--- title: "A pulcra HiSat2" author: Steven Roberts date: "`r format(Sys.time(), '%d %B, %Y')`" output: html_document: theme: readable highlight: zenburn toc: true toc_float: true number_sections: true code_folding: show code_download: true --- ```{r setup, include=FALSE} library(knitr) library(tidyverse) knitr::opts_chunk$set( echo = TRUE, # Display code chunks eval = FALSE, # Evaluate code chunks warning = FALSE, # Hide warnings message = FALSE, # Hide messages fig.width = 6, # Set plot width in inches fig.height = 4, # Set plot height in inches fig.align = "center" # Align plots to the center ) ``` #millipora genome prep ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2_extract_exons.py \ ../data/Amil/ncbi_dataset/data/GCF_013753865.1/genomic.gtf \ > ../output/04-Apulcra-hisat/m_exon.tab ``` ```{r, engine='bash'} head ../output/04-Apulcra-hisat/m_exon.tab ``` ```{r, engine='bash'} #!/bin/bash # This script will extract splice sites from the gtf file # This is the command to extract splice sites from the gtf file /home/shared/hisat2-2.2.1/hisat2_extract_splice_sites.py \ # This is the gtf file ../data/Amil/ncbi_dataset/data/GCF_013753865.1/genomic.gtf \ # This is the output file > ../output/04-Apulcra-hisat/m_splice_sites.tab ``` ``` "${programs_array[hisat2_build]}" \ "${genome_fasta}" \ "${genome_index_name}" \ --exon "${exons}" \ --ss "${splice_sites}" \ -p "${threads}" \ 2> hisat2-build_stats.txt ``` ```{r, engine='bash'} #!/bin/bash # hisat2-build is a program that builds a hisat2 index for the reference genome # ../data/Amil/ncbi_dataset/data/GCF_013753865.1/GCF_013753865.1_Amil_v2.1_genomic.fna is the reference genome # GCF_013753865.1_Amil_v2.1 is the name of the index # --exon ../output/04-Apulcra-hisat/m_exon.tab is the exon file # --ss ../output/04-Apulcra-hisat/m_splice_sites.tab is the splice site file # -p 40 is the number of threads # ../data/Amil/ncbi_dataset/data/GCF_013753865.1/genomic.gtf is the gtf file # 2> ../output/04-Apulcra-hisat/hisat2-build_stats.txt is the output file /home/shared/hisat2-2.2.1/hisat2-build \ ../data/Amil/ncbi_dataset/data/GCF_013753865.1/GCF_013753865.1_Amil_v2.1_genomic.fna \ GCF_013753865.1_Amil_v2.1 \ --exon ../output/04-Apulcra-hisat/m_exon.tab \ --ss ../output/04-Apulcra-hisat/m_splice_sites.tab \ -p 40 \ ../data/Amil/ncbi_dataset/data/GCF_013753865.1/genomic.gtf \ 2> ../output/04-Apulcra-hisat/hisat2-build_stats.txt ``` # Alignment ``` Hisat2 alignments "${programs_array[hisat2]}" \ -x "${genome_index_name}" \ -1 "${fastq_list_R1}" \ -2 "${fastq_list_R2}" \ -S "${sample_name}".sam \ 2> "${sample_name}"-hisat2_stats.txt ``` ```{r, engine='bash'} #!/bin/bash comment with GPT Studio # run hisat2 to align reads to the reference genome /home/shared/hisat2-2.2.1/hisat2 \ # specify the reference genome -x ../output/04-Apulcra-hisat/GCF_013753865.1_Amil_v2.1 \ -p 48 \ -1 ../data/SRR8601366_1.fastq \ -2 ../data/SRR8601366_2.fastq \ -S ../output/04-Apulcra-hisat/SRR8601366_mil.sam \ 2>&1 | tee ../output/04-Apulcra-hisat/hisat2_stats.txt ``` ### chatgpt comment test - same chunk as above this with ChatGPT not inline This R code is actually a command-line script written in Bash which can be run in the terminal or within an R script using the `system()` or `system2()` commands. It runs the program HISAT2 with specific options and parameters on paired-end RNA-seq data for a specific species of organism called *Acanthopleura granulata* (also known as *Acmaea intestinalis*) to align the sequencing reads to its reference genome. --- ChatGPT output: This code is running the HISAT2 alignment tool, which is used to align RNA sequencing reads to a reference genome. It is specifying the following options: `-x` specifies the reference genome index to use for the alignment. `-p` specifies the number of CPU cores to use in the alignment. `-1` and `-2` specify the input paired-end fastq read files to align. `-S` specifies the output SAM file to save the alignment results. `2>&1 | tee` specifies that both stdout and stderr are to be written to a file `../output/04-Apulcra-hisat/hisat2_stats.txt`, while also being printed to the console for the user to see. The code is assuming that the input fastq files are located at `../data/SRR8601366_1.fastq` and `../data/SRR8601366_2.fastq`, and that the reference genome index is located at `/home/shared/hisat2-2.2.1/hisat2`. The aligned output will be saved to a SAM file located at `../output/04-Apulcra-hisat/SRR8601366_mil.sam`. ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2 \ -x ../output/04-Apulcra-hisat/GCF_013753865.1_Amil_v2.1 \ -p 48 \ -1 ../data/SRR8601366_1.fastq \ -2 ../data/SRR8601366_2.fastq \ -S ../output/04-Apulcra-hisat/SRR8601366_mil.sam \ 2>&1 | tee ../output/04-Apulcra-hisat/hisat2_stats.txt ``` ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2 \ -x ../output/04-Apulcra-hisat/GCF_013753865.1_Amil_v2.1 \ -p 48 \ -1 ../data/SRR8601366_1.fastq \ -2 ../data/SRR8601366_2.fastq \ -S ../output/04-Apulcra-hisat/SRR8601366_mil.sam \ 2>&1 | tee ../output/04-Apulcra-hisat/hisat2_stats.txt ``` ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2 \ -x ../output/04-Apulcra-hisat/GCF_013753865.1_Amil_v2.1 \ -p 48 \ -1 ../data/SRR8601366_1.fastq \ -2 ../data/SRR8601366_2.fastq \ -S ../output/04-Apulcra-hisat/SRR8601366_mil.sam \ 2>&1 | tee ../output/04-Apulcra-hisat/hisat2_stats.txt ``` # Align with no splice sites ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2-build \ ../data/Amil/ncbi_dataset/data/GCF_013753865.1/GCF_013753865.1_Amil_v2.1_genomic.fna \ GCF_013753865.1_Amil_v2.1_nosplice \ -p 48 \ 2>&1 | tee ../output/04-Apulcra-hisat/hisat2-build_stats_nosplice.txt ``` ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2 \ -x ../output/04-Apulcra-hisat/GCF_013753865.1_Amil_v2.1_nosplice \ -p 48 \ -1 ../data/SRR8601366_1.fastq \ -2 ../data/SRR8601366_2.fastq \ -S ../output/04-Apulcra-hisat/SRR8601366_mil_nosplice.sam \ 2>&1 | tee ../output/04-Apulcra-hisat/hisat2_stats_nosplice.txt ``` # A hyacinthus ## no splice ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2-build \ ../data/Ahya/ncbi_dataset/data/GCA_020536085.1/GCA_020536085.1_Ahyacinthus.chrsV1_genomic.fna \ ../output/04-Apulcra-hisat/GCA_020536085.1_Ahya_nosplice \ -p 48 \ 2> ../output/04-Apulcra-hisat/hisat2-build_stats_Ahya_nosplice.txt ``` ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2 \ -x ../output/04-Apulcra-hisat/GCA_020536085.1_Ahya_nosplice \ -p 48 \ -1 ../data/SRR8601366_1.fastq \ -2 ../data/SRR8601366_2.fastq \ -S ../output/04-Apulcra-hisat/SRR8601366_hya_nosplice.sam \ 2>&1 | tee ../output/04-Apulcra-hisat/hisat2_stats_Ahya_nosplice.txt ``` # A digitera ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2_extract_exons.py \ ../data/Adig/ncbi_dataset/data/GCF_000222465.1/genomic.gtf \ > ../output/04-Apulcra-hisat/d_exon.tab ``` ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2_extract_splice_sites.py \ ../data/Adig/ncbi_dataset/data/GCF_000222465.1/genomic.gtf \ > ../output/04-Apulcra-hisat/d_splice_sites.tab ``` ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2-build \ ../data/Adig/ncbi_dataset/data/GCF_000222465.1/GCF_000222465.1_Adig_1.1_genomic.fna \ ../output/04-Apulcra-hisat/GCF_000222465.1_Adig \ --exon ../output/04-Apulcra-hisat/d_exon.tab \ --ss ../output/04-Apulcra-hisat/d_splice_sites.tab \ -p 48 \ ../data/Adig/ncbi_dataset/data/GCF_000222465.1/genomic.gtf \ 2> ../output/04-Apulcra-hisat/hisat2-build_stats_Adig.txt ``` ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2 \ -x ../output/04-Apulcra-hisat/GCF_000222465.1_Adig \ -p 48 \ -1 ../data/SRR8601366_1.fastq \ -2 ../data/SRR8601366_2.fastq \ -S ../output/04-Apulcra-hisat/SRR8601366_dig.sam \ 2>&1 | tee ../output/04-Apulcra-hisat/hisat2_stats_dig.txt ``` ## dig no splice ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2-build \ ../data/Adig/ncbi_dataset/data/GCF_000222465.1/GCF_000222465.1_Adig_1.1_genomic.fna \ ../output/04-Apulcra-hisat/GCF_000222465.1_Adig_nosplice \ -p 48 \ 2> ../output/04-Apulcra-hisat/hisat2-build_stats_Adig.txt ``` ```{r, engine='bash'} /home/shared/hisat2-2.2.1/hisat2 \ -x ../output/04-Apulcra-hisat/GCF_000222465.1_Adig_nosplice \ -p 48 \ -1 ../data/SRR8601366_1.fastq \ -2 ../data/SRR8601366_2.fastq \ -S ../output/04-Apulcra-hisat/SRR8601366_dig_nosplice.sam \ 2>&1 | tee ../output/04-Apulcra-hisat/hisat2_stats_dig_nosplice.txt ```