BICF Nanocourse: Gene Expression and Regulation (06/06/2019)

Your account and password are trainXX and passwordXX, respectively.

Today we are going to: First, we will log into a compute node:

  1. Open https://portal.biohpc.swmed.edu/accounts/login/?next=terminal/webgui/ using a web browser

  2. Input your training ID (trainXX) and password (passwordXX)

  3. Go to Cloud Services, then click Web Visualization

  4. Click the Connect via web link to launch your web visualization GUI

Note: The Linux commands shown in red need to be executed correctly in your home directory, while the other commands shown in black are optional for practice.

List of Linux commands

  1. Display the current working directory
    pwd

  2. Change directory to /usr
    cd /usr

  3. Change directory to /archive/nanocourse/gene_expr/trainXX , where trainXX is your account
    cd /archive/nanocourse/gene_expr/trainXX

  4. Change directory to your home directory
    cd ~

  5. Display files under the current directory
    ls -l

  6. Display the files to be used during this session
    ls -l /archive/nanocourse/gene_expr/shared/session1

  7. Make a shortcut to the course files
    ln -s /archive/nanocourse/gene_expr/shared/session1 session1

The file name and path to the real single-end sequencing reads for this workshop is:
 session1/reads/RNA.heart.e11.rep1.fastq.gz

To examine the file, you can optionally perform the following commands:

  1. Extract a compressed read file and redirect to a text file
    gzip -cd session1/reads/RNA.heart.e11.rep1.fastq.gz > RNA.heart.e11.rep1.fastq

  2. Display the text file; to stop the command, type ctrl+c
    cat RNA.heart.e11.rep1.fastq

  3. Display the first 10 lines of the read file
    head RNA.heart.e11.rep1.fastq

  4. Display the first page of the read file; type space to go to the next page, and type q to quit
    less RNA.heart.e11.rep1.fastq

  5. Display the first page of the read file directly on the compressed file using pipe |
    gzip -cd session1/reads/RNA.heart.e11.rep1.fastq.gz | less

  6. Count the number of lines in a read file
    wc -l RNA.heart.e11.rep1.fastq

  7. Divide the number of lines we calculated above by 4 to get the number of reads
    expr 113425724 / 4

Now we are going to run our alignment using the single-end reads against the mouse genome, mm10 (this alignment step using HISAT2 might take 15 to 20 minutes)

  1. We will use HISAT2 with a graph index to align reads as follows:
    session1/programs/hisat2 -p 8 -x session1/indexes/genome_snp_tran -U session1/reads/RNA.heart.e11.rep1.fastq.gz > heart.e11.sam

Next we will view the results of the alignment using samtools in two different formats - SAM (Sequence Alignment & Mapping) and BAM (Binary version of SAM):

  1. Look at the SAM file (use space to go to the next page and q to quit)
    less heart.e11.sam

  2. Convert the SAM file into a BAM file
    session1/programs/samtools view -@ 8 -bS heart.e11.sam > heart.e11.unsorted.bam

  3. Create a sorted BAM file
    session1/programs/samtools sort -@ 8 heart.e11.unsorted.bam -o heart.e11.sorted.bam

  4. Make an index for the sorted BAM file
    session1/programs/samtools index heart.e11.sorted.bam

  5. Look at the sorted alignments
    session1/programs/samtools view heart.e11.sorted.bam | less

  6. Look at alignments between 61,989,341 and 61,990,361 on Chromosome 15
    session1/programs/samtools view heart.e11.sorted.bam 15:61,989,341-61,990,361

  7. Look at bases of reads located at a particular locus to identify variants
    session1/programs/samtools mpileup -f session1/indexes/genome.fa -r 15:61,989,341-61,990,361 heart.e11.sorted.bam

Now we will look at the alignment using IGV (Integrative Genomics Viewer):

  1. Run IGV
    module add IGV/2.3.90
    igv.sh

  2. Load the genome using: Genomes -> Load from Server -> Select Mouse mm10

  3. Open the BAM file using File -> Load From File in IGV and choose heart.e11.sorted.bam