/Ab_4-uniprot_blastx_sep.tab", sep = '\t', header = FALSE)

spgo <- read.csv("https://gannet.fish.washington.edu/seashell/snaps/uniprot_table_r2023_01.tab", sep = '\t', header = TRUE)
```


joining




```{{r}}
left_join(bltabl, spgo,  by = c("V3" = "Entry")) %>%
  select(V1, V3, V13, Protein.names, Organism, Gene.Ontology..biological.process., Gene.Ontology.IDs) %>% mutate(V1 = str_replace_all(V1, 
            pattern = "solid0078_20110412_FRAG_BC_WHITE_WHITE_F3_QV_SE_trimmed", replacement = "Ab")) %>%
  write_delim("../output/blast_annot_go.tab", delim = '\t')
```